US20030191077A1 - Method and reagent for the treatment of asthma and allergic conditions

Info

Abstract

Description

Claims

US20030191077A1

Publication number: US20030191077A1
Application number: US10/230,006
Authority: US
Inventors: Kathy Fosnaugh; James McSwiggen
Original assignee: Ribozyme Pharmaceuticals Inc
Current assignee: Sirna Therapeutics Inc
Priority date: 2001-04-05
Filing date: 2002-08-28
Publication date: 2003-10-09
Also published as: US20060154271A1; US20070026394A1; EP1386004A2; US20050261212A1; WO2002081628A2; WO2002081628A8; US20030143732A1; EP1386004A4; US20030148507A1; US20030119017A1; WO2002081628A3; US7022828B2

The present invention relates to nucleic acid molecules, including antisense, enzymatic nucleic acid molecules, and RNA interference molecules, such as hammerhead ribozymes, DNAzymes, allozymes, siRNA, decoys and antisense, which modulate the expression of prostaglandin D2 (PTGDS), prostaglandin D2 receptor (PTGDR), and adenosine receptor genes.

PRIORITY

This application claims the benefit of U.S. Application Ser. No. 60/315,315, filed on Aug. 28, 2001, which is herein incorporated by reference in its entirity.[0001]

BACKGROUND OF THE INVENTION

The present invention relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to allergic response. Specifically, the invention provides compositions and methods for the treatment of diseases or conditions related to levels of factors involved in allergic conditions such as asthma, for example prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS) and adenosine A1 receptor (ADORA1). The discussion is provided only for understanding of the invention that follows. This summary is not an admission that any of the work described below is prior art to the claimed invention.

Asthma is a chronic inflammatory disorder of the lungs characterized by airflow obstruction, bronchial hyper-responsiveness, and airway inflammation. T-lymphocytes that produce TH2 cytokines and cosinophilic leukocytes infiltrate the airways. In the airway and in bronchial alveolar lavage (BAL) fluid of individuals with asthma, high concentrations of TH2 cytokines, interleukin-4 (IL-4), IL-5, and IL-13, are present along with increased levels of adenosine. In contrast to normal individuals, asthmatics respond to adenosine challenge with marked airway obstruction. Upon allergen challenge, mast cells are activated by cross-linked IgE-allergen complexes. Large amounts of prostaglandin D2 (PGD2), the major cyclooxygenase product of arachidonic acid are released. PGD2 is generated from PGH2 via the activity of prostaglandin D2 synthetase (PTGDS). PGD2 receptors and adenosine A1 receptors are present in the lungs and airway along with various other tissues in response to allergic stimuli (Howarth, 1997 , Allergy, 52, 12).

The significance of PGD2 as a mediator of allergic asthma has been established with the development of mice deficient in the PGD2 receptor (DP). DP is a heterotrimeric GTP-binding protein-coupled, rhodopsin-type receptor specific for PGD2 (Hirata et al., 1994 , PNAS USA., 91, 11192). These mice fail to develop airway hyperreactivity and have greatly reduced eosinophil infiltration and cytokine accumulation in response to allergens. Upon allergen challenge mice deficient in the prostaglandin D2 (PGD2) receptor (DP) did not develop airway hyperactivity. Cytokine, lymphocyte and eosinophil accumulation in the lungs were greatly reduced (Matsuoka et al., 2000, Science, 287, 2013). The DP −/− mice exhibited no behavioral, anatomic, or histological abnormalities. Primary immune response is not affected by DP disruption.

Asthma affects more than 100 million people worldwide and more than 17 million Americans (5% of the population). Since 1980 the incidence has more than doubled and deaths have tripled (5,000 deaths in 1995). Annual asthma-related healthcare costs in the US alone were estimated to exceed $14.5 billion in 2000. Current therapies such as inhalant anti-inflammatories and bronchodilators can be used to treat symptoms, however, these therapies do not prevent or cure asthma.

Sandberg et al., 2001 , Prog. Respir. Res., 31, 370-373, describes ribozyme therapy for asthma and COPD.

Sullivan et al., International U.S. Pat. No. 5,616,488, describes ribozymes targeting interleukin-5 for treatment and diagnosis of asthma and other inflammatory disorders.

Stinchcomb et al, International PCT Publication No. WO 95/23225, describes ribozymes and methods for inhibiting the expression of disease related genes including genes associated with asthma.

Nyce, International PCT Publication Nos. WO 00/62736, WO 00/09525, WO 99/13886, WO 98/23294, WO 96/40266 and U.S. Pat. No. 6,025,339 describe specific antisense oligonucleotides targeting certain mRNAs encoding particular adenosine receptors.

SUMMARY OF THE INVENTION

The invention features novel nucleic acid-based molecules, for example, enzymatic nucleic acid molecules, allozymes, antisense nucleic acids, 2-5A antisense chimeras, triplex forming oligonucleotides, decoy RNA, dsRNA, siRNA, aptamers, and antisense nucleic acids containing RNA cleaving chemical groups, and methods to modulate gene expression, for example, genes encoding prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), and adenosine receptors (AR) such as adenosine receptor A1, A2a, A2b, and A3. In particular, the instant invention features nucleic-acid based molecules and methods to modulate the expression of PTGDR, PTGDS, and adenosine A1 receptor (ADORA1).

In one embodiment, the invention features one or more nucleic acid-based molecules and methods that independently or in combination modulate the expression of gene(s) encoding prostaglandin D2 receptors (PTGDR), prostaglandin D2 synthetase (PTGDS) and adenosine receptors such as ADORA1. Specifically, the present invention features nucleic acid molecules that modulate the expression of prostaglandin D2 receptor (PTGDR) gene, for example Genbank Accession Nos. U31332 and U31099, prostaglandin D2 synthetase (PTGDS) gene, for example Genbank Accession No. NM _—000954, and Adenosine A1 receptor (ADORA1), for example Genbank Accession No. NM_—000674.

The description below of the various aspects and embodiments is provided with reference to the exemplary prostaglandin D2 receptor (PTGDR), prostaglandin D2 synthetase (PTGDS), and adenosine A1 receptor (ADORA1). However, the various aspects and embodiments are also directed to other genes that express prostaglandin proteins and other receptors involved in allergic reactions. Those additional genes can be analyzed for target sites using the methods described for PTGDS, PTGDR, and ADORA1. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.

In another embodiment, the invention features an enzymatic nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 228-454, 831-1206, 1438-1668, 1715-2057, and 2247-2666. In yet another embodiment, the invention features an enzymatic nucleic acid molecule comprising at least one binding arm wherein one or more of said binding arms comprises a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-227, 455-830, 1207-1437, 1669-1714, and 2058-2246.

In one embodiment, the invention features an antisense nucleic acid molecule comprising a sequence complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-227, 455-830, 1207-1437, 1669-1714, and 2058-2246.

In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention is adapted to treat asthma.

In one embodiment, an enzymatic nucleic acid molecule of the invention has an endonuclease activity to cleave RNA encoded by a PTGDS and/or PTGDR gene.

In another embodiment, an enzymatic nucleic acid molecule of the invention is in a hammerhead, Inozyme, Zinzyme, DNAzyme, Amberzyme, or G-cleaver configuration.

In another embodiment, an enzymatic nucleic acid molecule of the invention having a hammerhead configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 1-227. In yet another embodiment, an enzymatic nucleic acid molecule of invention having a hammerhead configuration comprises a sequence having SEQ ID NOs: 228-454.

In another embodiment, an enzymatic nucleic acid molecule of the invention having an Inozyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 455-830. In yet another embodiment, an enzymatic nucleic acid molecule of invention having an Inozyme configuration comprises a sequence having SEQ ID NOs: 831-1206.

In another embodiment, an enzymatic nucleic acid molecule of the invention having a Zinzyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 1207-1437. In yet another embodiment, an enzymatic nucleic acid molecule of invention having a Zinzyme configuration comprises a sequence having SEQ ID NOs: 1438-1668.

In another embodiment, an enzymatic nucleic acid molecule of the invention having a DNAzyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 1, 13, 55, 69, 74, 104, 112, 120, 123, 128, 131, 138, 147, 154, 157, 158, 169, 188, 192, 208, 221, 463, 475, 489, 505, 527, 541, 552, 554, 561, 563, 572, 591, 601, 605, 627, 637, 645, 652, 653, 661, 668, 669, 670, 676, 692, 699, 706, 719, 725, 732, 737, 741, 747, 763, 774, 782, 800, 805, 807, 816, 818, 823, 827, 828, 1207-1437, and 1669-1714. In yet another embodiment, an enzymatic nucleic acid molecule of invention having a DNAzyme configuration comprises a sequence having SEQ ID NOs: 1715-2057.

In another embodiment, an enzymatic nucleic acid molecule of the invention having an Amberzyme configuration comprises a sequence complementary to a sequence having SEQ ID NOs: 1207-1437, and 2058-2246. In yet another embodiment, an enzymatic nucleic acid molecule of invention having an Amberzyme configuration comprises a sequence having SEQ ID NOs: 2247-2666.

In one embodiment, an enzymatic nucleic acid molecule of the invention comprises between 8 and 100 bases complementary to the RNA of PTGDS, ADORA1 and/or PTGDR gene. In another embodiment, an enzymatic nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to a RNA molecule of a PTGDS or PTGDR gene.

In one embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention is chemically synthesized.

In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one 2′-sugar modification.

In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one nucleic acid base modification.

In another embodiment, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acids containing RNA cleaving chemical groups of the invention comprises at least one phosphate backbone modification.

In one embodiment, the invention features a mammalian cell, for example a human cell, including the enzymatic nucleic acid molecule of the invention.

In another embodiment, the invention features a method of reducing PTGDS, ADORA1 and/or PTGDR expression or activity in a cell, comprising contacting the cell with an enzymatic nucleic acid molecule of the invention, under conditions suitable for the reduction.

In another embodiment, the invention features a method of reducing PTGDS, ADORA1 and/or PTGDR expression or activity in a cell, comprising the step of contacting the cell with an antisense nucleic acid molecule of the invention under conditions suitable for the reduction.

In yet another embodiment, the invention features a method of treatment of a patient having a condition associated with the level of PTGDS, ADORA1 and/or PTGDR, comprising contacting cells of the patient with an enzymatic nucleic acid molecule of the invention, under conditions suitable for the treatment.

In one embodiment, the invention features a method of treatment of a patient having a condition associated with the level of PTGDS, ADORA1 and/or PTGDR, comprising contacting cells of the patient with an antisense nucleic acid molecule of the invention, under conditions suitable for the treatment.

In another embodiment, a method of treatment of a patient having a condition associated with the level of PTGDS, ADORA1 and/or PTGDR is featured, wherein the method further comprises the use of one or more drug therapies under conditions suitable for the treatment.

For example, in one embodiment, the invention features a method for treatment of asthma, allergic rhinitis, or atopic dermatitis under conditions suitable for the treatment.

In another embodiment, the invention features a method of cleaving a RNA molecule of PTGDS, ADORA1 and/or PTGDR gene comprising contacting an enzymatic nucleic acid molecule of the invention with a RNA molecule of a PTGDS, ADORA1 and/or PTGDR gene under conditions suitable for the cleavage, for example, wherein the cleavage is carried out in the presence of a divalent cation, such as Mg ²⁺.

In one embodiment, an enzymatic nucleic acid molecule of the invention comprises a cap structure, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end of the enzymatic nucleic acid molecule.

In another embodiment, an antisense nucleic acid molecule of the invention comprises a cap structure, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative, wherein the cap structure is at the 5′-end, or 3′-end, or both the 5′-end and the 3′-end of the antisense nucleic acid molecule.

In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one enzymatic nucleic acid molecule of the invention, in a manner which allows expression of the nucleic acid molecule.

In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the invention further comprises a sequence for an antisense nucleic acid molecule complementary to a RNA molecule of a PTGDS, ADORA1 and/or PTGDR gene.

In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more enzymatic nucleic acid molecules, which can be the same or different.

In another embodiment, the invention features a method for treatment of asthma, allergic rhinitis, or atopic dermatitis, comprising administering to a patient an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention, under conditions suitable for the treatment, including administering to the patient one or more other therapies, for example, inhalant anti-inflammatories, bronchodilators, adenosine inhibitors and adenosine A1 receptor inhibitors.

In one embodiment, the method of treatment features an enzymatic nucleic acid molecule or antisense nucleic acid molecule of the invention comprises at least five ribose residues, at least ten 2′-O-methyl modifications, and a 3′-end modification, such as a 3′-3′ inverted abasic moiety. In another embodiment, an enzymatic nucleic acid molecule or antisense nucleic acid molecule of the invention further comprises phosphorothioate linkages on at least three of the 5′ terminal nucleotides.

In another embodiment, the invention features a method of administering to a mammal, for example a human, an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention, comprising contacting the mammal with the nucleic acid molecule under conditions suitable for the administration, for example, in the presence of a delivery reagent such as a lipid, cationic lipid, phospholipid, or liposome.

In yet another embodiment, the invention features a method of administering to a mammal an enzymatic nucleic acid molecule, antisense nucleic acid molecule, 2-5A antisense chimera, triplex forming oligonucleotide, decoy RNA, dsRNA, siRNA, aptamer, or antisense nucleic acid containing RNA cleaving chemical groups of the invention in conjunction with a therapeutic agent, comprising contacting the mammal, for example a human, with the nucleic acid molecule and the therapeutic agent under conditions suitable for the administration.

In one embodiment, the invention features the use of an enzymatic nucleic acid molecule, which can be in a hammerhead, NCH, G-cleaver, Amberzyme, Zinzyme, and/or DNAzyme motif, to down-regulate the expression of a PTGDS, an ADORA1 and/or a PTGDR gene.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, such as PTGDS, ADORA1 and/or PTGDR proteins or PTGDS, ADORA1 and/or PTGDR subunit(s), is reduced below that observed in the absence of the nucleic acid molecules of the invention. In one embodiment, inhibition, down-regulation or reduction with an enzymatic nucleic acid molecule is below that level observed in the presence of an enzymatically inactive or attenuated molecule that is able to bind to the same site on the target RNA molecule, but is unable to cleave that RNA molecule. In another embodiment, inhibition, down-regulation, or reduction with antisense oligonucleotides is below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches. In another embodiment, inhibition, down-regulation, or reduction of PTGDS, ADORA1 and/or PTGDR with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.

By “up-regulate” is meant that the expression of a gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins, protein subunits, or activity of one or more proteins or protein subunits, such as PTGDS, ADORA1 and/or PTGDR proteins or PTGDS, ADORA1 and/or PTGDR subunits, is greater than that observed in the absence of the nucleic acid molecules of the invention. For example, the expression of a gene, such as PTGDS, ADORA1 and/or PTGDR gene, can be increased in order to treat, prevent, ameliorate, or modulate a pathological condition caused or exacerbated by an absence or low level of gene expression.

By “modulate” is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more protein subunits, or activity of one or more protein subunits is up-regulated or down-regulated, such that the expression, level, or activity is greater than or less than that observed in the absence of a nucleic acid molecule of the invention.

By “enzymatic nucleic acid molecule” it is meant a nucleic acid molecule that has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that is active to specifically cleave target a RNA molecule. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave a RNA molecule and thereby inactivate a target RNA molecule. These complementary regions allow sufficient hybridization of an enzymatic nucleic acid molecule to a target RNA molecule and thus permit cleavage. One hundred percent complementarity is preferred, but complementarity as low as 50-75% can also be useful in this invention (see for example Werner and Uhlenbeck, 1995 , Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). The nucleic acids can be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The specific enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site that is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al, U.S. Pat. No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).

By “nucleic acid molecule” as used herein is meant a molecule having nucleotides.

The nucleic acid can be single, double, or multiple stranded and can comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.

By “enzymatic portion” or “catalytic domain” is meant that portion/region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate (for example see FIGS. 1-4).

By “substrate binding arm” or “substrate binding domain” is meant that portion/region of a enzymatic nucleic acid that is able to interact, for example via complementarity (i.e., able to base-pair with), with a portion of its substrate. Such complementarity can be 100%, but can be less if desired. For example, as few as 10 bases out of 14 can be base-paired (see for example Werner and Uhlenbeck, 1995 , Nucleic Acids Research, 23, 2092-2096; Hammann et al, 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). Examples of such arms are shown generally in FIGS. 1-4. That is, these arms contain sequences within a enzymatic nucleic acid that are intended to bring enzymatic nucleic acid and target RNA together through complementary base-pairing interactions. The enzymatic nucleic acid of the invention can have binding arms that are contiguous or non-contiguous and can be of varying lengths. The length of the binding arm(s) can be greater than or equal to four nucleotides and of sufficient length to stably interact with a target RNA; in one embodiment they can be 12-100 nucleotides; in another embodiment they can be 14-24 nucleotides long (see for example Werner and Uhlenbeck, supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herranze et al., 1993, EMBO J., 12, 2567-73) or between 8 and 14 nucleotides long. If two binding arms are chosen, the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., four and four, five and five nucleotides, or six and six nucleotides, or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., three and five, six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).

By “Inozyme” or “NCH” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as NCH Rz in FIG. 1. Inozymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCH/, where N is a nucleotide, C is cytidine and H is adenosine, uridine or cytidine, and / represents the cleavage site. H is used interchangeably with X. Inozymes can also possess endonuclease activity to cleave RNA substrates having a cleavage triplet NCN/, where N is a nucleotide, C is cytidine, and / represents the cleavage site. “I” in FIG. 1 represents an Inosine nucleotide, including a ribo-Inosine or xylo-Inosine nucleoside.

By “G-cleaver” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described as G-cleaver Rz in FIG. 1. G-cleavers possess endonuclease activity to cleave RNA substrates having a cleavage triplet NYN/, where N is a nucleotide, Y is uridine or cytidine and/represents the cleavage site. G-cleavers can be chemically modified as is generally shown in FIG. 1.

By “amberzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG. 2. Amberzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet NG/N, where N is a nucleotide, G is guanosine, and/represents the cleavage site. Amberzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG. 2. In addition, differing nucleoside and/or non-nucleoside linkers can be used to substitute the 5′-gaaa-3′ loops shown in the figure. Amberzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.

By “zinzyme” motif or configuration is meant, an enzymatic nucleic acid molecule comprising a motif as is generally described in FIG. 3. Zinzymes possess endonuclease activity to cleave RNA substrates having a cleavage triplet including but not limited to YG/Y, where Y is uridine or cytidine, and G is guanosine and/represents the cleavage site.

Zinzymes can be chemically modified to increase nuclease stability through substitutions as are generally shown in FIG. 3, including substituting 2′-O-methyl guanosine nucleotides for guanosine nucleotides. In addition, differing nucleotide and/or non-nucleotide linkers can be used to substitute the 5′-gaaa-2′ loop shown in the figure. Zinzymes represent a non-limiting example of an enzymatic nucleic acid molecule that does not require a ribonucleotide (2′-OH) group within its own nucleic acid sequence for activity.

By ‘DNAzyme’ is meant, an enzymatic nucleic acid molecule that does not require the presence of a 2′-OH group within its own nucleic acid sequence for activity. In particular embodiments the enzymatic nucleic acid molecule can have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. DNAzymes can be synthesized chemically or expressed endogenously in vivo, by means of a single stranded DNA vector or equivalent thereof. An example of a DNAzyme is shown in FIG. 4 and is generally reviewed in Usman et al., U.S. Pat. No. 6,159,714; Chartrand et al., 1995 , NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655; Santoro et al., 1997, PNAS 94, 4262; Breaker, 1999, Nature Biotechnology, 17, 422-423; and Santoro et. al., 2000, J. Am. Chem. Soc., 122, 2433-39. The “10-23” DNAzyme motif is one particular type of DNAzyme that was evolved using in vitro selection (see Santoro et al., supra). Additional DNAzyme motifs can be selected for using techniques similar to those described in these references, and hence, are within the scope of the present invention.

By “sufficient length” is meant an oligonucleotide of greater than or equal to 3 nucleotides that is of a length great enough to provide the intended function under the expected condition. For example, for binding arms of enzymatic nucleic acid “sufficient length” means that the binding arm sequence is long enough to provide stable binding to a target site under the expected binding conditions. The binding arms are not so long as to prevent useful turnover of the nucleic acid molecule.

By “stably interact” is meant interaction of the oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions) that is sufficient to the intended purpose (e.g., cleavage of target RNA by an enzyme).

By “equivalent” RNA to PTGDS is meant to include RNA molecules having homology (partial or complete) to RNA molecules encoding PTGDS proteins or encoding proteins with similar function as PTGDS proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites. The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.

By “equivalent” RNA to PTGDR is meant to include RNA molecules having homology (partial or complete) to RNA molecules encoding PTGDR proteins or encoding proteins with similar function as PTGDR proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites. The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.

By “equivalent” RNA to ADORA1 is meant to include RNA molecules having homology (partial or complete) to RNA molecule encoding ADORA1 proteins or encoding proteins with similar function as ADORA1 proteins in various organisms, including human, rodent, primate, rabbit, pig, plants, protozoans, fungi, and other microorganisms and parasites. The equivalent RNA sequence can also include in addition to the coding region, regions such as 5′-untranslated region, 3′-untranslated region, introns, intron-exon junction and the like.

By “homology” is meant the nucleotide sequence of two or more nucleic acid molecules is partially or completely identical.

By “antisense nucleic acid”, it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat. No. 5,849,902). Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both. For a review of current antisense strategies, see Schmajuk et al., 1999, J. Biol. Chem., 274, 21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al., 1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol., 313, 3-45; Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad. Pharmacol., 40, 1-49. In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA. Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.

By “RNase H activating region” is meant a region (generally greater than or equal to 4-25 nucleotides in length, and in one embodiment from 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al., U.S. Pat. No. 5,849,902; Arrow et al., U.S. Pat. No. 5,989,912). The RNase H enzyme binds to the nucleic acid molecule-target RNA complex and cleaves the target RNA sequence. The RNase H activating region comprises, for example, phosphodiester, phosphorothioate (at least four of the nucleotides are phosphorothiote substitutions; and in another embodiment, 4-11 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5′-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof. In addition to one or more backbone chemistries described above, the RNase H activating region can also comprise a variety of sugar chemistries. For example, the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry. Those skilled in the art will recognize that the foregoing are non-limiting examples and that any combination of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of RNase H enzyme is within the scope of the definition of the RNase H activating region and the instant invention.

By “2-5A antisense chimera” is meant an antisense oligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylate residue. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and Torrence, 1998, Pharmacol. Ther., 78, 55-113).

By “triplex forming oligonucleotides” is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al., 2000, Biochim. Biophys. Acta, 1489, 181-206).

By “gene” it is meant a nucleic acid that encodes an RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.

“Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another RNA molecule by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., enzymatic nucleic acid cleavage, antisense or triple helix inhibition. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987 , CSHSymp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.

By “RNA” is meant a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” or “2′-OH” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribo-furanose moiety.

By “decoy RNA” is meant an RNA molecule or aptamer that is designed to preferentially bind to a predetermined ligand. Such binding can result in the inhibition or activation of a target molecule. The decoy RNA or aptamer can compete with a naturally occurring binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a “decoy” and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990, Cell, 63, 601-608). This is but a specific example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, see for example Gold et al., 1995 , Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628. Similarly, a decoy RNA can be designed to bind to a D2 receptor and block the binding of PTGDS or a decoy RNA can be designed to bind to PTGDS and prevent interaction with the D2 receptor.

The term “double stranded RNA” or “dsRNA” as used herein refers to a double stranded RNA molecule capable of RNA interference, including short interfering RNA “siRNA” (see, e.g., Bass, 2001 , Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498).

The term “allozyme” as used herein refers to an allosteric enzymatic nucleic acid molecule, see, e.g., George et al., U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington, International PCT publication No. WO 00/24931, Breaker et al., International PCT Publication Nos. WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No. WO 99/29842. The term “2-5A chimera” as used herein refers to an oligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylate residue. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and Torrence, 1998, Pharmacol. Ther., 78, 55-113).

The term “triplex forming oligonucleotides” as used herein refers to an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al., 2000, Biochim. Biophys. Acta, 1489, 181-206).

Several varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid that is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor of gene expression, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.

The enzymatic nucleic acid molecule that cleave the specified sites in PTGDS, ADORA1 and PTGDR-specific RNAs represent a novel therapeutic approach to treat a variety of allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of PTGDS, ADORA1 and/or PTGDR expression.

In one embodiment of the inventions described herein, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but can also be formed in the motif of a hepatitis delta virus, group I intron, group II intron or RNase P RNA (in association with an RNA guide sequence), Neurospora VS RNA, DNAzymes, NCH cleaving motifs, or G-cleavers. Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al., 1992 , AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al., 1990 Nucleic Acids Res. 18, 299; Chowrira & McSwiggen, U.S. Pat. No. 5,631,359; of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNase P motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J 14, 363); Group II introns are described by Griffin et al., 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al., International PCT Publication No. WO 96/22689; of the Group I intron by Cech et al., U.S. Pat. No. 4,987,071 and of DNAzymes by Usman et al., International PCT Publication No. WO 95/11304; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655; Santoro et al., 1997, PNAS 94, 4262, and Beigelman et al., International PCT publication No. WO 99/55857. NCH cleaving motifs are described in Ludwig & Sproat, International PCT Publication No. WO 98/58058; and G-cleavers are described in Kore et al., 1998, Nucleic Acids Research 26, 4116-4120 and Eckstein et al., International PCT Publication No. WO 99/16871. Additional motifs such as the Aptazyme (Breaker et al., WO 98/43993), Amberzyme (Class I motif; FIG. 2; Beigelman et al., U.S. Ser. No. 09/301,511) and Zinzyme (FIG. 3) (Beigelman et al., U.S. Ser. No. 09/301,511), all included by reference herein including drawings, can also be used in the present invention. These specific motifs or configurations are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule (Cech et al., U.S. Pat. No. 4,987,071).

In one embodiment of the present invention, a nucleic acid molecule of the instant invention can be between 12 and 100 nucleotides in length. Exemplary enzymatic nucleic acid molecules of the invention are shown in Table III-VII. For example, enzymatic nucleic acid molecules of the invention can be between 15 and 50 nucleotides in length, and in another embodiment between 25 and 40 nucleotides in length, e.g., 34, 36, or 38 nucleotides in length (for example see Jarvis et al., 1996 , J. Biol. Chem., 271, 29107-29112). Exemplary DNAzymes of the invention are can between 15 and 40 nucleotides in length, and in one embodiment, between 25 and 35 nucleotides in length, e.g., 29, 30, 31, or 32 nucleotides in length (see, e.g., Santoro et al., 1998, Biochemistry, 37, 13330-13342; Chartrand et al., 1995, Nucleic Acids Research, 23, 4092-4096). Exemplary antisense molecules of the invention can be between 15 and 75 nucleotides in length, and in one embodiment between 20 and 35 nucleotides in length, e.g., 25, 26, 27, or 28 nucleotides in length (see for example Woolf et al., 1992, PNAS., 89, 7305-7309; Milner et al., 1997, Nature Biotechnology, 15, 537-541). Exemplary triplex forming oligonucleotide molecules of the invention are between 10 and 40 nucleotides in length, and in one embodiment are between 12 and 25 nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides in length (see for example Maher et al., 1990, Biochemistry, 29, 8820-8826; Strobel and Dervan, 1990, Science, 249, 73-75). Those skilled in the art will recognize that all that is required is for the nucleic acid molecule to be of length and conformation sufficient and suitable for the nucleic acid molecule to catalyze a reaction contemplated herein. The length of the nucleic acid molecules of the instant invention are not limiting within the general limits stated.

In one embodiment, a nucleic acid molecule that modulates, for example, down-regulates, PTGDS replication or expression comprises between 8 and 100 bases complementary to a RNA molecule of PTGDS. In another embodiment, a nucleic acid molecule that modulates PTGDS replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of PTGDS.

In another embodiment, a nucleic acid molecule that modulates, for example, down-regulates, PTGDR replication or expression comprises between 8 and 100 bases complementary to a RNA molecule of PTGDR. In another embodiment, a nucleic acid molecule that modulates PTGDR replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of PTGDR.

In another embodiment, a nucleic acid molecule that modulates, for example, down-regulates, ADORA1 replication or expression comprises between 8 and 100 bases complementary to a RNA molecule of ADORA1. In another embodiment, a nucleic acid molecule that modulates ADORA1 replication or expression comprises between 14 and 24 bases complementary to a RNA molecule of ADORA1.

The invention provides a method for producing a class of nucleic acid-based gene modulating agents that exhibit a high degree of specificity for the RNA of a desired target. For example, the enzymatic nucleic acid molecule is can be targeted to a highly conserved sequence region of target RNAs encoding PTGDS, ADORA1 and/or PTGDR (e.g., PTGDS, ADORA1 and/or PTGDR genes) such that specific treatment of a disease or condition can be provided with either one or several nucleic acid molecules of the invention. Such nucleic acid molecules can be delivered exogenously to specific tissue or cellular targets as required. Alternatively, the nucleic acid molecules (e.g., ribozymes and antisense) can be expressed from DNA and/or RNA vectors that are delivered to specific cells.

As used in herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism. The cell can, for example, be in vitro, e.g., in cell culture, or present in a multicellular organism, including, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell may be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).

By “PTGDR proteins” is meant, a protein receptor or a mutant protein or peptide derivative thereof, having prostaglandin D2 receptor activity, for example, having the ability to bind prostaglandin D2 and/or having GTP-binding protein coupled activity.

By “PTGDS proteins” is meant, a prostaglandin synthetase protein or a mutant protein or peptide derivative thereof, having prostaglandin D2 synthetase activity, for example, having the ability to convert PGH2 to PGD2.

By “highly conserved sequence region” is meant, a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.

Nucleic acid-based inhibitors of PTGDS, ADORA1 and PTGDR expression are useful for the prevention and/or treatment of allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and any other diseases or conditions that are related to or will respond to the levels of PTGDS, ADORA1 and/or PTGDR in a cell or tissue, alone or in combination with other therapies. The reduction of PTGDS, ADORA1 and/or PTGDR expression (specifically PTGDS, ADORA1 and/or PTGDR gene RNA levels) and thus reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.

The nucleic acid-based inhibitors of the invention can be added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues, for example by pulmonary delivery of an aerosol formulation with an inhaler or nebulizer. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through inhalation, injection or infusion pump, with or without their incorporation in biopolymers. In preferred embodiments, the enzymatic nucleic acid inhibitors comprise sequences that are complementary to the substrate sequences in Tables III to VII. Examples of such enzymatic nucleic acid molecules also are shown in Tables III to VII. Examples of such enzymatic nucleic acid molecules consist essentially of sequences defined in these tables.

In another embodiment, the invention features antisense nucleic acid molecules and 2-5A chimera including sequences complementary to the substrate sequences shown in Tables III to VII. Such nucleic acid molecules can include sequences as shown for the binding arms of the enzymatic nucleic acid molecules in Tables III to VII. Similarly, triplex molecules can be provided targeted to the corresponding DNA target regions, and containing the DNA equivalent of a target sequence or a sequence complementary to the specified target (substrate) sequence. Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.

By “consists essentially of” is meant that the active nucleic acid molecule of the invention, for example, an enzymatic nucleic acid molecule, contains an enzymatic center or core equivalent to those in the examples, and binding arms able to bind RNA such that cleavage at the target site occurs. Other sequences can be present that do not interfere with such cleavage. Thus, a core region can, for example, include one or more loop, stem-loop structure, or linker which does not prevent enzymatic activity. Thus, the underlined regions in the sequences in Tables III and IV can be such a loop, stem-loop, nucleotide linker, and/or non-nucleotide linker and can be represented generally as sequence “X”. For example, a core sequence for a hammerhead enzymatic nucleic acid can comprise a conserved sequence, such as 5′-CUGAUGAG-3′ and 5′-CGAA-3′ connected by “X”, where X is 5′- GCCGUUAGGC-3′ (SEQ ID NO: 2678), or any other Stem II region known in the art, or a nucleotide and/or non-nucleotide linker. Similarly, for other nucleic acid molecules of the instant invention, such as Inozyme, G-cleaver, amberzyme, zinzyme, DNAzyme, antisense, 2-5A antisense, triplex forming nucleic acid, and decoy nucleic acids, other sequences or non-nucleotide linkers can be present that do not interfere with the function of the nucleic acid molecule.

Sequence X can be a linker of ≧2 nucleotides in length, including 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides can be internally base-paired to form a stem of ≧2 base pairs. Alternatively or in addition, sequence X can be a non-nucleotide linker. In yet another embodiment, the nucleotide linker X can be a nucleic acid aptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer (TAR) and others (for a review see Gold et al., 1995 , Annu. Rev. Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World, ed. Gesteland and Atkins, pp. 511, CSH Laboratory Press). A “nucleic acid aptamer” as used herein is meant to indicate a nucleic acid sequence capable of interacting with a ligand. The ligand can be any natural or a synthetic molecule, including but not limited to a resin, metabolites, nucleosides, nucleotides, drugs, toxins, transition state analogs, peptides, lipids, proteins, amino acids, nucleic acid molecules, hormones, carbohydrates, receptors, cells, viruses, bacteria and others.

In yet another embodiment, the non-nucleotide linker X is as defined herein. The term “non-nucleotide” as used herein include either abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds. Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. A “non-nucleotide” further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. Thus, in a preferred embodiment, the invention features an enzymatic nucleic acid molecule having one or more non-nucleotide moieties, and having enzymatic activity to cleave an RNA or DNA molecule.

In another aspect of the invention, enzymatic nucleic acid molecules or antisense molecules that interact with target RNA molecules and down-regulate PTGDS, ADORA1 and/or PTGDR (e.g., PTGDS, ADORA1 and/or PTGDR gene) activity are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. Enzymatic nucleic acid molecule or antisense expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the enzymatic nucleic acid molecules or antisense can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of enzymatic nucleic acid molecules or antisense. Such vectors can be repeatedly administered as necessary. Once expressed, the enzymatic nucleic acid molecules or antisense bind to the target RNA and down-regulate its function or expression. Delivery of enzymatic nucleic acid molecule or antisense expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell. Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector.

By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

By “patient” is meant an organism, which is a donor or recipient of explanted cells, or the cells themselves. “Patient” also refers to an organism to which the nucleic acid molecules of the invention can be administered. A patient can be a mammal or mammalian cells. In one embodiment, a patient is a human or human cells.

By “enhanced enzymatic activity” is meant to include activity measured in cells and/or in vivo where the activity is a reflection of both the catalytic activity and the stability of the nucleic acid molecules of the invention. In this invention, the product of these properties can be increased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme. In some cases, the activity or stability of the nucleic acid molecule can be decreased (i.e., less than ten-fold), but the overall activity of the nucleic acid molecule is enhanced, in vivo.

The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed above. For example, to treat a disease or condition associated with the levels of PTGDS, ADORA1 and/or PTGDR, the patient can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

In a further embodiment, the described molecules, such as antisense or enzymatic nucleic acid molecules, can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules can be used in combination with one or more known therapeutic agents to treat allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of PTGDS, ADORA1 and/or PTGDR expression.

In another embodiment, the invention features nucleic acid-based inhibitors (e.g., enzymatic nucleic acid molecules (e.g., ribozymes), antisense nucleic acids, 2-5A antisense chimeras, triplex DNA, antisense nucleic acids containing RNA cleaving chemical groups) and methods for their use to down regulate or inhibit the expression of genes (e.g., PTGDS, ADORA1 and/or PTGDR) capable of progression and/or maintenance allergic diseases or conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and/or other allergic or inflammatory diseases and conditions which respond to the modulation of PTGDS, ADORA1 and/or PTGDR expression.

By “comprising” is meant including, but not limited to, whatever follows the word “comprising”. Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows examples of chemically stabilized ribozyme motifs. HH Rz, represents hammerhead ribozyme motif (Usman et al., 1996[0105] , Curr. Op. Struct. Bio., 1, 527); NCH Rz represents the NCH ribozyme motif (Ludwig & Sproat, International PCT Publication No. WO 98/58058); G-Cleaver, represents G-cleaver ribozyme motif (Kore et al., 1998, Nucleic Acids Research 26, 4116-4120, Eckstein et al., International PCT publication No. WO 99/16871). N or n, represent independently a nucleotide that can be same or different and have complementarity to each other; rI, represents ribo-Inosine nucleotide; arrow indicates the site of cleavage within the target. Position 4 of the HH Rz and the NCH Rz is shown as having 2′-C-allyl modification, but those skilled in the art will recognize that this position can be modified with other modifications well known in the art, so long as such modifications do not significantly inhibit the activity of the ribozyme.
FIG. 2 shows an example of the Amberzyme ribozyme motif that is chemically stabilized (see for example Beigelman et al., International PCT publication No. WO 99/55857). [0106]
FIG. 3 shows an example of the Zinzyme A ribozyme motif that is chemically stabilized (see for example Beigelman et al., Beigelman et al., International PCT publication No. WO 99/55857). [0107]
FIG. 4 shows an example of a specific DNAzyme motif, commonly referred to as the “10-23 motif”, as described by Santoro et al., 1997[0108] , PNAS, 94, 4262.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Nucleic Acid Molecules and Mechanism of Action [0109]
Antisense: Antisense molecules can be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides and primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, Nov 1994[0110] , BioPharm, 20-33). The antisense oligonucleotide binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme. Antisense molecules can also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).
In addition, binding of single stranded DNA to RNA can result in nuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke, supra). To date, the only backbone modified DNA chemistry which act as substrates for RNase H are phosphorothioates, phosphorodithioates, and borontrifluoridates. Recently it has been reported that 2′-arabino and 2′-fluoro arabino-containing oligos can also activate RNase H activity. [0111]
A number of antisense molecules have been described that utilize novel configurations of chemically modified nucleotides, secondary structure, and/or RNase H substrate domains (Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., International PCT Publication No. WO 99/54459; Hartmann et al., U.S. S No. 60/101,174, filed on Sep. 21, 1998) all of these are incorporated by reference herein in their entirety. [0112]
In addition, antisense deoxyoligoribonucleotides can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. Antisense DNA can be expressed via the use of a single stranded DNA intracellular expression vector or equivalents and variations thereof. [0113]
Triplex Forming Oligonucleotides (TFO): Single stranded DNA can be designed to bind to genomic DNA in a sequence specific manner. TFOs are comprised of pyrimidine-rich oligonucleotides which bind DNA helices through Hoogsteen Base-pairing (Wu-Pong, supra). The resulting triple helix composed of the DNA sense, DNA antisense, and TFO disrupts RNA synthesis by RNA polymerase. The TFO mechanism can result in gene expression or cell death since binding can be irreversible (Mukhopadhyay & Roth, supra). [0114]
2-5A Antisense Chimera: The 2-5A system is an interferon mediated mechanism for RNA degradation found in higher vertebrates (Mitra et al., 1996[0115] , Proc Nat Acad Sci USA 93, 6780-6785). Two types of enzymes, 2-5A synthetase and RNase L, are required for RNA cleavage. The 2-5A synthetases require double stranded RNA to form 2′-5′ oligoadenylates (2-5A). 2-5A then acts as an allosteric effector for utilizing RNase L, which has the ability to cleave single stranded RNA. The ability to form 2-5A structures with double stranded RNA makes this system particularly useful for inhibition of viral replication.
(2′-5′) oligoadenylate structures can be covalently linked to antisense molecules to form chimeric oligonucleotides capable of RNA cleavage (Torrence, supra). These molecules putatively bind and activate a 2-5A dependent RNase, the oligonucleotide/enzyme complex then binds to a target RNA molecule which can then be cleaved by the RNase enzyme. [0116]
Enzymatic Nucleic Acid: Several varieties of naturally-occurring enzymatic RNAs are presently known. In addition, several in vitro selection (evolution) strategies (Orgel, 1979, [0117] Proc. R. Soc. London, B 205, 435) have been used to evolve new nucleic acid catalysts capable of catalyzing cleavage and ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB J, 9, 1183; Breaker, 1996, Curr. Op. Biotech., 7, 442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94, 4262; Tang et al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra; Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish et al., 1997, Biochemistry 36, 6495; all of these are incorporated by reference herein). Each can catalyze a series of reactions including the hydrolysis of phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions.
The enzymatic nature of an enzymatic nucleic acid molecule has significant advantages, one advantage being that the concentration of enzymatic nucleic acid molecule necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the enzymatic nucleic acid molecule to act enzymatically. Thus, a single enzymatic nucleic acid molecule is able to cleave many molecules of target RNA. In addition, the enzymatic nucleic acid molecule is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can be chosen to completely eliminate catalytic activity of a enzymatic nucleic acid molecule. [0118]
Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. With the proper design, such enzymatic nucleic acid molecules can be targeted to RNA transcripts, and achieve efficient cleavage in vitro (Zaug et al., 324[0119] , Nature 429 1986; Uhlenbeck, 1987 Nature 328, 596; Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Dreyfus, 1988, Einstein Quart. J. Bio. Med., 6, 92; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989; Santoro et al., 1997 supra).
Because of their sequence specificity, trans-cleaving enzymatic nucleic acid molecules can be used as therapeutic agents for human disease (Usman & McSwiggen, 1995 [0120] Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the RNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited (Warashina et al., 1999, Chemistry and Biology, 6, 237-250).
Enzymatic nucleic acid molecules of the invention that are allosterically regulated (“allozymes”) can be used to down-regulate PTGDS and/or PTGDR expression. These allosteric enzymatic nucleic acids or allozymes (see for example George et al., U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington, International PCT publication No. WO 00/24931, Breaker et al., International PCT Publication Nos. WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No. WO 99/29842) are designed to respond to a signaling agent, for example, mutant PTGDS and/or PTGDR protein, wild-type PTGDS and/or PTGDR protein, mutant PTGDS and/or PTGDR RNA, wild-type PTGDS and/or PTGDR RNA, other proteins and/or RNAs involved in PTGDS or PTGDR signal transduction, compounds, metals, polymers, molecules and/or drugs that are targeted to PTGDS and/or PTGDR expressing cells etc., which in turn modulates the activity of the enzymatic nucleic acid molecule. In response to interaction with a predetermined signaling agent, the allosteric enzymatic nucleic acid molecule's activity is activated or inhibited such that the expression of a particular target is selectively down-regulated. The target can comprise wild-type PTGDS, ADORA1 and/or PTGDR, mutant PTGDS, ADORA1 and/or PTGDR, and/or a predetermined component of the PTGDS, ADORA1 or PTGDR signal transduction pathway. In a specific example, allosteric enzymatic nucleic acid molecules that are activated by interaction with a RNA encoding a PTGDR protein are used as therapeutic agents in vivo. The presence of RNA encoding the PTGDS protein activates the allosteric enzymatic nucleic acid molecule that subsequently cleaves the RNA encoding a PTGDR protein resulting in the inhibition of PTGDR protein expression. In this manner, cells that express both PTGDS and PTGDR protein are selectively targeted. [0121]
In another non-limiting example, an allozyme can be activated by a PTGDS or PTGDR protein, peptide, or mutant polypeptide that causes the allozyme to inhibit the expression of PTGDS or PTGDR gene, by, for example, cleaving RNA encoded by PTGDS or PTGDR gene. In this non-limiting example, the allozyme acts as a decoy to inhibit the function of PTGDS or PTGDR and also inhibit the expression of PTGDS or PTGDR once activated by the PTGDS or PTGDR protein. [0122]
Target Sites [0123]
Targets for useful enzymatic nucleic acid molecules and antisense nucleic acids can be determined as disclosed in Draper et al., WO 93/23569; Sullivan et al, WO 93/23057; Thompson et al., WO 94/02595; Draper et al., WO 95/04818; McSwiggen et al., U.S. Pat. No. 5,525,468, and hereby incorporated by reference herein in totality. Other examples include the following PCT applications, which concern inactivation of expression of disease-related genes: WO 95/23225, WO 95/13380, WO 94/02595, incorporated by reference herein. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Enzymatic nucleic acid molecules and antisense to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. The sequences of human PTGDR RNAs were screened for optimal enzymatic nucleic acid and antisense target sites using a computer-folding algorithm. Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme, or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified. These sites are shown in Tables III to VII (all sequences are 5′ to 3′ in the tables; underlined regions can be any sequence “X” or linker X, the actual sequence is not relevant here). The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of enzymatic nucleic acid molecule. While human sequences can be screened and enzymatic nucleic acid molecule and/or antisense thereafter designed, as discussed in Stinchcomb et al., WO 95/23225, mouse targeted enzymatic nucleic acid molecules can be useful to test efficacy of action of the enzymatic nucleic acid molecule and/or antisense prior to testing in humans. [0124]
Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified. The nucleic acid molecules are individually analyzed by computer folding (Jaeger et al., 1989 [0125] Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the sequences fold into the appropriate secondary structure. Those nucleic acid molecules with unfavorable intramolecular interactions such as between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity.
Antisense, hammerhead, DNAzyme, NCH, amberzyme, zinzyme or G-Cleaver enzymatic nucleic acid molecule binding/cleavage sites were identified and were designed to anneal to various sites in the RNA target. The binding arms are complementary to the target site sequences described above. The nucleic acid molecules were chemically synthesized. The method of synthesis used follows the procedure for normal DNA/RNA synthesis as described below and in Usman et al., 1987 [0126] J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990 Nucleic Acids Res., 18, 5433; and Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684; Caruthers et al., 1992, Methods in Enzymology 211,3-19.
Synthesis of Nucleic acid Molecules [0127]
Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small refers to nucleic acid motifs less than about 100 nucleotides in length, and in one embodiment less than about 80 nucleotides in length, and in another embodiment less than about 50 nucleotides in length; e.g., antisense oligonucleotides, hammerhead or the NCH ribozymes) can be used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized. [0128]
Oligonucleotides (e.g., antisense GeneBlocs) are synthesized using protocols known in the art as described in Caruthers et al., 1992[0129] , Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45 sec coupling step for 2′-deoxy nucleotides. Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by calorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methylimidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.
Deprotection of the antisense oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H[0130] ₂O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
The method of synthesis used for normal RNA including certain enzymatic nucleic acid molecules follows the procedure as described in Usman et al., 1987, [0131] J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides. Table II outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include; detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methylimidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.
Deprotection of the RNA is performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 h, the oligomer is quenched with 1.5 M NH[0132] ₄HCO₃.
Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65° C. for 15 min. The vial is brought to r.t. TEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 min. The sample is cooled at −20° C. and then quenched with 1.5 M NH[0133] ₄HCO₃.
For purification of the trityl-on oligomers, the quenched NH[0134] ₄HCO₃solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing, the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 min. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.
Inactive hammerhead ribozymes or binding attenuated control (BAC) oligonucleotides are synthesized by substituting a U for G[0135] ₅and a U for A14 (numbering from Hertel, K. J., et al., 1992, Nucleic Acids Res., 20, 3252). Similarly, one or more nucleotide substitutions can be introduced in other enzymatic nucleic acid molecules to inactivate the molecule and such molecules can serve as a negative control.
The average stepwise coupling yields are typically >98% (Wincott et al., 1995 [0136] Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96 well format, all that is important is the ratio of chemicals used in the reaction.
Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example by ligation (Moore et al., 1992[0137] , Science 256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204).
The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992[0138] , TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., Supra, the totality of which is hereby incorporated herein by reference) and are re-suspended in water.
The sequences of the nucleic acid molecules, including enzymatic nucleic acid molecules and antisense, that are chemically synthesized, are shown in Tables III-VII. The sequences of the enzymatic nucleic acid constructs that are chemically synthesized are complementary to the Substrate sequences shown in Tables III-VII. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the enzymatic nucleic acid (all but the binding arms) is altered to affect activity. The enzymatic nucleic acid construct sequences listed in Tables III-VII can be formed of ribonucleotides or other nucleotides or non-nucleotides. Such enzymatic nucleic acid molecules with enzymatic activity are equivalent to the enzymatic nucleic acid molecules described specifically in the Tables. [0139]
Optimizing Activity of the Nucleic Acid Molecule of the Invention. [0140]
Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 [0141] Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; and Burgin et al., supra; all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules herein). Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired. (All these publications are hereby incorporated by reference herein).
There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994[0142] , Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. S No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into ribozymes without inhibiting catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the nucleic acid molecules of the instant invention.
While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorothioate, and/or 5′-methylphosphonate linkages improves stability, too many of these modifications can cause some toxicity. Therefore when designing nucleic acid molecules the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity resulting in increased efficacy and higher specificity of these molecules. [0143]
Nucleic acid molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Thus, in a cell and/or in vivo the activity may not be significantly lowered. Therapeutic nucleic acid molecules delivered exogenously are optimally stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Clearly, nucleic acid molecules must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995 [0144] Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211,3-19 (incorporated by reference herein) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.
In one embodiment, nucleic acid molecules of the invention include one or more G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein modifications result in the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998[0145] , J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substation within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules of the invention can enable both enhanced affinity and specificity to nucleic acid targets.
Therapeutic nucleic acid molecules (e.g., enzymatic nucleic acid molecules and antisense nucleic acid molecules) delivered exogenously are optimally stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. These nucleic acid molecules should be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above. [0146]
In another embodiment, the invention features conjugates and/or complexes of nucleic acid molecules targeting PTGDS, PTGDR, and/or adenosine receptors. Compositions and conjugates are used to facilitate delivery of molecules into a biological system, such as cells. The conjugates provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel agents for the delivery of molecules, including but not limited to small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules. [0147]
The term “biodegradable nucleic acid linker molecule” as used herein, refers to a nucleic acid molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule. The stability of the biodegradable nucleic acid linker molecule can be modulated by using various combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, for example 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus based linkage, for example a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications. [0148]
The term “biodegradable” as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation. [0149]
The term “biologically active molecule” as used herein, refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siRNA, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers. [0150]
The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups. [0151]
In another embodiment, nucleic acid catalysts having chemical modifications that maintain or enhance enzymatic activity are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the activity of the nucleic acid may not be significantly lowered. As exemplified herein such enzymatic nucleic acids are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al., 1996[0152] , Biochemistry, 35, 14090). Such enzymatic nucleic acids herein are said to “maintain” the enzymatic activity of an all RNA ribozyme or all DNA DNAzyme.
In another aspect the nucleic acid molecules comprise a 5′ and/or a 3′-cap structure. [0153]
By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see for example Wincott et al., WO 97/26270, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present on both terminus. In non-limiting examples, the 5′-cap includes inverted abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Wincott et al., International PCT publication No. WO 97/26270, incorporated by reference herein). [0154]
In another embodiment the 3′-cap includes, for example 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or [0155] non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).
By the term “non-nucleotide” is meant any group or compound thatcan be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. [0156]
An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. The alkyl group can have, for example, 1 to 12 carbons. In one embodiment of the invention, the alkyl group is a lower alkyl of from 1 to 7 carbons. In another embodiment the alkyl group is 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) can be hydroxyl, cyano, alkoxy, ═O, ═S, NO[0157] ₂or N(CH₃)₂, amino, or SH. The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. The alkenyl group can have, for example, 1 to 12 carbons. In one embodiment of the invention the alkenyl group can be a lower alkenyl of from 1 to 7 carbons. In another embodiment the alkenyl group can be 1 to 4 carbons. The alkenyl group can be substituted or unsubstituted. When substituted the substituted group(s) can be, for example, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. The alkynyl group can have, for example, 1 to 12 carbons. In one embodiment of the invention, the alkynyl group is a lower alkynyl of from 1 to 7 carbons. In another embodiment of the invention, the alkynyl group is 1 to 4 carbons. The alkynyl group can be substituted or unsubstituted. When substituted the substituted group(s) can be, for example, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino or SH.
Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group which has at least one ring having a conjugated p electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which can be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylaryl or hydrogen. [0158]
By “nucleotide” is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, for example, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule. [0159]
By “nucleoside” is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1′ position of a nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group. The nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, modified nucleosides, non-natural nucleosides, non-standard nucleosides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleoside bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule. [0160]
In one embodiment, the invention features modified enzymatic nucleic acid molecules with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications see Hunziker and Leumann, 1995[0161] , Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39. These references are hereby incorporated by reference herein.
By “abasic” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1′ position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribose derivative (for more details see Wincott et al., International PCT publication No. WO 97/26270). [0162]
By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1′ carbon of β-D-ribo-furanose. [0163]
By “modified nucleoside” is meant any nucleotide base that contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate. In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH[0164] ₂or 2′-O-NH₂, which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., WO 98/28317, respectively, which are both incorporated by reference in their entireties.
Various modifications to nucleic acid (e.g., antisense and ribozyme) structure can be made to enhance the utility of these molecules. For example, such modifications can enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, including e.g., enhancing penetration of cellular membranes and conferring the ability to recognize and bind to targeted cells. [0165]
Use of the nucleic acid-based molecules of the invention can lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs) and/or other chemical or biological molecules). The treatment of patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules. Therapies can be devised which include a mixture of enzymatic nucleic acid molecules (including different enzymatic nucleic acid molecule motifs), antisense and/or 2-5A chimera molecules to one or more targets to alleviate symptoms of a disease. [0166]
Administration of Nucleic Acid Molecules [0167]
A nucleid acid molecule of the invention can be adapted for use to treat asthma and other related diseases and conditions described herein. For example, a nucleic acid molecule can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992[0168] , Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995 which are both incorporated herein by reference. Sullivan et al., PCT WO 94/02595, further describes the general methods for delivery of enzymatic RNA molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. The nucleic acid molecules or the invention are administered via pulmonary delivery, such as by inhalation of an aerosol or spray dried formulation administered by an inhalation device or nebulizer. Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Other routes of delivery include, but are not limited to oral (tablet or pill form) and/or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1158). Other approaches include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. For a comprehensive review on drug delivery strategies including CNS delivery, see Ho et al., 1999, Curr. Opin. Mol. Ther., 1, 336-343 and Jain, Drug Delivery Systems: Technologies and Commercial Opportunities, Decision Resources, 1998 and Groothuis et al., 1997, J NeuroVirol., 3, 387-400. More detailed descriptions of nucleic acid delivery and administration are provided in Sullivan et al., supra, Draper et al., PCT WO93/23569, Beigelman et al., PCT WO99/05094, and Klimuk et al., PCT WO99/04819 all of which have been incorporated by reference herein.
The molecules of the instant invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, or all of the symptoms) of a disease state in a patient. [0169]
The negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the other compositions known in the art. [0170]
The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid. [0171]
A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., local administration or systemic administration, into a cell or patient, including, for example, a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect. [0172]
By “local administration” is meant in vivo local absorption or accumulation of drugs in the specific tissue, organ, or compartment of the body. Administration routes that can lead to local absorption include, without limitations: inhalation, direct injection, or dermatological applications. [0173]
By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the desired compound, e.g., nucleic acids, to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention, for example PEG or phospholipids conjugates, can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A nucleic acid formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells. [0174]
Both local and systemic administration approaches can be used to administer nucleic acid molecules of the invention for the treatment of asthma or related conditions. In one embodiment, the nucleic acid molecule or formulation comprising the nucleic acid molecule is administered to a patient with an inhaler or nebulizer, providing rapid local uptake of the nucleic acid molecules into relevant pulmonary tissues. In another embodiment, the nucleic acid molecule or formulation comprising the nucleic acid molecule is administered to a patient systemically, for example by intravenous or subcutaneous injection, providing sustained uptake of the nucleic acid molecules into relevant bodily tissues. [0175]
By pharmaceutically acceptable formulation is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: PEG conjugated nucleic acids, phospholipid conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues, for exaple the CNS (Jolliet-Riant and Tillement, 1999[0176] , Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies, including CNS delivery of the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058. All these references are hereby incorporated herein by reference.
The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). Nucleic acid molecules of the invention can also comprise covalently attached PEG molecules of various molecular weights. These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. [0177] Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392; all of which are incorporated by reference herein). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen. All of these references are incorporated by reference herein.
The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in [0178] Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985) hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.
A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, or all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer. [0179]
The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. [0180]
Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed. [0181]
Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. [0182]
Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin. [0183]
Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid. [0184]
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present. [0185]
Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents. [0186]
Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. [0187]
The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols. [0188]
Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle. [0189]
Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient. [0190]
It is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy. [0191]
For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water. [0192]
The nucleic acid molecules of the present invention can also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects. [0193]
Alternatively, certain of the nucleic acid molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985[0194] , Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45; all of these references are hereby incorporated in their totalities by reference herein). Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al, PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856; all of these references are hereby incorporated in their totalities by reference herein). Gene therapy approaches specific to the CNS are described by Blesch et al., 2000, Drug News Perspect., 13, 269-280; Peterson et al., 2000, Cent. Nerv. Syst. Dis., 485-508; Peel and Klein, 2000, J. Neurosci. Methods, 98, 95-104; Hagihara et al., 2000, Gene Ther., 7, 759-763; and Herrlinger et al., 2000, Methods Mol. Med., 35, 287-312. AAV-mediated delivery of nucleic acid to cells of the nervous system is further described by Kaplitt et al., U.S. Pat. No. 6,180,613.
In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996[0195] , TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. Ribozyme expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the nucleic acid molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the nucleic acid molecule binds to the target mRNA. Delivery of nucleic acid molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).
In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the instant invention is disclosed. The nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operable linked in a manner that allows expression of that nucleic acid molecule. [0196]
In another aspect the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); c) a nucleic acid sequence encoding at least one of the nucleic acid catalyst of the instant invention; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner that allows expression and/or delivery of said nucleic acid molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the nucleic acid catalyst of the invention; and/or an intron (intervening sequences). [0197]
Transcription of the nucleic acid molecule sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990[0198] , Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37). All of these references are incorporated by reference herein. Several investigators have demonstrated that nucleic acid molecules, such as ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J, 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S. A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736; all of these publications are incorporated by reference herein). The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).
In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention, in a manner that allows expression of that nucleic acid molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner that allows expression and/or delivery of said nucleic acid molecule. [0199]
In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3′-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said open reading frame and said termination region, in a manner that allows expression and/or delivery of said nucleic acid molecule. In yet another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. [0200]
In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a nucleic acid sequence encoding at least one said nucleic acid molecule, wherein said sequence is operably linked to the 3′-end of said open reading frame; and wherein said sequence is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule. [0201]

EXAMPLES

The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention. [0202]
The following examples demonstrate the selection and design of Antisense, hammerhead, DNAzyme, NCH, Amberzyme, Zinzyme, or G-Cleaver ribozyme molecules and binding/cleavage sites within PTGDS and/or PTGDR RNA. [0203]

Example 1

Identification of Potential Target Sites in Human PTGDS, ADORA1 and PTGDR RNA

The sequence of human PTGDS, ADORA1 and PTGDR genes are screened for accessible sites using a computer-folding algorithm. Regions of the RNA that do not form secondary folding structures and contained potential enzymatic nucleic acid molecule and/or antisense binding/cleavage sites are identified. The sequences of PTGDR binding/cleavage sites are shown in Tables III-VII. [0204]

Example 2

Selection of Enzymatic Nucleic Acid Cleavage Sites in Human PTGDS, ADORA1 and PTGDR RNA

Enzymatic nucleic acid molecule target sites are chosen by analyzing sequences of Human PTGDS (Genbank accession No: NM 000954), ADORA1 (Genbank accession No: NM[0205] _—000674) and PTGDR gene (Genbank accession Nos: U31332 and U31099) and prioritizing the sites on the basis of folding. Enzymatic nucleic acid molecules are designed that can bind each target and are individually analyzed by computer folding (Christoffersen et al., 1994 J. Mol. Struc. Theochem, 311, 273; Jaeger et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 7706) to assess whether the enzymatic nucleic acid molecule sequences fold into the appropriate secondary structure. Those enzymatic nucleic acid molecules with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. As noted below, varying binding arm lengths can be chosen to optimize activity. Generally, at least 4 bases on each arm are able to bind to, or otherwise interact with, the target RNA.

Example 3

Chemical Synthesis and Purification of Ribozymes and Antisense for Efficient Cleavage and/or blocking of PTGDS, ADORA1 and PTGDR RNA

Enzymatic nucleic acid molecules and antisense constructs are designed to anneal to various sites in the RNA message. The binding arms of the enzymatic nucleic acid molecules are complementary to the target site sequences described above, while the antisense constructs are fully complementary to the target site sequences described above. The enzymatic nucleic acid molecules and antisense constructs were chemically synthesized. The method of synthesis used followed the procedure for normal RNA synthesis as described above and in Usman et al., (1987 J. Am. Chem. Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids Res., 18, 5433) and Wincott et al., supra, and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. The average stepwise coupling yields were typically >98%. [0206]
Enzymatic nucleic acid molecules and antisense constructs are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). Enzymatic nucleic acid molecules and antisense constructs are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al., supra; the totality of which is hereby incorporated herein by reference) and are resuspended in water. The sequences of the chemically synthesized enzymatic nucleic acid molecules used in this study are shown below in Table III-VII. The sequences of the chemically synthesized antisense constructs used in this study are complementary sequences to the Substrate sequences shown below as in Table III-VII. [0207]

Example 4

Enzymatic Nucleic Acid Molecule Cleavage of PTGDS, ADORA1 and PTGDR RNA Target in vitro

Enzymatic nucleic acid molecules targeted to the human PTGDS, ADORA1 and PTGDR RNA are designed and synthesized as described above. These enzymatic nucleic acid molecules can be tested for cleavage activity in vitro, for example, using the following procedure. The target sequences and the nucleotide location within the PTGDR RNA are given in Tables III-VII. [0208]
Cleavage Reactions: Full-length or partially full-length, internally-labeled target RNA for enzymatic nucleic acid molecule cleavage assay is prepared by in vitro transcription in the presence of [a-[0209] ³²P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as substrate RNA without further purification. Alternately, substrates are 5′-³²P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a 2×concentration of purified enzymatic nucleic acid molecule in enzymatic nucleic acid molecule cleavage buffer (50 mM Tris-HCl, pH 7.5 at 37° C., 10 mM MgCl₂) and the cleavage reaction was initiated by adding the 2×enzymatic nucleic acid molecule mix to an equal volume of substrate RNA (maximum of 1-5 nM) that was also pre-warmed in cleavage buffer. As an initial screen, assays are carried out for 1 hour at 37° C. using a final concentration of either 40 nM or 1 mM enzymatic nucleic acid molecule, i.e., enzymatic nucleic acid molecule excess. The reaction is quenched by the addition of an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is heated to 95° C. for 2 minutes, quick chilled and loaded onto a denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage products generated by enzymatic nucleic acid molecule cleavage are visualized on an autoradiograph of the gel. The percentage of cleavage is determined by Phosphor Imager® quantitation of bands representing the intact substrate and the cleavage products.

Example 5

In vivo Models used to Evaluate the Down-Regulation of PTGDS, ADORA1 and PTGDR Gene Expression

Animal Models [0210]
Evaluating the efficacy of anti-PTGDS, ADORA-1 and/or PTGDR agents in animal models is an important prerequisite to human clinical trials. Matsuoka et al., 2000[0211] , Science, 287, 2012-2016, describe a useful asthma animal model having generating mice deficient in the PTGDR receptor. Sensitization and aerosol challenge of homozygous (PTGDR−/−) mice with ovalbumin was shown to induce increases in the serum concentration of immunoglobin E (IgE), an allergic mediator that activates mast cells, similar to wild-type mice subjected to the same conditions. The concentration of TH2 cytokines and the degree of lymphocyte lung infiltration in the OVA challenged PTGDR −/− mice was shown to be greatly reduced compared to wild type mice. In addition, the PTGDR −/− mice showed only marginal eosinophil infiltration and failed to develop airway hyperreactivity. Similarly, this model can be used to evaluate mice that are treated with nucleic acid molecules of the invention and can furthermore be used as a positive control in determining the response of mice treated with nucleic acid molecules of the invention by using such factors as airway obstruction, lung capacity, and bronchiolar alveolar lavage (BAL) fluid in the evaluation.
Cell Culture [0212]
Two human cell lines, NPE cells and NCB-20 cells are known to express PTGDR. Cloned human PTGDR has been expressed in CHO and COS7 cells and used in various studies. These PTGDR expressing lung cell lines can be used in cell culture assays to evaluate nucleic acid molecules of the invention. A primary endpoint in these experiments would be the RT-PCR analysis of PTGDR mRNA expression in PTGDR expressing cells. In addition, ligand binding assays can be developed where binding of PTGDS can be evaluated in response to treatment with nucleic acid molecules of the invention. [0213]
Indications [0214]
The present body of knowledge in PTGDS, ADORA1 and PTGDR research indicates the need for methods to assay PTGDS, ADORA1 and PTGDR activity and for compounds that can regulate PTGDS, ADORA1 and PTGDR expression for research, diagnostic, and therapeutic use. As described herein, the nucleic acid molecules of the present invention can be used in assays to diagnose disease state related of PTGDS, ADORA1 and/or PTGDR levels. In addition, the nucleic acid molecules can be used to treat disease state related to PTGDS, ADORA1 and/or PTGDR levels. [0215]
Particular degenerative and disease states that can be associated with PTGDS, ADORA1 and PTGDR levels include, but are not limited to allergic diseases and conditions, including but not limited to asthma, allergic rhinitis, atopic dermatitis, and any other diseases or conditions that are related to or will respond to the levels of PTGDS, ADORA1 and/or PTGDR in a cell or tissue, alone or in combination with other therapies. [0216]
The use of anti-inflammatories, bronchodilators, adenosine inhibitors and adenosine A1 receptor inhibitors are examples of other treatments or therapies can be combined with the nucleic acid molecules of the invention. Those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. enzymatic nucleic acid molecules and antisense molecules) are hence within the scope of the instant invention. [0217]
Diagnostic Uses [0218]
The nucleic acid molecules of this invention (e.g., enzymatic nucleic acid molecules) can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of PTGDS, ADORA1 and/or PTGDR RNA in a cell. The close relationship between enzymatic nucleic acid molecule activity and the structure of the target RNA allows the detection of mutations in any region of the molecule that alters the base-pairing and three-dimensional structure of the target RNA. By using multiple enzymatic nucleic acid molecules described in this invention, one can map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with enzymatic nucleic acid molecules can be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments can lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple enzymatic nucleic acid molecules targeted to different genes, enzymatic nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of enzymatic nucleic acid molecules and/or other chemical or biological molecules). Other in vitro uses of enzymatic nucleic acid molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with PTGDS, ADORA1 or PTGDR-related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with an enzymatic nucleic acid molecule using standard methodology. [0219]
In a specific example, enzymatic nucleic acid molecules which cleave only wild-type or mutant forms of the target RNA are used for the assay. The first enzymatic nucleic acid molecule is used to identify wild-type RNA present in the sample and the second enzymatic nucleic acid molecule is used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both enzymatic nucleic acid molecules to demonstrate the relative enzymatic nucleic acid molecule efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus each analysis requires two enzymatic nucleic acid molecules, two substrates and one unknown sample which is combined into six reactions. The presence of cleavage products is determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., PTGDS/PTGDR) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively. The use of enzymatic nucleic acid molecules in diagnostic applications contemplated by the instant invention is described, for example, in George et al., U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington, International PCT publication No. WO 00/24931, Breaker et al., International PCT Publication Nos. WO 00/26226 and 98/27104, and Sullenger et al., International PCT publication No. WO 99/29842. [0220]
Additional Uses [0221]
Potential uses of sequence-specific enzymatic nucleic acid molecules of the instant invention can have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans et al., 1975 [0222] Ann. Rev. Biochem. 44:273). For example, the pattern of restriction fragments can be used to establish sequence relationships between two related RNAs, and large RNAs can be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the enzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknown sequence. Applicant has described the use of nucleic acid molecules to down-regulate gene expression of target genes in bacterial, microbial, fungal, viral, and eukaryotic systems including plant, or mammalian cells.
All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. [0223]
One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims. [0224]
It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. [0225]
The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” can be replaced with either of the other two terms. The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims. [0226]
In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group. [0227]

Other embodiments are within the following claims.

TABLE I


Characteristics of naturally occurring ribozymes

Group I Introns

Size: ˜150 to >1000 nucleotides.

Requires a U in the target sequence immediately 5′ of the cleavage site.

Binds 4-6 nucleotides at the 5′-side of the cleavage site.

Reaction mechanism: attack by the 3′-OH of guanosine to generate cleavage

products with 3′-OH and 5′-guanosine.

Additional protein cofactors required in some cases to help folding and

maintenance of the active structure.

Over 300 known members of this class. Found as an intervening sequence in

Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-

green algae, and others.

Major structural features largely established though phylogenetic comparisons,

mutagenesis, and biochemical studies [ⁱ,ⁱⁱ].

Complete kinetic framework established for one ribozyme [ⁱⁱⁱ,^iv,^v,^vi].

Studies of ribozyme folding and substrate docking underway [^vii,^viii,^ix].

Chemical modification investigation of important residues well established [^x,^xi].

The small (4-6 nt) binding site may make this ribozyme too non-specific for

targeted RNA cleavage, however, the Tetrahymena group I intron has been used

to repair a “defective” β-galactosidase message by the ligation of new β-

galactosidase sequences onto the defective message [^xii].

RNAse P RNA (M1 RNA)

Size: ˜290 to 400 nucleotides.

RNA portion of a ubiquitous ribonucleoprotein enzyme.

Cleaves tRNA precursors to form mature tRNA [^xiii].

Reaction mechanism: possible attack by M²⁺-OH to generate cleavage products

with 3′-OH and 5′-phosphate.

RNAse P is found throughout the prokaryotes and eukaryotes. The RNA subunit

has been sequenced from bacteria, yeast, rodents, and primates.

Recruitment of endogenous RNAse P for therapeutic applications is possible

through hybridization of an External Guide Sequence (EGS) to the target RNA

[^xiv,^xv]

Important phosphate and 2′OH contacts recently identified [^xvi,^xvii]

Group II Introns

Size: >1000 nucleotides.

Trans cleavage of target RNAs recently demonstrated [^xviii,^xix].

Sequence requirements not fully determined.

Reaction mechanism: 2′-OH of an internal adenosine generates cleavage products

with 3′-OH and a “lariat” RNA containing a 3′-5′ and a 2′-5′ branch point.

Only natural ribozyme with demonstrated participation in DNA cleavage [^xx,^xxi] in

addition to RNA cleavage and ligation.

Major structural features largely established through phylogenetic comparisons

[^xxii].

Important 2′OH contacts beginning to be identified [^xxiii]

Kinetic framework under development [^xxiv]

Neurospora VS RNA

Size: ˜144 nucleotides.

Trans cleavage of hairpin target RNAs recently demonstrated [^xxv].

Sequence requirements not fully determined.

Reaction mechanism: attack by 2′-OH 5′to the scissile bond to generate cleavage

products with 2′,3′-cyclic phosphate and 5′-OH ends.

Binding sites and structural requirements not fully determined.

Only 1 known member of this class. Found in Neurospora VS RNA.

Hammerhead Ribozyme

(see text for references)

Size: ˜13 to 40 nucleotides.

Requires the target sequence UH immediately 5′ of the cleavage site.

Binds a variable number nucleotides on both sides of the cleavage site.

Reaction mechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavage

products with 2′,3′-cyclic phosphate and 5′-OH ends.

14 known members of this class. Found in a number of plant pathogens

(virusoids) that use RNA as the infectious agent.

Essential structural features largely defined, including 2 crystal structures [^xxvi,^xxvii]

Minimal ligation activity demonstrated (for engineering through in vitro selection)

[^xxviii]

Complete kinetic framework established for two or more ribozymes [^xxix].

Chemical modification investigation of important residues well established [^xxx].

Hairpin Ribozyme

Size: ˜50 nucleotides.

Requires the target sequence GUC immediately 3′of the cleavage site.

Binds 4-6 nucleotides at the 5′-side of the cleavage site and a variable number to

the 3′-side of the cleavage site.

products with 2′,3′-cyclic phosphate and 5′-OH ends.

3 known members of this class. Found in three plant pathogen (satellite RNAs of

the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus)

which uses RNA as the infectious agent.

Essential structural features largely defined [^xxxi,^xxxii,^xxxiii,^xxxiv]

Ligation activity (in addition to cleavage activity) makes ribozyme amenable to

engineering through in vitro selection [^xxxv]

Complete kinetic framework established for one ribozyme [^xxxvi].

Chemical modification investigation of important residues begun [^xxxvii,^xxxviii].

Hepatitis Delta Virus (HDV) Ribozyme

Size: ˜60 nucleotides.

Trans cleavage of target RNAs demonstrated [^xxxix].

Binding sites and structural requirements not fully determined, although no

sequences 5′ of cleavage site are required. Folded ribozyme contains a pseudoknot

structure [^xl].

products with 2′,3′-cyclic phosphate and 5′-OH ends.

Only 2 known members of this class. Found in human HDV.

Circular form of HDV is active and shows increased nuclease stability [^xli]

A. 2.5 μmol Synthesis Cycle ABI 394 Instrument

Phosphoramidites	6.5	163	μL	45 sec	2.5	min	7.5	min
S-Ethyl Tetrazole	23.8	238	μL	45 sec	2.5	min	7.5	min
Acetic Anhydride	100	233	μL	5 sec	5	sec	5	sec
N-Methyl	186	233	μL	5 sec	5	sec	5	sec
Imidazole
TCA	176	2.3	mL	21 sec	21	sec	21	sec
Iodine	11.2	1.7	mL	45 sec	45	sec	45	sec
Beaucage	12.9	645	μL	100 sec	300	sec	300	sec

Acetonitrile

NA

6.67

mL

NA

B. 0.2 μmol Synthesis Cycle ABI 394 Instrument

Phosphoramidites	15	31	μL	45 sec	233	sec	465	sec
S-Ethyl Tetrazole	38.7	31	μL	45 sec	233	min	465	sec
Acetic Anhydride	655	124	μL	5 sec	5	sec	5	sec
N-Methyl	1245	124	μL	5 sec	5	sec	5	sec
Imidazole
TCA	700	732	μL	10 sec	10	sec	10	sec
Iodine	20.6	244	μL	15 sec	15	sec	15	sec
Beaucage	7.7	232	μL	100 sec	300	sec	300	sec

Acetonitrile	NA	2.64	mL	NA	NA	NA

C. 0.2 μmol Synthesis Cycle 96 well Instrument

	Equivalents: DNA/	Amount: DNA/2′-O-		Wait Time* 2′-O-	Wait Time*
Reagent	2′-O-methyl/Ribo	methyl/Ribo	Wait Time* DNA	methyl	Ribo

Phosphoramidites	22/33/66	40/60/120	μL	60 sec	180	sec	360 sec
S-Ethyl Tetrazole	70/105/210	40/60/120	μL	60 sec	180	min	360 sec
Acetic Anhydride	265/265/265	50/50/50	μL	10 sec	10	sec	10 sec
N-Methyl	502/502/502	50/50/50	μL	10 sec	10	sec	10 sec
Imidazole
TCA	238/475/475	250/500/500	μL	15 sec	15	sec	15 sec
Iodine	6.8/6.8/6.8	80/80/80	μL	30 sec	30	sec	30 sec
Beaucage	34/51/51	80/120/120		100 sec	200	sec	200 sec

Acetonitrile	NA	1150/1150/1150	μL	NA	NA	NA

TABLE III


Human PTGDR Hammerhead Ribozyme and Substrate Sequence

		Seq		Seq
Pos	Substrate	ID	Hammerhead Ribozyme	ID

12	UUCUGGCU A UUUUCCUC	1	GAGGAAAA CUGAUGAGGCCGUUAGGCCGAA AGCCAGAA	228

14	CUGGCUAU U UUCCUCCU	2	AGGAGGAA CUGAUGAGGCCGUUAGGCCGAA AUAGCCAG	229

15	UGGCUAUU U UCCUCCUG	3	CAGGAGGA CUGAUGAGGCCGUUAGGCCGAA AAUAGCCA	230

16	GGCUAUUU U CCUCCUGC	4	GCAGGAGG CUGAUGAGGCCGUUAGGCCGAA AAAUAGCC	231

17	GCUAUUUU C CUCCUGCC	5	GGCAGGAG CUGAUGAGGCCGUUAGGCCGAA AAAAUAGC	232

20	AUUUUCCU C CUGCCGUU	6	AACGGCAG CUGAUGAGGCCGUUAGGCCGAA AGGAAAAU	233

28	CCUGCCGU U CCGACUCG	7	CGAGUCGG CUGAUGAGGCCGUUAGGCCGAA ACGGCAGG	234

29	CUGCCGUU C CGACUCGG	8	CCGAGUCG CUGAUGAGGCCGUUAGGCCGAA AACGGCAG	235

35	UUCCGACU C GGCACCAG	9	CUGGUGCC CUGAUGAGGCCGUUAGGCCGAA AGUCGGAA	236

47	ACCAGAGU C UGUCUCUA	10	UAGAGACA CUGAUGAGGCCGUUAGGCCGAA ACUCUGGU	237

51	GAGUCUGU C UCUACUGA	11	UCAGUAGA CUGAUGAGGCCGUUAGGCCGAA ACAGACUC	238

53	GUCUGUCU C UACUGAGA	12	UCUCAGUA CUGAUGAGGCCGUUAGGCCGAA AGACAGAC	239

55	CUGUCUCU A CUGAGAAC	13	GUUCUCAG CUGAUGAGGCCGUUAGGCCGAA AGAGACAG	240

73	CAGCGCGU C AGGGCCGA	14	UCGGCCCU CUGAUGAGGCCGUUAGGCCGAA ACGCGCUG	241

85	GCCGAGCU C UUCACUGG	15	CCAGUGAA CUGAUGAGGCCGUUAGGCCGAA AGCUCGGC	242

87	CGAGCUCU U CACUGGCC	16	GGCCAGUG CUGAUGAGGCCGUUAGGCCGAA AGAGCUCG	243

88	GAGCUCUU C ACUGGCCU	17	AGGCCAGU CUGAUGAGGCCGUUAGGCCGAA AAGAGCUC	244

100	GGCCUGCU C CGCGCUCU	18	AGAGCGCG CUGAUGAGGCCGUUAGGCCGAA AGCAGGCC	245

107	UCCGCGCU C UUCAAUGC	19	GCAUUGAA CUGAUGAGGCCGUUAGGCCGAA AGCGCGGA	246

109	CGCGCUCU U CAAUGCCA	20	UGGCAUUG CUGAUGAGGCCGUUAGGCCGAA AGAGCGCG	247

110	GCGCUCUU C AAUGCCAG	21	CUGGCAUU CUGAUGAGGCCGUUAGGCCGAA AAGAGCGC	248

130	CAGGCGCU C ACCCUGCA	22	UGCAGGGU CUGAUGAGGCCGUUAGGCCGAA AGCGCCUG	249

145	CAGAGCGU C CCGCCUCU	23	AGAGGCGG CUGAUGAGGCCGUUAGGCCGAA ACGCUCUG	250

152	UCCCGCCU C UCAAAGAG	24	CUCUUUGA CUGAUGAGGCCGUUAGGCCGAA AGGCGGGA	251

154	CCGCCUCU C AAAGAGGG	25	CCCUCUUU CUGAUGAGGCCGUUAGGCCGAA AGAGGCGG	252

178	CCGCGAGU U UAGAUAGG	26	CCUAUCUA CUGAUGAGGCCGUUAGGCCGAA ACUCGCGG	253

179	CGCGAGUU U AGAUAGGA	27	UCCUAUCU CUGAUGAGGCCGUUAGGCCGAA AACUCGCG	254

180	GCGAGUUU A GAUAGGAG	28	CUCCUAUC CUGAUGAGGCCGUUAGGCCGAA AAACUCGC	255

184	GUUUAGAU A GGAGGUUC	29	GAACCUCC CUGAUGAGGCCGUUAGGCCGAA AUCUAAAC	256

191	UAGGAGGU U CCUGCCGU	30	ACGGCAGG CUGAUGAGGCCGUUAGGCCGAA ACCUCCUA	257

192	AGGAGGUU C CUGCCGUG	31	CACGGCAG CUGAUGAGGCCGUUAGGCCGAA AACCUCCU	258

220	GCCGCCCU C GGAGCUUU	32	AAAGCUCC CUGAUGAGGCCGUUAGGCCGAA AGGGCGGC	259

227	UCGGAGCU U UUUCUGUG	33	CACAGAAA CUGAUGAGGCCGUUAGGCCGAA AGCUCCGA	260

228	CGGAGCUU U UUCUGUGG	34	CCACAGAA CUGAUGAGGCCGUUAGGCCGAA AAGCUCCG	261

229	GGAGCUUU U UCUGUGGC	35	GCCACAGA CUGAUGAGGCCGUUAGGCCGAA AAAGCUCC	262

230	GAGCUUUU U CUGUGGCG	36	CGCCACAG CUGAUGAGGCCGUUAGGCCGAA AAAAGCUC	263

231	AGCUUUUU C UGUGGCGC	37	GCGCCACA CUGAUGAGGCCGUUAGGCCGAA AAAAAGCU	264

244	GCGCAGCU U CUCCGCCC	38	GGGCGGAG CUGAUGAGGCCGUUAGGCCGAA AGCUGCGC	265

245	CGCAGCUU C UCCGCCCG	39	CGGGCGGA CUGAUGAGGCCGUUAGGCCGAA AAGCUGCG	266

247	CAGCUUCU C CGCCCGAG	40	CUCGGGCG CUGAUGAGGCCGUUAGGCCGAA AGAAGCUG	267

280	CGGGGGCU C CUUAGCAC	41	GUGCUAAG CUGAUGAGGCCGUUAGGCCGAA AGCCCCCG	268

283	GGGCUCCU U AGCACCCG	42	CGGGUGCU CUGAUGAGGCCGUUAGGCCGAA AGGAGCCC	269

284	GGCUCCUU A GCACCCGG	43	CCGGGUGC CUGAUGAGGCCGUUAGGCCGAA AAGGAGCC	270

306	GGGGCCCU C GCCCUUCC	44	GGAAGGGC CUGAUGAGGCCGUUAGGCCGAA AGGGCCCC	271

312	CUCGCCCU U CCGCAGCC	45	GGCUGCGG CUGAUGAGGCCGUUAGGCCGAA AGGGCGAG	272

313	UCGCCCUU C CGCAGCCU	46	AGGCUGCG CUGAUGAGGCCGUUAGGCCGAA AAGGGCGA	273

322	CGCAGCCU U CACUCCAG	47	CUGGAGUG CUGAUGAGGCCGUUAGGCCGAA AGGCUGCG	274

323	GCAGCCUU C ACUCCAGC	48	GCUGGAGU CUGAUGAGGCCGUUAGGCCGAA AAGGCUGC	275

327	CCUUCACU C CAGCCCUC	49	GAGGGCUG CUGAUGAGGCCGUUAGGCCCAA AGUGAAGG	276

335	CCAGCCCU C UGCUCCCG	50	CGUGAGCA CUGAUGAGGCCGUUACGCCGAA AGGGCUGG	277

340	CCUCUGCU C CCGCACGC	51	GCGUGCGG CUGAUGAGGCCGUUAGGCCGAA AGCAGAGG	278

357	CAUGAAGU C GCCGUUCU	52	AGAACGGC CUGAUGAGGCCGUUAGGCCGAA ACUUCAUG	279

363	GUCGCCGU U CUACCGCU	53	ACCCGUAG CUGAUGAGGCCGUUAGGCCGAA ACGGCGAC	280

364	UCGCCGUU C UACCGCUG	54	CAGCGGUA CUGAUGAGGCCGUUAGGCCGAA AACGCCGA	281

366	GCCGUUCU A CCGCUGCC	55	GGCAGCGG CUGAUGAGGCCGUUAGGCCGAA AGAACGGC	282

387	CACCACCU C UCUCGAAA	56	UUUCCACA CUGAUGAGGCCGUUAGGCCGAA AGGUGGUG	283

405	AGGCAACU C GGCGGUGA	57	UCACCGCC CUGAUGAGGCCGUUAGGCCGAA AGUUGCCU	284

427	GCGGUGCU C UUCAGCAC	58	GUGCUGAA CUGAUGAGGCCGUUAGGCCGAA AGCACCCC	285

429	GGUGCUCU U CAGCACCG	59	CGGUGCUG CUGAUGAGGCCUUUAGGCCGAA AGAGCACC	286

430	GUGCUCUU C AGCACCGG	60	CCGGUGCU CUGAUGAGGCCGUUAGGCCGAA AAGAGCAC	287

442	ACCGGCCU C CUGGGCAA	61	UUGCCCAG CUGAUGAGGCCGUUAGGCCGAA AGGCCGGU	288

480	GGCCCGCU C GGGGCUGG	62	CCAGCCCC CUGAUGAGGCCGUUAGGCCGAA AGCGCGCC	289

498	GUGGUGCU C GCGGCGUC	63	GACGCCGC CUGAUGAGGCCGUUAGGCCGAA AGCACCAC	290

506	CGCGGCGU C CACUGCGC	64	GCGCAGUG CUGAUGAGGCCGUUAGGCCCAA ACGCCGCG	291

525	GCUGCCCU C GGUCUUCU	65	AGAAGACC CUGAUGAGGCCGUUAGGCCGAA AGGGCAGC	292

529	CCCUCGGU C UUCUACAU	66	AUGUAGAA CUGAUGAGGCCGUUAGGCCGAA ACCGAGGG	293

531	CUCGGUCU U CUACAUGC	67	GCAUGUAG CUGAUGAGGCCCUUACGCCGAA AGACCGAG	294

532	UCGGUCUU C UACAUGCU	68	AGCAUGUA CUGAUGAGGCCGUUAGGCCGAA AAGACCGA	295

534	GGUCUUCU A CAUGCUGG	69	CCAGCAUG CUGAUGAGGCCGUUAGCCCGAA AGAAGACC	296

559	CUGACGGU C ACCGACUU	70	AAGUCGGU CUGAUGAGGCCGUUAGGCCGAA ACCGUCAG	297

567	CACCGACU U GCUGGGCA	71	UCCCCAGC CUGAUCAGCCCGUUAGGCCCAA AGUCGGUG	298

583	AAGUGCCU C CUAAGCCC	72	GGGCUUAG CUGAUGAGGCCGUUAGGCCGAA AGGCACUU	299

586	UGCCUCCU A AGCCCGGU	73	ACCGGGCU CUGAUGAGGCCCUUAGGCCGAA ACGAGGCA	300

609	GGCUGCCU A CGCUCAGA	74	UCUGAGCG CUGAUGAGGCCGUUAGGCCGAA AGGCAGCC	301

614	CCUACCCU C AGAACCGG	75	CCGGUUCU CUGAUGAGGCCGUUAGGCCGAA AGCGUAGG	302

626	ACCGGAGU C UGCGGGUG	76	CACCCCCA CUGAUGAGGCCCUUAGGCCGAA ACUCCCCU	303

637	CGCGUGCU U CCGCCCGC	77	GCGGGCGC CUGAUGAGGCCGUUAGGCCGAA AGCACCCG	304

648	GCCCGCAU U GGACAACU	78	AGUUGUCC CUGAUGAGGCCGUUAGGCCGAA AUGCGGGC	305

657	CGACAACU C GUUGUGCC	79	GGCACAAC CUGAUGAGGCCGUUAGGCCGAA AGUUGUCC	306

660	CAACUCGU U GUGCCAAG	80	CUUGGCAC CUGAUGAGGCCGUUAGGCCGAA ACGAGUUG	307

672	CCAAGCCU U CGCCUUCU	81	AGAAGGCG CUGAUGAGGCCGUUAGGCCGAA AGGCUUGG 308

673	CAAGCCUU C GCCUUCUU	82	AAGAAGGC CUGAUGAGGCCGUUAGGCCGAA AAGGCUUG	309

678	CUUCGCCU U CUUCAUGU	83	ACAUGAAG CUGAUGAGGCCGUUAGGCCGAA AGGCGAAG	310

679	UUCGCCUU C UUCAUGUC	84	GACAUGAA CUGAUGAGGCCGUUAGGCCGAA AAGGCGAA	311

681	CGCCUUCU U CAUGUCCU	85	AGGACAUC CUGAUGACCCCGUUAGGCCGAA ACAAGGCC	312

682	GCCUUCUU C AUGUCCUU	86	AAGGACAU CUGAUGAGGCCGUUAGGCCGAA AAGAAGGC	313

687	CUUCAUGU C CUUCUUUG	87	CAAAGAAG CUGAUGAGGCCGUUAGGCCGAA ACAUGAAG	314

690	CAUGUCCU U CUUUGGGC	88	GCCCAAAG CUGAUGAGGCCGUUAGGCCGAA AGGACAUG	315

691	AUGUCCUU C UUUGGGCU	89	AGCCCAAA CUGAUGAGGCCGUUAGGCCGAA AAGGACAU	316

693	GUCCUUCU U UGGGCUCU	90	AGAGCCCA CUGAUGAGGCCGUUAGGCCGAA AGAAGGAC	317

694	UCCUUCUU U GGGCUCUC	91	GAGAGCCC CUGAUGAGGCCGUUAGGCCGAA AAGAAGGA	318

700	UUUGGGCU C UCCUCGAC	92	GUCGAGGA CUGAUGAGGCCGUUAGGCCGAA AGCCCAAA	319

702	UGGGCUCU C CUCGACAC	93	GUGUCGAG CUGAUGAGGCCGUUAGGCCGAA AGAGCCCA	320

705	GCUCUCCU C GACACUGC	94	GCAGUGUC CUGAUGAGGCCGUUAGGCCGAA AGGAGAGC	321

718	CUGCAACU C CUGGCCAU	95	AUGGCCAG CUGAUGAGGCCGUUAGGCCGAA AGUUGCAG	322

745	UGCUGGCU C UCCCUAGG	96	CCUAGGGA CUGAUGAGGCCGUUAGGCCGAA AGCCAGCA	323

747	CUGGCUCU C CCUAGGGC	97	GCCCUAGG CUGAUGAGGCCGUUAGGCCGAA AGAGCCAG	324

751	CUCUCCCU A GGGCACCC	98	GGGUGCCC CUGAUGAGGCCGUUAGGCCGAA AGGGAGAG	325

761	GGCACCCU U UCUUCUAC	99	GUAGAAGA CUGAUGAGGCCGUUAGGCCGAA AGGGUGCC	326

762	GCACCCUU U CUUCUACC	100	GGUAGAAG CUGAUGAGGCCGUUAGGCCGAA AAGGGUGC	327

763	CACCCUUU C UUCUACCC	101	CGGUAGAA CUGAUGAGCCCGUUAGGCCGAA AAAGGGUG	328

765	CCCUUUCU U CUACCGAC	102	GUCGGUAG CUGAUGAGGCCGUUAGGCCGAA AGAAAGGG	329

766	CCUUUCUU C UACCGACG	103	CGUCGGUA CUGAUGAGGCCGUUAGGCCGAA AAGAAAGG	330

768	UUUCUUCU A CCGACGGC	104	GCCGUCGG CUCAUGAGGCCUUUAGGCCGAA AGAAGAAA	331

781	CGCCACAU C ACCCUGCG	105	CGCAGGGU CUGAUGAGGCCGUUAGGCCGAA AUGUGCCG	332

825	GAGCGCCU U CUCCCUGG	106	CCAGGGAG CUGAUCAGCCCGUUAGGCCGAA AGGCGCUC	333

826	AGCGCCUU C UCCCUGGC	107	GCCAGGGA CUGAUGAGGCCGUUAGGCCGAA AAGGCGCU	334

828	CGCCUUCU C CCUGGCUU	108	AAGCCACG CUGAUGAGGCCGUUAGGCCGAA AGAAGGCG	335

836	CCCUGGCU U UCUGCGCG	109	CGCGCAGA CUGAUGAGGCCGUUAGGCCGAA AUCCAGGG	336

837	CCUGGCUU U CUGCGCGC	110	GCGCGCAG CUGAUCACGCCGUUAGGCCGAA AAGCCAGG	337

838	CUGGCUUU C UGCGCGCU	111	AGCGCGCA CUGAUGAGGCCGUUAGGCCGAA AAAGCCAG	338

847	UGCGCGCU A CCUUUCAU	112	AUGAAAGC CUCAUGAGGCCGUUACGCCGAA AGCGCGCA	339

851	CGCUACCU U UCAUCGCC	113	GCCCAUGA CUGAUGAGGCCGUUAGGCCGAA AGGUAGCG	340

852	GCUACCUU U CAUGGGCU	114	AGCCCAUG CUGAUGAGGCCGUUAGGCCGAA AAGGUAGC	341

853	CUACCUUU C AUGGGCUU	115	AAGCCCAU CUGAUGAGGCCGUUAGGCCGAA AAAGGUAG	342

861	CAUGGGCU U CGGGAAGU	116	ACUUCCCG CUGAUGAGGCCGUUAGGCCGAA AGCCCAUG	343

862	AUGGGCUU C GGGAAGUU	117	AACUUCCC CUGAUGAGGCCGUUAGGCCGAA AAGCCCAU	344

870	CGGGAAGU U CGUGCAGU	118	ACUGCACG CUGAUGAGGCCGUUAGGCCGAA ACUUCCCG	345

871	GGGAAGUU C GUGCAGUA	119	UACUGCAC CUGAUGAGGCCGUUAGGCCGAA AACUUCCC	346

879	CGUGCAGU A CUGCCCCG	120	CGGGGCAG CUGAUGAGGCCGUUAGGCCGAA ACUGCACG	347

900	CUGGUGCU U UAUCCAGA	121	UCUGGAUA CUGAUGAGGCCGUUAGGCCGAA AGCACCAG	348

901	UGGUGCUU U AUCCAGAU	122	AUCUGGAU CUGAUGAGGCCGUUAGGCCGAA AAGCACCA	349

902	GGUGCUUU A UCCAGAUG	123	CAUCUGGA CUGAUGAGGCCGUUAGGCCGAA AAAGCACC	350

904	UGCUUUAU C CAGAUGGU	124	ACCAUCUG CUGAUGAGGCCGUUAGGCCGAA AUAAAGCA	351

913	CAGAUGGU C CACGAGGA	125	UCCUCGUG CUGAUGAGGCCGUUAGGCCGAA ACCAUCUG	352

927	GGAGGGCU C GCUGUCGG	126	CCGACAGC CUGAUGAGGCCGUUAGGCCGAA AGCCCUCC	353

933	CUCGCUGU C GGUGCUGG	127	CCAGCACC CUGAUGAGGCCGUUAGGCCGAA ACAGCGAG	354

945	GCUGGGGU A CUCUGUGC	128	GCACAGAG CUGAUGAGGCCGUUAGGCCGAA ACCCCAGC	355

948	GGGGUACU C UGUGCUCU	129	AGAGCACA CUGAUGAGGCCGUUAGGCCGAA AGUACCCC	356

955	UCUGUGCU C UACUCCAG	130	CUGGAGUA CUGAUGAGGCCGUUAGGCCGAA AGCACAGA	357

957	UGUGCUCU A CUCCAGCC	131	GGCUGGAG CUGAUGAGGCCGUUAGGCCGAA AGAGCACA	358

960	GCUCUACU C CAGCCUCA	132	UGAGGCUG CUGAUGAGGCCGUUAGGCCGAA AGUAGAGC	359

967	UCCAGCCU C AUGGCGCU	133	AGCGCCAU CUGAUGAGGCCGUUAGGCCGAA AGGCUGGA	360

982	CUGCUGGU C CUCGCCAC	134	GUGGOGAG CUGAUGAGGCCGUUAGGCCGAA ACCAGCAG	361

985	CUGGUCCU C GCCACCGU	135	ACGGUGGC CUGAUGAGGCCGUUAGGCCGAA AGGACCAG	362

1006	UGCAACCU C GGCGCCAU	136	AUGGCGCC CUGAUGAGGCCGUUAGGCCGAA AGGUUGCA	363

1024	CGCAACCU C UAUGCGAU	137	AUCGCAUA CUGAUGAGGCCGUUAGGCCGAA AGGUUGCG	364

1026	CAACCUCU A UGCGAUGC	138	GCAUCGCA CUGAUGAGGCCGUUAGGCCGAA AGAGGUUG	365

1062	CCCGCGCU C CUCCACCA	139	UGGUGCAG CUGAUGAGGCCGUUAGGCCGAA AGCGCGGG	366

1110	GGAAGCGU C CCCUCAGC	140	GCUGAGGG CUGAUGAGGCCGUUAGGCCGAA ACGCUUCC	367

1115	CGUCCCCU C AGCCCCUG	141	CAGGGGCU CUGAUGAGGCCGUUAGGCCGAA AGGGGACG	368

1136	AGCUGGAU C ACCUCCUG	142	CAGGAGGU CUGAUGAGGCCGUUAGGCCGAA AUCCAGCU	369

1141	GAUCACCU C CUGCUGCU	143	AGCAGCAG CUGAUGAGGCCGUUAGGCCGAA AGGUGAUC	370

1168	ACCGUGCU C UUCACUAU	144	AUAGUGAA CUGAUGAGGCCGUUAGGCCGAA AGCACGGU	371

1170	CGUGCUCU U CACUAUGU	145	ACAUAGUG CUGAUGAGGCCGUUAGGCCGAA AGAGCACG	372

1171	GUGCUCUU C ACUAUGUG	146	CACAUAGU CUGAUGAGGCCGUUAGGCCGAA AAGAGCAC	373

1175	UCUUCACU A UGUGUUCU	147	AGAACACA CUGAUGAGGCCGUUAGGCCGAA AGUGAAGA	374

1181	CUAUGUGU U CUCUGCCC	148	GGGCAGAG CUGAUGAGGCCGUUAGGCCGAA ACACAUAG	375

1182	UAUGUGUU C UCUGCCCG	149	CGGGCAGA CUGAUGAGGCCGUUAGGCCGAA AACACAUA	376

1184	UGUGUUCU C UGCCCGUA	150	UACGGGCA CUGAUGAGCCCGUUAGGCCGAA AGAACACA	377

1192	CUGCCCGU A AUUUAUCG	151	CGAUAAAU CUGAUGAGGCCGUUAGGCCGAA ACGGGCAC	378

1195	CCCGUAAU U UAUCGCGC	152	GCGCGAUA CUGAUGAGGCCGUUAGGCCGAA AUUACGGG	379

1196	CCGUAAUU U AUCGCGCU	153	AGCGCGAU CUCAUGAGGCCGUUAGGCCGAA AAUUACGG	380

1197	CGUAAUUU A UCGCUCUU	154	AAGCGCGA CUGAUGAGGCCGUUAGGCCGAA AAAUUACG	381

1199	UAAUUUAU C GCGCUUAC	155	GUAAGCGC CUGAUGAGGCCGUUAGGCCGAA AUAAAUUA 382

1205	AUCGCGCU U ACUAUGGA	156	UCCAUAGU CUGAUGAGGCCGUUAGGCCGAA AGCGCGAU	383

1206	UCGCGCUU A CUAUGGAG	157	CUCCAUAG CUGAUGAGGCCGUUAGGCCGAA AAGCGCGA	384

1209	CGCUUACU A UGGAGCAU	158	AUGCUCCA CUGAUGAGGCCGUUAGGCCGAA AGUAAGCG	385

1218	UGGAGCAU U UAAGGAUG	159	CAUCCUUA CUGAUGAGGCCCUUAGGCCGAA AUGCUCCA	386

1219	GGAGCAUU U AAGGAUGU	160	ACAUCCUU CUGAUGAGGCCGUUAGGCCGAA AAUGCUCC	387

1220	GAGCAUUU A AGGAUGUC	161	GACAUCCU CUGAUGAGGCCGUUAGGCCGAA AAAUGCUC	388

1228	AAGGAUGU C AAGGAGAA	162	UUCUCCUU CUGAUGAGGCCGUUAGGCCGAA ACAUCCUC	389

1248	CAGGACCU C UGAAGAAG	163	CUUCUUCA CUGAUGAGGCCGUUAGGCCGAA AGGUCCUG	390

1267	GAAGACCU C CGAGCCUU	164	AAGGCUCG CUGAUGAGGCCGUUAGGCCGAA AGGUCUUC	391

1275	CCGAGCCU U GCGAUUUC	165	GAAAUCGC CUGAUGAGGCCGUUAGGCCGAA AGGCUCGG	392

1281	CUUGCGAU U UCUAUCUG	166	CAGAUAGA CUGAUGAGGCCGUUAGGCCGAA AUCGCAAG	393

1282	UUGCGAUU U CUAUCUGU	167	ACAGAUAG CUGAUGAGGCCGUUAGGCCGAA AAUCGCAA	394

1283	UGCGAUUU C UAUCUGUG	168	CACAGAUA CUGAUGAGGCCGUUAGGCCGAA AAAUCGCA	395

1285	CGAUUUCU A UCUGUGAU	169	AUCACAGA CUGAUGAGGCCGUUAGGCCGAA AGAAAUCG	396

1287	AUUUCUAU C UGUGAUUU	170	AAAUCACA CUGAUGAGGCCGUUAGGCCGAA AUAGAAAU	397

1294	UCUGUGAU U UCAAUUGU	171	ACAAUUGA CUGAUGAGGCCGUUAGGCCGAA AUCACAGA	398

1295	CUGUGAUU U CAAUUGUG	172	CACAAUUG CUGAUGAGGCCGUUAGGCCGAA AAUCACAG	399

1296	UGUGAUUU C AAUUGUGG	173C	CACAAUU CUGAUGAGGCCGUUAGGCCGAA AAAUCACA	400

1300	AUUUCAAU U GUGGACCC	174	GGGUCCAC CUGAUGAGGCCGUUAGGCCGAA AUUGAAAU	401

1310	UGGACCCU U GGAUUUUU	175	AAAAAUCC CUGAUGAGGCCGUUAGGCCGAA AGGGUCCA	402

1315	CCUUGGAU U UUUAUCAU	176	AUGAUAAA CUGAUGAGGCCGUUAGGCCGAA AUCCAAGG	403

1316	CUUGGAUU U UUAUCAUU	177	AAUGAUAA CUGAUGAGGCCGUUAGGCCGAA AAUCCAAG	404

1317	UUGGAUUU U UAUCAUUU	178	AAAUGAUA CUGAUGAGGCCGUUAGGCCGAA AAAUCCAA	405

1318	UGGAUUUU U AUCAUUUU	179	AAAAUGAU CUGAUGAGGCCGUUAGGCCGAA AAAAUCCA	406

1319	GGAUUUUU A UCAUUUUC	180	GAAAAUGA CUGAUGAGGCCGUUAGGCCGAA AAAAAUCC	407

1321	AUUUUUAU C AUUUUCAG	181	CUGAAAAU CUGAUGAGGCCGUUAGGCCGAA AUAAAAAU	408

1324	UUUAUCAU U UUCAGAUC	182	GAUCUGAA CUGAUGAGGCCGUUAGGCCGAA AUGAUAAA	409

1325	UUAUCAUU U UCAGAUCU	183	AGAUCUGA CUGAUGAGGCCGUUAGGCCGAA AAUGAUAA	410

1326	UAUCAUUU U CAGAUCUC	184	GAGAUCUG CUGAUGAGGCCGUUAGGCCGAA AAAUGAUA	411

1327	AUCAUUUU C AGAUCUCC	185	GGAGAUCU CUGAUGAGGCCGUUAGGCCGAA AAAAUGAU	412

1332	UUUCAGAU C UCCAGUAU	186	AUACUGGA CUGAUGAGGCCGUUAGGCCGAA AUCUGAAA	413

1334	UCAGAUCU C CAGUAUUU	187	AAAUACUG CUGAUGAGGCCGUUAGGCCGAA AGAUCUGA	414

1339	UCUCCAGU A UUUCGGAU	188	AUCCGAAA CUGAUGAGGCCGUUAGGCCGAA ACUGGAGA	415

1341	UCCAGUAU U UCGGAUAU	189	AUAUCCGA CUGAUGAGGCCGUUAGGCCGAA AUACUGGA	416

1342	CCAGUAUU U CGGAUAUU	190	AAUAUCCG CUGAUGAGGCCGUUAGGCCGAA AAUACUGG	417

1343	CAGUAUUU C GGAUAUUU	191	AAAUAUCC CUGAUGAGGCCGUUAGGCCGAA AAAUACUG	418

1348	UUUCGGAU A UUUUUUCA	192	UGAAAAAA CUGAUGAGGCCGUUAGGCCGAA AUCCGAAA	419

1350	UCGGAUAU U UUUUCACA	193	UGUGAAAA CUGAUGAGGCCGUUAGGCCGAA AUAUCCGA	420

1351	CGGAUAUU U UUUCACAA	194	UUGUGAAA CUGAUGAGGCCGUUAGGCCGAA AAUAUCCG	421

1352	GGAUAUUU U UUCACAAG	195	CUUGUGAA CUGAUGAGGCCGUUAGGCCGAA AAAUAUCC	422

1353	GAUAUUUU U UCACAAGA	196	UCUUGUGA CUGAUGAGGCCGUUAGGCCGAA AAAAUAUC	423

1354	AUAUUUUU U CACAAGAU	197	AUCUUGUG CUGAUGAGGCCGUUAGGCCGAA AAAAAUAU	424

1355	UAUUUUUU C ACAAGAUU	198	AAUCUUGU CUGAUGAGGCCGUUAGGCCGAA AAAAAAUA	425

1363	CACAAGAU U UUCAUUAG	199	CUAAUGAA CUGAUGAGGCCGUUAGGCCGAA AUCUUGUG	426

1364	ACAAGAUU U UCAUUAGA	200	UCUAAUGA CUGAUGAGGCCGUUAGGCCGAA AAUCUUGU	427

1365	CAAGAUUU U CAUUAGAC	201	GUCUAAUG CUGAUGAGGCCGUUAGGCCGAA AAAUCUUG	428

1366	AAGAUUUU C AUUAGACC	202	CGUCUAAU CUGAUGAGGCCGUUACGCCGAA AAAAUCUU	429

1369	AUUUUCAU U AGACCUCU	203	AGAGGUCU CUGAUGAGGCCGUUAGGCCGAA AUGAAAAU 430

1370	UUUUCAUU A GACCUCUU	204	AAGAGGUC CUGAUGAGGCCGUUAGGCCGAA AAUGAAAA 431

1376	UUAGACCU C UUAGGUAC	205	GUACCUAA CUGAUGAGGCCGUUAGGCCGAA AGGUCUAA	432

1378	AGACCUCU U AGGUACAG	206	CUGUACCU CUGAUGAGGCCGUUAGGCCGAA AGAGGUCU	433

1379	GACCUCUU A GGUACAGG	207	CCUGUACC CUGAUGAGGCCGUUAGGCCGAA AAGAGGUC	434

1383	UCUUAGGU A CAGGAGCC	208	GGCUCCUG CUGAUGAGGCCGUUAGGCCGAA ACCUAAGA	435

1403	GCAGCAAU U CCACUAAC	209	GUUAGUGG CUGAUGAGGCCGUUAGGCCGAA AUUGCUGC	436

1404	CAGCAAUU C CACUAACA	210	UGUUAGUG CUGAUGAGGCCGUUAGGCCGAA AAUUGCUG	437

1409	AUUCCACU A ACAUGGAA	211	UUCCAUGU CUGAUGAGGCCGUUAGGCCGAA AGUGGAAU	438

1419	CAUGGAAU C CAGUCUGU	212	ACAGACUG CUGAUGAGGCCGUUAGGCCGAA AUUCCAUG	439

1424	AAUCCAGU C UGUGACAG	213	CUGUCACA CUGAUGAGGCCGUUAGGCCGAA ACUGGAUU	440

1436	GACAGUGU U UUUCACUC	214	GAGUGAAA CUGAUGAGGCCGUUAGGCCGAA ACACUGUC	441

1437	ACAGUGUU U UUCACUCU	215	AGAGUGAA CUGAUGAGGCCGUUAGGCCGAA AACACUGU	442

1438	CAGUGUUU U UCACUCUG	216	CAGAGUGA CUGAUGAGGCCGUUAGGCCGAA AAACACUG	443

1439	AGUGUUUU U CACUCUGU	217	ACAGAGUG CUGAUGAGGCCGUUAGGCCGAA AAAACACU	444

1440	GUGUUUUU C ACUCUGUG	218	CACAGAGU CUGAUGAGGCCGUUAGGCCGAA AAAAACAC	445

1444	UUUUCACU C UGUGGUAA	219	UUACCACA CUGAUGAGGCCGUUAGGCCGAA AGUGAAAA	446

1451	UCUGUGGU A AGCUGAGG	220	CCUCAGCU CUGAUGAGGCCGUUAGGCCGAA ACCACAGA	447

1463	UGAGGAAU A UGUCACAU	221	AUGUGACA CUGAUGAGGCCGUUAGGCCGAA AUUCCUCA	448

1467	GAAUAUGU C ACAUUUUC	222	GAAAAUGU CUGAUGAGGCCGUUAGGCCGAA ACAUAUUC	449

1472	UGUCACAU U UUCAGUCA	223	UGACUGAA CUGAUGAGGCCGUUAGGCCGAA AUGUGACA	450

1473	GUCACAUU U UCAGUCAA	224	UUGACUGA CUGAUGAGGCCGUUAGGCCGAA AAUGUGAC	451

1474	UCACAUUU U CAGUCAAA	225	UUUGACUG CUGAUGAGGCCGUUAGGCCGAA AAAUGUGA	452

1475	CACAUUUU C AGUCAAAG	226	CUUUGACU CUGAUGAGGCCGUUAGGCCGAA AAAAUGUG	453

1479	UUUUCAGU C AAAGAACC	227	GGUUCUUU CUGAUGAGGCCGUUAGGCCGAA ACUGAAAA	454

TABLE IV


Human PTGDR Inozyme and Substrate Sequence

		Seq		Seq
Pos	Substrate	ID	Inozyme	ID

11	AUUCUGGC U AUUUUCCU	455	AGGAAAAU CUGAUGAGGCCGUUAGGCCGAA ICCAGAAU	831

18	CUAUUUUC C UCCUGCCG	456	CGGCAGGA CUGAUGAGGCCGUUAGGCCGAA IAAAAUAG	832

19	UAUUUUCC U CCUGCCGU	457	ACGGCAGG CUGAUGAGGCCGUUAGGCCGAA IGAAAAUA	833

21	UUUUCCUC C UGCCGUUC	458	GAACGGCA CUGAUGAGGCCGUUAGGCCGAA IAGGAAAA	834

22	UUUCCUCC U GCCGUUCC	459	GGAACGGC CUGAUGAGGCCGUUAGGCCGAA IGAGGAAA	835

25	CCUCCUGC C GUUCCGAC	460	GUCGGAAC CUGAUCACGCCGUUAGGCCCAA ICAGGAGG	836

30	UGCCGUUC C GACUCCUC	461	GCCGAGUC CUGAUGAGGCCGUUAGGCCGAA IAACGGCA	837

34	GUUCCGAC U CGGCACCA	462	UGGUGCCG CUGAUGAGGCCGUUAGGCCGAA IUCGGAAC	838

39	GACUCGGC A CCAGAGUC	463	GACUCUGG CUGAUGAGGCCGUUAGGCCGAA ICCGAGUC	839

41	CUCGGCAC C AGAGUCUG	464	CAGACUCU CUGAUGAGGCCGUUAGGCCGAA IUGCCGAG	840

42	UCGGCACC A GAGUCUGU	465	ACAGACUC CUGAUGAGGCCGUUAGGCCGAA IGUGCCGA	841

48	CCAGAGUC U GUCUCUAC	466	GUAGAGAC CUGAUGAGGCCGUUAGGCCGAA IACUCUGG	842

52	AGUCUGUC U CUACUGAG	467	CUCAGUAG CUGAUGAGGCCGUUAGGCCGAA IACAGACU	843

54	UCUGUCUC U ACUGAGAA	468	UUCUCAGU CUGAUGAGGCCGUUAGGCCGAA IAGACAGA	844

57	GUCUCUAC U GAGAACGC	469	GCGUUCUC CUGAUGAGGCCGUUAGGCCGAA IUAGAGAC	845

66	GAGAACGC A GCGCGUCA	470	UGACGCGC CUGAUGAGGCCGUUAGGCCGAA ICGUUCUC	846

74	AGCGCGUC A GGGCCGAG	471	CUCGGCCC CUGAUGAGGCCGUUAGGCCGAA IACGCGCU	847

79	GUCAGGGC C GAGCUCUU	472	AAGAGCUC CUGAUGAGGCCGUUAGGCCGAA ICCCUGAC	848

84	GGCCGAGC U CUUCACUG	473	CAGUGAAG CUGAUGAGGCCGUUAGGCCGAA ICUCGGCC	849

86	CCGAGCUC U UCACUGGC	474	GCCAGUGA CUGAUGAGGCCGUUAGGCCGAA IAGCUCGG	850

89	AGCUCUUC A CUGGCCUG	475	CAGGCCAG CUGAUGAGGCCGUUAGGCCGAA IAAGAGCU	851

91	CUCUUCAC U GGCCUGCU	476	AGCAGGCC CUGAUGAGGCCGUUAGGCCGAA IUGAAGAG	852

95	UCACUGGC C UGCUCCGC	477	GCGGAGCA CUGAUGAGGCCGUUAGGCGGAA ICCAGUGA	853

96	CACUGGCC U GCUCCGCG	478	CGCGGAGC CUGAUGAGGCCGUUAGGCCGAA IGCCAGUG	854

99	UGGCCUGC U CCGCGCUC	479	GAGCGCGG CUGAUGAGGCCGUUAGGCCGAA ICAGGCCA	855

101	GCCUGCUC C GCGCUCUU	480	AAGAGCGC CUGAUGAGGCCGUUAGGCCGAA IAGCAGGC	856

106	CUCCGCGC U CUUCAAUG	481	CAUUGAAG CUGAUGAGGCCGUUAGGCCGAA ICGCGGAG	857

108	CCGCGCUC U UCAAUGCC	482	GGCAUUGA CUGAUGAGGCCGUUAGGCCGAA IAGCGCGG	858

111	CGCUCUUC A AUGCCAGC	483	GCUGGCAU CUGAUGAGGCCGUUAGGCCGAA IAAGAGCG	859

116	UUCAAUGC C AGCGCCAG	484	CUGGCGCU CUGAUGAGGCCGUUAGGCCGAA ICAUUGAA	860

117	UCAAUGCC A GCGCCAGG	485	CCUGGCGC CUGAUGAGGCCGUUAGGCCGAA IGCAUUGA	861

122	GCCAGCGC C AGGCGCUC	486	GAGCGCCU CUGAUGAGGCCGUUAGGCCGAA ICGCUGGC	862

123	CCAGCGCC A GGCGCUCA	487	UGAGCGCC CUGAUGAGGCCGUUAGGCCGAA IGCGCUGG	863

129	CCAGGCGC U CACCCUGC	488	GCAGGGUG CUGAUGAGGCCGUUAGGCCGAA ICGCCUGG	864

131	AGGCGCUC A CCCUGCAG	489	CUGCAGGG CUGAUGAGGCCGUUAGGCCGAA IAGCGCCU	865

133	GCGCUCAC C CUGCAGAG	490	CUCUGCAG CUGAUGAGGCCGUUAGGCCGAA IUGAGCGC	866

134	CGCUCACC C UGCAGAGC	491	GCUCUGCA CUGAUGAGGCCGUUAGGCCGAA IGUGAGCG	867

135	GCUCACCC U GCAGAGCG	492	CGCUCUGC CUGAUGAGGCCGUUAGGCCGAA IGGUGAGC	868

138	CACCCUGC A GAGCGUCC	493	GGACGCUC CUGAUGAGGCCGUUAGGCCGAA ICAGGGUG	869

146	AGAGCGUC C CGCCUCUC	494	GAGAGGCG CUGAUGAGGCCGUUAGGCCGAA IACGCUCU	870

147	GAGCGUCC C GCCUCUCA	495	UGAGAGGC CUGAUGAGGCCGUUAGGCCGAA IGACGCUC	871

150	CGUCCCGC C UCUCAAAG	496	CUUUGAGA CUGAUGAGGCCGUUAGGCCGAA ICGGGACG	872

151	GUCCCGCC U CUCAAAGA	497	UCUUUGAG CUGAUGAGGCCGUUAGGCCGAA IGCGGGAC	873

153	CCCGCCUC U CAAAGAGG	498	CCUCUUUG CUGAUGAGGCCGUUAGGCCGAA IAGGCGGG	874

155	CGCCUCUC A AAGAGGGG	499	CCCCUCUU CUGAUGAGGCCGUUAGGCCGAA IAGAGGCG	875

170	GGUGUGAC C CGCGAGUU	500	AACUCGCG CUGAUGAGGCCGUUAGGCCGAA IUCACACC	876

171	GUGUGACC C GCGAGUUU	501	AAACUCGC CUGAUGAGGCCGUUAGGCCGAA IGUCACAC	877

193	GGAGGUUC C UGCCCUCG	502	CCACGGCA CUCAUGAGGCCGUUAGGCCGAA IAACCUCC	878

194	GACGUUCC U GCCGUGGG	503	CCCACGGC CUGAUGAGGCCGUUAGGCCGAA IGAACCUC	879

197	GUUCCUGC C GUGGGGAA	504	UUCCCCAC CUGAUGAGGCCGUUAGGCCGAA ICAGGAAC	880

207	UGCGGAAC A CCCCGCCG	505	CCGCGGGG CUGAUGAGGCCGUUAGGCCGAA IUUCCCCA	881

209	GGGAACAC C CCGCCGCC	506	GGCGGCGG CUGAUGAGGCCGUUAGGCCGAA IUGUUCCC	882

210	GGAACACC C CGCCGCCC	507	GGGCCGCG CUGAUGAGGCCGUUAGGCCGAA IGUGUUCC	883

211	GAACACCC C GCCGCCCU	508	AGGGCGGC CUCAUGAGGCCGUUAGGCCGAA IGGUGUUC	884

214	CACCCCGC C GCCCUCGG	509	CCGAGGGC CUGAUGAGGCCGUUAGGCCGAA ICGGGGUG	885

217	CCCGCCCC C CUCGGAGC	510	GCUCCGAG CUGAUGAGGCCGUUAGGCCGAA ICGGCGGG	886

218	CCGCCGCC C UCGGAGCU	511	AGCUCCGA CUGAUGAGGCCGUUAGGCCGAA IGCGGCGG	887

219	CGCCGCCC U CGGAGCUU	512	AAGCUCCG CUGAUGAGGCCGUUAGGCCGAA IGGCGGCG	888

226	CUCGGAGC U UUUUCUGU	513	ACAGAAAA CUGAUGAGGCCGUUAGGCCGAA ICUCCGAG	889

232	GCUUUUUC U GUGGCGCA	514	UGCGCCAC CUGAUGAGGCCGUUAGGCCGAA IAAAAAGC	890

240	UGUGGCGC A GCUUCUCC	515	GGAGAAGC CUGAUGAGGCCGUUAGGCCGAA ICGCCACA	891

243	GGCGCAGC U UCUCCGCC	516	GGCGGAGA CUGAUGAGGCCGUUAGGCCGAA ICUGCGCC	892

246	GCAGCUUC U CCGCCCGA	517	UCGGGCGG CUGAUGAGGCCGUUAGGCCGAA IAAGCUGC	893

248	AGCUUCUC C GCCCGAGC	518	GCUCGGGC CUGAUGAGGCCGUUAGGCCGAA IAGAAGCU	894

251	UUCUCCGC C CGAGCCGC	519	GCGGCUCG CUGAUGAGGCCGUUAGGCCGAA ICGGAGAA	895

252	UCUCCGCC C GAGCCGCG	520	CGCGGCUC CUGAUGAGGCCGUUAGGCCGAA ICCUGAGA	896

257	GCCCGAGC C GCGCGCGG	521	CCGCGCGC CUGAUGAGGCCGUUAGGCCGAA ICUCGGGC	897

269	CGCGGAGC U GCCGGGGG	522	CCCCCGGC CUGAUGAGGCCGUUAGGCCGAA ICUCCGCG	898

272	GGAGCUGC C GGGGGCUC	523	GAGCCCCC CUGAUGAGGCCGUUAGGCCGAA ICAGCUCC	899

279	CCGGGGGC U CCUUAGCA	524	UGCUAAGG CUGAUGAGGCCGUUAGGCCGAA ICCCCCGG	900

281	GGGGGCUC C UUAGCACC	525	GGUGCUAA CUGAUGAGGCCGUUAGGCCGAA IAGCCCCC	901

282	GGGGCUCC U UAGCACCC	526	GGGUGCUA CUGAUGAGGCCGUUAGGCCGAA IGAGCCCC	902

287	UCCUUAGC A CCCGGGCG	527	CGCCCGGG CUGAUGAGGCCGUUAGGCCGAA ICUAAGGA	903

289	CUUAGCAC C CGGGCGCC	528	GGCGCCCG CUGAUGAGGCCGUUAGGCCGAA IUGCUAAG	904

290	UUAGCACC C GGGCGCCG	529	CGGCGCCC CUGAUGAGGCCGUUAGGCCGAA IGUGCUAA	905

297	CCGGGCGC C GGGGCCCU	530	AGGGCCCC CUGAUGAGGCCGUUAGGCCGAA ICGCCCGG	906

303	GCCGGGGC C CUCGCCCU	531	AGGGCGAG CUGAUGAGGCCGUUAGGCCGAA ICCCCGGC	907

304	CCGGGGCC C UCGCCCUU	532	AAGGGCGA CUGAUGAGGCCGUUAGGCCGAA IGCCCCGG	908

305	CGGGGCCC U CGCCCUUC	533	GAAGGGCG CUGAUGAGGCCGUUAGGCCGAA IGGCCCCG	909

309	GCCCUCGC C CUUCCGCA	534	UGCGGAAG CUGAUGAGGCCGUUAGGCCGAA ICGAGGGC	910

310	CCCUCGCC C UUCCGCAG	535	CUGCGGAA CUGAUGAGGCCGUUAGGCCGAA IGCGAGGG	911

311	CCUCGCCC U UCCGCAGC	536	GCUGCGGA CUGAUGAGGCCGUUAGGCCGAA IGGCGAGG	912

314	CGCCCUUC C GCAGCCUU	537	AAGGCUGC CUGAUGAGGCCGUUAGGCCGAA IAAGGGCG	913

317	CCUUCCGC A GCCUUCAC	538	GUGAAGGC CUGAUGAGGCCGUUAGGCCGAA ICGGAAGG	914

320	UCCGCAGC C UUCACUCC	539	GGAGUGAA CUGAUGAGGCCGUUAGGCCGAA ICUGCGGA	915

321	CCGCAGCC U UCACUCCA	540	UGGAGUGA CUGAUGAGGCCGUUAGGCCGAA IGCUGCGG	916

324	CAGCCUUC A CUCCAGCC	541	GGCUGGAG CUGAUGAGGCCGUUAGGCCGAA IAAGGCUG	917

326	GCCUUCAC U CCAGCCCU	542	AGGGCUGG CUGAUGAGGCCGUUAGGCCGAA IUGAAGGC	918

328	CUUCACUC C AGCCCUCU	543	AGAGGGCU CUGAUGAGGCCGUUAGGCCGAA IAGUGAAG	919

329	UUCACUCC A GCCCUCUG	544	CAGAGGGC CUGAUGAGGCCGUUAGGCCGAA IGAGUGAA	920

332	ACUCCAGC C CUCUGCUC	545	GAGCAGAG CUGAUGAGGCCGUUAGGCCGAA ICUGGAGU	921

333	CUCCAGCC C UCUGCUCC	546	GGAGCAGA CUGAUGAGGCCGUUAGGCCGAA IGCUGGAG	922

334	UCCAGCCC U CUGCUCCC	547	GGGAGCAG CUGAUGAGGCCGUUAGGCCGAA IGGCUGGA	923

336	CAGCCCUC U GCUCCCGC	548	GCGGGAGC CUGAUGAGGCCGUUAGGCCGAA IAGGGCUG	924

339	CCCUCUGC U CCCGCACG	549	CGUGCGGG CUGAUGAGGCCGUUAGGCCGAA ICAGAGGG	925

341	CUCUGCUC C CGCACGCC	550	GGCGUGCG CUGAUGAGGCCGUUAGGCCGAA IAGCAGAG	926

342	UCUGCUCC C GCACGCCA	551	UGGCGUGC CUGAUGAGGCCGUUAGGCCGAA IGAGCAGA	927

345	GCUCCCGC A CGCCAUGA	552	UCAUGGCG CUGAUGAGGCCGUUAGGCCGAA ICGGGAGC	928

349	CCGCACGC C AUGAAGUC	553	GACUUCAU CUGAUGACCCCGUUAGGCCGAA ICGUGCGC	929

350	CGCACGCC A UGAAGUCG	554	CGACUUCA CUGAUGAGGCCGUUAGGCCGAA IGCGUGCG	930

360	GAAGUCGC C GUUCUACC	555	GGUAGAAC CUGAUGAGGCCGUUAGGCCGAA ICGACUUC	931

365	CGCCGUUC U ACCGCUGC	556	GCACCGGU CUGAUGAGGCCGUUAGGCCGAA IAACGGCG	932

368	CGUUCUAC C GCUGCCAG	557	CUGGCAGC CUGAUGAGGCCGUUAGGCCGAA IUAGAACG	933

371	UCUACCUC U GCCAGAAC	558	GUUCUGGC CUGAUGAGGCCGUUAGGCCGAA ICOGUAGA	934

374	ACCGCUGC C AGAACACC	559	GGUGUUCU CUGAUGAGGCCGUUAGGCCGAA ICAGCGGU	935

375	CCGCUGCC A GAACACCA	560	UGGUGUUC CUGAUGAGGCCGUUAGGCCGAA IGCAGCGG	936

380	GCCAGAAC A CCACCUCU	561	AGAGGUGG CUGAUGAGGCCGUUAGGCCGAA IUUCUGGC	937

382	CAGAACAC C ACCUCUGU	562	ACAGAGGU CUGAUGAGGCCGUUAGGCCGAA IUGUUCUG	938

383	AGAACACC A CCUCUGUG	563	CACAGAGG CUGAUGAGGCCGUUAGGCCGAA IGUGUUCU	939

385	AACACCAC C UCUGUGGA	564	UCCACAGA CUGAUGAGGCCGUUAGGCCGAA IUGGUGUU	940

386	ACACCACC U CUGUGGAA	565	UUCCACAG CUGAUGAGGCCGUUAGGCCGAA IGUGGUGU	941

388	ACCACCUC U GUGGAAAA	566	UUUUCCAC CUGAUGAGGCCGUUAGGCCGAA IAGGUGGU	942

401	AAAAAGGC A ACUCGGCG	567	CGCCGAGU CUGAUGAGGCCGUUAGGCCGAA ICCUUUUU	943

404	AAGGCAAC U CGGCGGUG	568	CACCGCCG CUGAUGAGGCCGUUAGGCCGAA IUUGCCUU	944

426	CGGGGUGC U CUUCAGCA	569	UGCUGAAG CUGAUGAGGCCGUUAGGCCGAA ICACCCCG	945

428	GGGUGCUC U UCAGCACC	570	GGUGCUGA CUGAUGAGGCCGUUAGGCCGAA IAGCACCC	946

431	UGCUCUUC A GCACCGGC	571	GCCGGUGC CUGAUGAGGCCGUUAGGCCGAA IAAGAGCA	947

434	UCUUCAGC A CCGGCCUC	572	GAGGCCGG CUGAUGAGGCCGUUAGGCCGAA ICUGAAGA	948

436	UUCAGCAC C GGCCUCCU	573	AGGAGGCC CUGAUGAGGCCGUUAGGCCGAA IUGCUGAA	949

440	GCACCGGC C UCCUGGGC	574	GCCCAGGA CUGAUGAGGCCGUUAGGCCGAA ICCGGUGC	950

441	CACCGGCC U CCUGGGCA	575	UGCCCAGG CUGAUGAGGCCGUUAGGCCGAA IGCCGGUG	951

443	CCGGCCUC C UGGGCAAC	576	GUUGCCCA CUGAUGAGGCCGUUAGGCCGAA IAGGCCGG	952

444	CGGCCUCC U GGGCAACC	577	GGUUGCCC CUGAUGAGGCCGUUAGGCCGAA IGAGGCCG	953

449	UCCUGGGC A ACCUGCUG	578	CAGCAGGU CUGAUGAGGCCGUUAGGCCGAA ICCCAGGA	954

452	UGGGCAAC C UGCUGGCC	579	GGCCAGCA CUGAUGAGGCCGUUAGGCCGAA IUUGCCCA	955

453	GGGCAACC U GCUGGCCC	580	GGGCCAGC CUGAUGAGGCCGUUAGGCCGAA IGUUGCCC	956

456	CAACCUGC U GGCCCUGG	581	CCAGGGCC CUGAUGAGGCCGUUAGGCCGAA ICAGGUUG	957

460	CUGCUGGC C CUGGGGCU	582	AGCCCCAG CUGAUGAGGCCGUUAGGCCGAA ICCAGCAG	958

461	UGCUGGCC C UGGGGCUG	583	CAGCCCCA CUGAUGAGGCCGUUAGGCCGAA IGCCAGCA	959

462	GCUGGCCC U GGGGCUGC	584	GCAGCCCC CUGAUGAGGCCGUUAGGCCGAA IGGCCAGC	960

468	CCUGGGGC U GCUGGCGC	585	GCGCCAGC CUGAUGAGGCCGUUAGGCCGAA ICCCCAGG	961

471	GGGGCUGC U GGCGCGCU	586	AGCGCGCC CUGAUGAGGCCGUUAGGCCGAA ICAGCCCC	962

479	UGGCGCGC U CGGGGCUG	587	CAGCCCCG CUGAUGAGGCCGUUAGGCCGAA ICGCGCCA	963

486	CUCGGGGC U GGGGUGGU	588	ACCACCCC CUGAUGAGGCCGUUAGGCCGAA ICCCCGAG	964

497	GGUGGUGC U CGCGGCGU	589	ACGCCGCG CUGAUGAGGCCGUUAGGCCGAA ICACCACC	965

507	GCGGCGUC C ACUGCGCC	590	GGCGCAGU CUGAUGAGGCCGUUAGGCCGAA IACGCCGC	966

508	CGGCGUCC A CUGCGCCC	591	GGGCGCAG CUGAUGAGGCCGUUAGGCCGAA IGACGCCG	967

510	GCGUCCAC U GCGCCCGC	592	GCGGGCGC CUGAUGAGGCCGUUAGGCCGAA IUGGACGC	968

515	CACUGCGC C CGCUGCCC	593	GGGCAGCG CUGAUGAGGCCGUUAGGCCGAA ICGCAGUG	969

516	ACUGCGCC C GCUGCCCU	594	AGGGCAGC CUGAUGAGGCCGUUAGGCCGAA IGCGCAGU	970

519	GCGCCCGC U GCCCUCGG	595	CCGAGGGC CUGAUGAGGCCGUUAGGCCGAA ICGGGCGC	971

522	CCCGCUGC C CUCGGUCU	596	AGACCGAG CUGAUGAGGCCGUUAGGCCGAA ICAGCGGG	972

523	CCGCUGCC C UCGGUCUU	597	AAGACCGA CUGAUGAGGCCGUUAGGCCGAA IGCAGCGG	973

524	CGCUGCCC U CGGUCUUC	598	GAAGACCG CUGAUGAGGCCGUUAGGCCGAA IGGCAGCG	974

530	CCUCGGUC U UCUACAUG	599	CAUGUAGA CUGAUGAGGCCGUUAGGCCGAA IACCGAGG	975

533	CGGUCUUC U ACAUGCUG	600	CAGCAUGU CUGAUGAGGCCGUUAGGCCGAA IAAGACCG	976

536	UCUUCUAC A UGCUGGUG	601	CACCAGCA CUGAUGAGGCCGUUAGGCCGAA IUAGAAGA	977

540	CUACAUGC U GGUGUGUG	602	CACACACC CUGAUGAGGCCGUUAGGCCGAA ICAUGUAG	978

551	UGUGUGGC C UGACGGUC	603	GACCGUCA CUGAUGAGGCCGUUAGGCCGAA ICCACACA	979

552	GUGUGGCC U GACGGUCA	604	UGACCGUC CUGAUGAGGCCGUUAGGCCGAA IGCCACAC	980

560	UGACGGUC A CCGACUUG	605	CAAGUCGG CUGAUGAGGCCGUUAGGCCGAA IACCGUCA	981

562	ACGGUCAC C GACUUCCU	606	AGCAAGUC CUGAUCAGGCCGUUAGGCCGAA IUGACCGU	982

566	UCACCGAC U UGCUGGGC	607	GCCCAGCA CUGAUGAGGCCGUUAGGCCGAA IUCCGUGA	983

570	CGACUUCC U GCCCAAGU	608	ACUUGCCC CUGAUGAGGCCGUUAGGCCGAA ICAAGUCG	984

575	UGCUGGGC A AGUGCCUC	609	GAGGCACU CUGAUGAGGCCGUUAGGCCGAA ICCCAGCA	985

581	CCAAGUGC C UCCUAAGC	610	GCUUACGA CUGAUGAGGCCGUUAGGCCGAA ICACUUGC	986

582	CAAGUGCC U CCUAAGCC	611	GGCUUAGG CUGAUCACGCCGUUAGGCCGAA IGCACUUG	987

584	AGUGCCUC C UAAGCCCG	612	CGGGCUUA CUGAUGAGGCCGUUAGGCCGAA IAGGCACU	988

585	GUGCCUCC U AAGCCCGG	613	CCGGGCUU CUGAUGAGGCCGUUAGGCCGAA IGAGGCAC	989

590	UCCUAAGC C CGGUGGUG	614	CACCACCG CUGAUGAGGCCGUUAGGCCGAA ICUUAGGA	990

591	CCUAAGCC C CGUGGUGC	615	GCACCACC CUGAUGAGGCCGUUAGGCCGAA IGCUUAGG	991

600	GGUGGUGC U GGCUGCCU	616	AGGCAGCC CUGAUGAGGCCGUUAGGCCGAA ICACCACC	992

604	GUGCUCGC U CCCUACGC	617	GCGUAGGC CUCAUCAGGCCUUUAGGCCGAA ICCAGCAC	993

607	CUGGCUGC C UACGCUCA	618	UGAGCGUA CUGAUGAGGCCGUUAGGCCCAA ICAGCCAG	994

608	UCGCUGCC U ACGCUCAG	619	CUGACCGU CUGAUGAGGCCGUUAGGCCGAA IGCAGCCA	995

613	GCCUACGC U CAGAACCC	620	CGGUUCUG CUGAUGAGGCCGUUAGGCCGAA ICGUAGGC	996

615	CUACGCUC A GAACCGGA	621	UCCGGUUC CUGAUGAGGCCGUUAGGCCGAA IAGCGUAG	997

620	CUCAGAAC C GGAGUCUG	622	CAGACUCC CUGAUGAGGCCGUUAGGCCGAA IUUCUGAG	998

627	CCGGAGUC U GCGGGUGC	623	GCACCCGC CUGAUGAGGCCGUUAGGCCGAA IACUCCGG	999

636	GCCGGUGC U UGCGCCCG	624	CGGGCCCA CUGAUGAGGCCGUUAGGCCGAA ICACCCCC	1000

642	GCUUGCGC C CGCAUUGG	625	CCAAUGCG CUGAUGAGGCCGUUAGGCCGAA ICGCAAGC	1001

643	CUUCCGCC C GCAUUGGA	626	UCCAAUGC CUGAUGAGGCCGUUAGGCCGAA IGCGCAAC	1002

646	GCGCCCGC A UUGGACAA	627	UUGUCCAA CUGAUGAGGCCGUUACGCCGAA ICGGGCGC	1003

653	CAUUGCAC A ACUCGUUG	628	CAACGAGU CUGAUGAGGCCGUUAGGCCGAA IUCCAAUG	1004

656	UGGACAAC U CGUUGUGC	629	GCACAACG CUGAUGAGGCCGUUAGGCCGAA IUUGUCCA	1005

665	CGUUGUGC C AAGCCUUC	630	CAAGCCUU CUGAUGAGGCCGUUAGGCCGAA ICACAACG	1006

666	GUUGUGCC A AGCCUUCG	631	CGAAGGCU CUGAUGAGGCCGUUAGGCCGAA IGCACAAC	1007

670	UGCCAAGC C UUCGCCUU	632	AAGGCGAA CUGAUGAGGCCGUUACGCCCAA ICUUGGCA	1008

671	GCCAAGCC U UCGCCUUC	633	GAACGCGA CUGAUGAGGCCGUUAGGCCGAA IGCUUCGC	1009

676	GCCUUCGC C UUCUUCAU	634	AUGAAGAA CUGAUGAGCCCGUUAGGCCGAA ICGAAGGC	1010

677	CCUUCGCC U UCUUCAUG	635	CAUGAAGA CUGAUGAGGCCGUUAGGCCGAA IGCGAAGG	1011

680	UCGCCUUC U UCAUGUCC	636	GGACAUGA CUGAUGAGGCCGUUAGGCCGAA IAAGGCGA	1012

683	CCUUCUUC A UGUCCUUC	637	GAAGGACA CUGAUGAGGCCGUUAGGCCGAA IAAGAAGG	1013

688	UUCAUGUC C UUCUUUGG	638	CCAAAGAA CUGAUGAGGCCGUUAGGCCGAA IACAUGAA	1014

689	UCAUGUCC U UCUUUGGG	639	CCCAAAGA CUGAUGAGGCCGUUAGGCCGAA IGACAUGA	1015

692	UGUCCUUC U UUGGGCUC	640	GAGCCCAA CUGAUGAGGCCGUUAGGCCGAA IAAGGACA	1016

699	CUUUGGGC U CUCCUCCA	641	UCGAGGAG CUGAUGAGGCCGUUAGGCCGAA ICCCAAAG	1017

701	UUGGGCUC U CCUCGACA	642	UGUCGAGG CUGAUGAGGCCGUUAGGCCGAA IAGCCCAA	1018

703	GGGCUCUC C UCGACACU	643	AGUGUCGA CUGAUGAGGCCGUUAGGCCGAA IAGAGCCC	1019

704	GGCUCUCC U CGACACUG	644	CAGUGUCG CUGAUGAGGCCGUUAGGCCGAA IGAGAGCC	1020

709	UCCUCGAC A CUGCAACU	645	AGUUGCAG CUGAUGAGGCCGUUAGGCCGAA IUCGAGGA	1021

721	CUCGACAC U GCAACUCC	646	GGAGUUGC CUGAUGAGGCCGUUAGGCCGAA IUGUCGAG	1022

714	GACACUGG A ACUCCUGG	647	CCAGGAGU CUGAUGAGGCCGUUAGGCCGAA ICAGUGUC	1023

717	ACUGCAAC U CCUGGCCA	648	UGGCCAGG CUGAUGAGGCCGUUAGGCCGAA IUUGCAGU	1024

719	UGCAACUC C UGGCCAUG	649	CAUGGCCA CUGAUGAGGCCGUUAGGCCGAA IAGUUGCA	1025

720	GCAACUCC U GGCCAUGG	650	CCAUGGCC CUGAUGAGGCCGUUAGGCCGAA IGAGUUGC	1026

724	CUCCUGGC C AUGGCACU	651	AGUGCCAU CUGAUGAGGCCGUUAGGCCGAA ICCAGGAG	1027

725	UCCUGGCC A UGGCACUG	652	CAGUGCCA CUGAUGAGGCCGUUAGGCCGAA IGCCAGGA	1028

730	GCCAUGGC A CUGGAGUG	653	CACUCCAG CUGAUGAGGCCGUUAGGCCGAA ICCAUGGC	1029

732	CAUGGCAC U GGAGUGCU	654	AGCACUCC CUGAUGAGGCCGUUAGGCCGAA IUGCCAUG	1030

740	UCGAGUGC U GGCUCUCC	655	GGAGAGCC CUGAUGAGGCCGUUAGGCCGAA ICACUCCA	1031

744	GUGCUGGC U CUCCCUAG	656	CUAGGGAG CUGAUGAGGCCGUUAGGCCGAA ICCAGCAC	1032

746	GCUGCCUC U CCCUAGGG	657	CCCUAGGG CUGAUGAGGCCGUUAGGCCGAA IAGCCAGC	1033

748	UGGCUCUC C CUAGOCCA	658	UGCCCUAG CUGAUGAGGCCGUUAGGCCGAA IAGAGCCA	1034

749	GGCUCUCC C UAGGGCAC	659	GUCCCCUA CUGAUGAGGCCGUUAGGCCGAA IGAGAGCC	1035

750	GCUCUCCC U AGGGCACC	660	GGUGCCCU CUGAUGAGGCCGUUAGGCCGAA IGGAGAUC	1036

756	CCUAGGGC A CCCUUIJCU	661	AGAAAGGG CUGAUGAGGCCGUUAGGCCGAA ICCCUAGG	1037

758	UAGGGCAC C CUUUCUUC6	62	GAAGAAAG CUGAUGAGGCCGUUAGGCCGAA IUGCCCUA	1038

759	AGGGCACC C UUUCUUCU	663	AGAAGAAA CUGAUGAGGCCGUUAGGCCGAA IGUGCCCU	1039

760	GGGCACCC U UUCUUCUA	664	UAGAAGAA CUGAUGAGGCCGUUAGGCCGAA IGGUGCCC	1040

764	ACCCUUUC U UCUACCGA	665	UCGGUAGA CUGAUGAGGCCGUUAGGCCGAA TAAAGGGU	1041

767	CUUUCUUC U ACCGACGG	666	CCGUCGGU CUGAUGAGGCCGUUAGGCCGAA IAAGAAAG	1042

770	UCUUCUAC C GACGGCAC	667	GUGCCGUC CUGAUGAGCCCGUUAGGCCGAA IUAGAAGA	1043

777	CCGACGGC A CAUCACCC	668	GGGUGAUG CUGAUGAGGCCGUUAGGCCGAA ICCGUCGG	1044

779	GACGGCAC A UCACCCUG	669	CAGGGUGA CUGAUGAGGCCGUUAGGCCGAA IUGCCGUC	1045

782	GGCACAUC A CCCUGCGC	670	GCGCAGGG CUGAUGAGGCCGUUAGGCCGAA IAUGUGCC	1046

784	CACAUCAC C CUGCGCCU	671	AGGCGCAG CUGAUGAGGCCGUUAGGCCGAA TUGAUGUG	1047

785	ACAUCACC C UGCGCCUG	672	CAGGCGCA CUGAUGAGGCCGUUAGGCCGAA IGUGAUGU	1048

786	CAUCACCC U GCGCCUGG	673	CCAGGCGC CUGAUGAGGCCGUUAGGCCGAA IGGUGAUG	1049

791	CCCUGCGC C UGGGCGCA	674	UGCGCCCA CUGAUGAGGCCGUUAGGCCGAA ICGCAGGG	1050

792	CCUGCGCC U GGGCGCAC	675	GUGCGCCC CUGAUGAGGCCGUUAGGCCGAA IGCGCAGG	1051

799	CUGGGCGC A CUGGUGGC	676	GCCACCAG CUGAUGAGGCCGUUAGGCCGAA ICGCCCAG	1052

801	GGGCGCAC U GGUGGCCC	677	GGGCCACC CUGAUGAGGCCGUUAGGCCGAA IUGCGCCC	1053

808	CUGGUGGC C CCGGUGGU	678	ACCACCGG CUGAUGAGGCCGUUAGGCCGAA ICCACCAG	1054

809	UGGUGGCC C CGGUGGUG	679	CACCACCG CUGAUGAGGCCGUUAGGCCGAA IGCCACCA	1055

810	GGUGGCCC C GGUGGUGA	680	UCACCACC CUGAUGAGGCCGUUAGGCCGAA IGGCCACC	1056

823	GUGAGCGC C UUCUCCCU	681	AGGGAGAA CUGAUGAGGCCGUUAGGCCGAA ICOCUCAC	1057

824	UGAGCGCC U UCUCCCUG	682	CAUGGAGA CUGAUGAGGCCGUUAGGCCGAA IGCGCUCA	1058

827	GCGCCUUC U CCCUGGCU	683	AGCCAGGG CUGAUGAGGCCGUUAGGCCGAA IAAGGCGC	1059

829	GCCUUCUC C CUGGCUUU	684	AAAGCCAG CUGAUGAGGCCGUUAGGCCGAA IAGAAGGC	1060

830	CCUUCUCC C UGGCUUUC	685	GAAAGCCA CUGAUGAGGCCGUUAGGCCGAA IGAGAAGG	1061

831	CUUCUCCC U GGCUUUCU	686	AGAAAGCC CUGAUGAGGCCGUUAGGCCGAA IGGAGAAG	1062

835	UCCCUGGC U UUCUGCGC	687	GCGCAGAA CUGAUGAGGCCGUUAGGCCGAA ICCAGGGA	1063

839	UGGCUUUC U GCGCGCUA	688	UAGCGCGC CUGAUGAGGCCGUUAGGCCGAA IAAAGCCA	1064

846	CUGCGCGC U ACCUUUCA	689	UGAAAGGU CUGAUGAGGCCGUUAGGCCGAA ICGCGCAG	1065

849	CGCGCUAC C UUUCAUGG	690	CCAUGAAA CUGAUGAGGCCGUUAGGCCGAA IUAGCGCG	1066

850	GCGCUACC U UUCAUGGG	691	CCCAUGAA CUGAUGAGGCCGUUAGGCCGAA IGUAGCGC	1067

854	UACCUUUC A UGGGCUUC	692	GAAGCCCA CUGAUGAGGCCGUUAGGCCGAA IAAAGGUA	1068

860	UCAUGGGC U UCGGGAAG	693	CUUCCCGA CUGAUGAGGCCGUUAGGCCGAA ICCCAUGA	1069

876	GUUCGUGC A GUACUGCC	694	GGCAGUAC CUGAUGAGGCCGUUAGGCCGAA ICACGAAC	1070

881	UGCAGUAC U GCCCCGGC	695	GCCGGGGC CUGAUGAGGCCGUUAGGCCGAA IUACUGCA	1071

884	AGUACUGC C CCGGCACC	696	GGUGCCGG CUGAUGAGGCCGUUAGGCCGAA ICAGUACU	1072

885	GUACUGCC C CGGCACCU	697	AGGUGCCG CUGAUGAGGCCGUUAGGCCGAA IGCAGUAC	1073

886	UACUGCCC C GGCACCUG	698	CAGGUGCC CUGAUGAGGCCGUUAGGCCGAA IGGCAGUA	1074

890	GCCCCGGC A CCUGGUGC	699	GCACCAGG CUGAUGAGGCCGUUAGGCCGAA ICCGGGGC	1075

892	CCCGGCAC C UGGUGCUU	700	AAGCACCA CUGAUGAGGCCGUUAGGCCGAA IUGCCGGG	1076

893	CCGGCACC U GGUGCLRTU	701	AAAGCACC CUGAUGAGGCCGUUAGGCCGAA IGUGCCGG	1077

899	CCUGGUGC U UUAUCCAG	702	CUGGAUAA CUGAUGAGGCCGUUAGGCCGAA ICACCAGG	1078

905	GCUUUAUC C AGAUGGUC	703	GACCAUCU CUGAUGAGGCCGUUAGGCCGAA IAUAAAGC	1079

906	CUUUAUCC A GAUGGUCC	704	GGACCAUC CUGAUGAGGCCGUUAGGCCGAA IGAUAAAG	1080

914	AGAUGGUC C ACGAGGAG	705	CUCCUCGU CUGAUGAGGCCGUUAGGCCGAA IACCAUCU	1081

915	GAUCGUCC A CCAGGAGG	706	CCUCCUCG CUGAUGAGGCCGUUAGGCCGAA IGACCAUC	1082

926	AGGAGGGC U CGCUGUCG	707	CGACAGCG CUGAUGAGGCCGUUAGGCCGAA ICCCUCCU	1083

930	GGGCUCGC U GUCGGUGC	708	GCACCGAC CUGAUGAGGCCGUUAGGCCGAA ICGAGCCC	1084

939	GUCGGUGC U GGGGUACU	709	AGUACCCC CUGAUGAGGCCGUUAGGCCGAA ICACCGAC	1085

947	UGGGGUAC U CUGUCCUC	710	GAGCACAG CUGAUGAGGCCGUUAGGCCGAA IUACCCCA	1086

949	GOGUACUC U GUGCUCUA	711	UAGAGCAC CUGAUGAGGCCGUUAGGCCGAA IAGUACCC	1087

954	CUCUGUGC U CUACUCCA	712	UGGAGUAG CUGAUGAGGCCGUUAGGCCGAA ICACAGAG	1088

956	CUGUCCUC U ACUCCAGC	713	GCUGGAGU CUGAUGAGGCCGUUAGGCCGAA IAGCACAG	1089

959	UGCUCUAC U CCAGCCUC	714	GAGGCUGG CUGAUGAGGCCGUUAGGCCGAA IUAGAGCA	1090

961	CUCUACUC C AGCCUCAU	715	AUGAGGCU CUGAUGAGGCCGUUAGGCCGAA IAGUAGAG	1091

962	UCUACUCC A GCCUCAUG	716	CAUGAGGC CUGAUGAGGCCGUUAGGCCGAA IGAGUAGA	1092

965	ACUCCAGC C UCAUGGCG	717	CGCCAUGA CUGAUGAGGCCGUUAGGCCGAA ICUGGAGU	1093

966	CUCCAGCC U CAUGGCGC	718	GCGCCAUG CUGAUGAGGCCGUUAGGCCGAA IGCUGGAG	1094

968	CCAGCCUC A UGGCGCUG	719	CAGCGCCA CUGAUGAGGCCGUUAGGCCGAA IAGGCUGG	1095

975	CAUGGCGC U GCUGGUCC	720	GGACCAGC CUGAUGAGGCCGUUAGGCCGAA ICGCCAUG	1096

978	GGCGCUGC U GGUCCUCG	721	CGAGGACC CUGAUGAGGCCGUUAGGCCGAA ICAGCGCC	1097

983	UCCUGGUC C UCGCCACC	722	GGUGGCGA CUGAUGAGGCCGUUAGGCCGAA IACCAGCA	1098

984	GCUGGUCC U CGCCACCG	723	CGGUGGCG CUGAUGAGGCCGUUAGGCCGAA IGACCAGC	1099

988	GUCCUCGC C ACCGUGCU	724	AGCACGGU CUGAUGAGGCCGUUAGGCCGAA ICCAGGAC	1100

989	UCCUCGCC A CCGUGCUG	725	CAGCACGG CUGAUGAGGCCGUUAGGCCGAA IGCGAGGA	1101

991	CUCGCCAC C GUGCUGUG	726	CACAGCAC CUGAUGAGGCCGUUAGGCCGAA IUGGCGAG	1102

996	CACCGUGC U GUGCAACC	727	GGUUGCAC CUGAUGAGGCCGUUAGGCCGAA ICACGGUG	1103

1001	UGCUGUGC A ACCUCGGC	728	GCCGAGGU CUGAUGAGGCCGUUAGGCCGAA ICACAGCA	1104

1004	UGUGCAAC C UCGGCGCC	729	GGCGCCGA CUGAUGAGGCCGUUAGGCCGAA IUUGCACA	1105

1005	GUGCAACC U CGGCGCCA	730	UGGCGCCG CUGAUGAGGCCGUUAGGCCGAA IGUUGCAC	1106

1012	CUCGGCGC C AUGCGCAA	731	UUGCGCAU CUGAUGAGGCCGUUAGGCCGAA ICGCCGAG	1107

1013	UCGGCGCC A UGCGCAAC	732	GUUGCGCA CUGAUGAGGCCGUUAGGCCGAA IGCGCCGA	1108

1019	CCAUGCGC A ACCUCUAU	733	AUAGAGGU CUGAUGAGGCCGUUAGGCCGAA ICGCAUGG	1109

1022	UGCGCAAC C UCUAUGCG	734	CGCAUAGA CUGAUGAGGCCGUUAGGCCGAA IUUGCGCA	1110

1023	GCGCAACC U CUAUGCGA	735	UCGCAUAG CUGAUGAGGCCGUUAGGCCGAA IGUUGCGC	1111

1025	GCAACCUC U AUGCGAUG	736	CAUCGCAU CUGAUGAGGCCGUUAGGCCGAA IAGGUUGC	1112

1035	UGCGAUGC A CCGGCGGC	737	GCCGCCGG CUGAUGAGGCCGUUAGGCCGAA ICAUCGCA	1113

1037	CGAUGCAC C GGCGGCUG	738	CAGCCGCC CUGAUGAGGCCGUUAGGCCGAA IUGCAUCG	1114

1044	CCGGCGGC U GCAGCGGC	739	GCCGCUGC CUGAUGAGGCCGUUAGGCCGAA ICCGCCGG	1115

1047	GCGGCUGC A GCGGCACC	740	GGUGCCGC CUGAUGAGGCCGUUAGGCCGAA ICAGCCGC	1116

1053	GCAGCGGC A CCCGCGCU	741	AGCGCGGG CUGAUGAGGCCGUUAGGCCGAA ICCGCUGC	1117

1055	AGCGGCAC C CGCGCUCC	742	GGAGCGCG CUGAUGAGGCCGUUAGGCCGAA IUGCCGCU	1118

1056	GCGGCACC C GCGCUCCU	743	AGGAGCGC CUGAUGAGGCCGUUAGGCCGAA IGUGCCGC	1119

1061	ACCCGCGC U CCUGCACC	744	GGUGCAGG CUGAUGAGGCCGUUAGGCCGAA ICGCGGGU	1120

1063	CCGCGCUC C UCCACCAG	745	CUGGUCCA CUGAUGAGGCCGUUAGGCCGAA IAGCGCGG	1121

1064	CGCGCUCC U GCACCAGG	746	CCUGGUGC CUGAUGAGGCCGUUAGGCCGAA IGAGCGCG	1122

1067	GCUCCUGC A CCAGGGAC	747	GUCCCUGG CUGAUGAGGCCGUUAGGCCGAA ICAGGAGC	1123

1069	UCCUCCAC C AGGGACUG	748	CAGUCCCU CUGAUGAGGCCGUUAGGCCGAA IUCCAGGA	1124

1070	CCUGCACC A GGGACUGU	749	ACAGUCCC CUGAUGAGGCCGUUAGGCCGAA IGUGCAGG	1125

1076	CCAGGGAC U GUGCCGAG	750	CUCGGCAC CUGAUGAGGCCGUUAGGCCGAA IUCCCUGG	1126

1081	GACUGUGC C GAGCCGCG	751	CGCGGCUC CUGAUGAGGCCGUUAGGCCGAA ICACAGUC	1127

1086	UGCCGAGC C GCGCGCGG	752	CCGCGCGC CUGAUGAGGCCGUUAGGCCGAA ICUCGGCA	1128

1111	GAAGCGUC C CCUCAGCC	753	GGCUGAGG CUGAUGAGGCCGUUAGGCCGAA IACGCUUC	1129

1112	AAGCGUCC C CUCAGCCC	754	GGGCUGAG CUGAUGAGGCCGUUAGGCCGAA IGACGCUU	1130

1113	AGCGUCCC C UCAGCCCC	755	GGGGCUGA CUGAUGAGGCCGUUAGGCCGAA IGGACGCU	1131

1114	GCGUCCCC U CAGCCCCU	756	AGGGGCUG CUGAUGAGGCCGUUAGGCCGAA IGGGACGC	1132

1116	GUCCCCUC A GCCCCUGG	757	CCAGGGGC CUGAUGAGGCCGUUAGGCCGAA IAGGGGAC	1133

1119	CCCUCAGC C CCUCGAGG	758	CCUCCAGG CUGAUGACGCCGUUAGGCCGAA ICUGAGGG	1134

1120	CCUCAGCC C CUGGAGGA	759	UCCUCCAG CUGAUGAGGCCGUUAGGCCGAA IGCUGAGG	1135

1121	CUCAGCCC C UGGAGGAG	760	CUCCUCCA CUGAUGAGGCCGUUAGGCCGAA IGGCUGAG	1136

1122	UCAGCCCC U GGAGGAGC	761	OCUCCUCO CUGAUGAGGCCGUUAGGCCGAA IGGGCUGA	1137

1131	GGAGGAGC U GGAUCACC	762	GGUGAUCC CUGAUGAGGCCGUUAGGCCGAA ICUCCUCC	1138

1137	GCUGGAUC A CCUCCUGC	763	GCAGGAGG CUGAUGAGGCCGUUAGGCCGAA IAUCCAGC	1139

1139	UGGAUCAC C UCCUGCUG	764	CAGCAGGA CUGAUGAGGCCGUUAGGCCGAA IUGAUCCA	1140

1140	GGAUCACC U CCUGCUGC	765	GCAGCAGG CUGAUGAGGCCGUUAGGCCGAA TGUGAUCC	1141

1142	AUCACCUC C UGCUGCUG	766	CAGCAGCA CUGAUGAGGCCGUUAGGCCGAA IAGGUGAU	1142

1143	UCACCUCC U GCUGCUGG	767	CCAGCAGC CUGAUGAGGCCGUUAGGCCGAA IGAGGUGA	1143

1146	CCUCCUGC U GCUGGCGC	768	GCGCCAGC CUGAUGAGGCCGUUAGGCCGAA ICAGGAGG	1144

1149	CCUGCUGC U GGCGCUGA	769	UCAGCGCC CUGAUGAGGCCGUUAGGCCGAA ICAGCAGG	1145

1155	GCUGGCGC U GAUGACCG	770	CGGUCAUC CUGAUGAGGCCGUUAGGCCGAA ICGCCAGC	1146

1162	CUGAUGAC C GUGCUCUU	771	AAGAGCAC CUGAUGAGGCCGUUAGGCCGAA IUCAUCAG	1147

1167	GACCGUGC U CUUCACUA	772	UAGUGAAG CUGAUGAGGCCGUUAGGCCGAA ICACGGUC	1148

1169	CCGUGCUC U UCACUAUG	773	CAUAGUGA CUGAUGAGGCCGUUAGGCCGAA IAGCACGG	1149

1172	UGCUCUUC A CUAUGUGU	774	ACACAUAG CUGAUGAGGCCGUUAGGCCGAA IAAGAGCA	1150

1174	CUCUUCAC U AUGUGUUC	775	GAACACAU CUGAUGAGGCCGUUAGGCCGAA IUGAAGAG	1151

1183	AUGUGUUC U CUGCCCGU	776	ACGGGCAG CUGAUGAGGCCGUUAGGCCGAA IAACACAU	1152

1185	GUGUUCUC U GCCCGUAA	777	UUACGGGC CUGAUGAGGCCGUUAGGCCGAA IAGAACAC	1153

1188	UUCUCUGC C CGUAAUUU	778	AAAUUACG CUGAUGAGGCCGUUAGGCCGAA ICAGAGAA	1154

1189	UCUCUGCC C GUAAUUUA	779	UAAAUUAC CUGAUGAGGCCGUUAGGCCGAA IGCAGAGA	1155

1204	UAUCGCGC U UACUAUGG	780	CCAUAGUA CUGAUGAGGCCGUUAGGCCGAA ICGCGAUA	1156

1208	GCGCUUAC U AUGGAGCA	781	UGCUCCAU CUGAUGAGGCCGUUAGGCCGAA IUAAGCGC	1157

1216	UAUGGAGC A UUUAAGGA	782	UCCUUAAA CUGAUGAGGCCGUUAGGCCGAA ICUCCAUA	1158

1229	AGGAUGUC A AGGAGAAA	783	UUUCUCCU CUGAUGAGGCCGUUAGGCCGAA IACAUCCU	1159

1241	AGAAAAAC A GGACCUCU	784	AGAGGUCC CUGAUGAGGCCGUUAGGCCGAA IUUUUUCU	1160

1246	AACAGGAC C UCUGAAGA	785	UCUUCAGA CUGAUGAGGCCGUUAGGCCGAA IUCCUGUU	1161

1247	ACAGGACC U CUGAAGAA	786	UUCUUCAG CUGAUGAGGCCGUUAGGCCGAA IGUCCUGU	1162

1249	AGGACCUC U GAAGAAGC	787	GCUUCUUC CUGAUGAGGCCGUUAGGCCGAA IAGGUCCU	1163

1258	GAAGAAGC A GAAGACCU	788	AGGUCUUC CUGAUGAGGCCGUUAGGCCGAA ICUUCUUC	1164

1265	CAGAAGAC C UCCGAGCC	789	GGCUCGGA CUGAUGAGGCCGUUAGGCCGAA IUCUUCUG	1165

1266	AGAAGACC U CCGAGCCU	790	AGGCUCGG CUGAUGAGGCCGUUAGGCCGAA IGUCUUCU	1166

1268	AAGACCUC C GAGCCUUG	791	CAAGGCUC CUGAUGAGGCCGUUAGGCCGAA IAGGUCUU	1167

1273	CUCCGAGC C UUGCGAUU	792	AAUCGCAA CUGAUGAGGCCGUUAGGCCGAA ICUCGGAG	1168

1274	UCCGAGCC U UUGCGAUU	793	AAAUCGCA CUGAUGAGGCCGUUAGGCCGAA IGCUCGGA	1169

1284	GCGAUUUC U AUCUGUGA	794	UCACAGAU CUGAUGAGGCCGUUAGGCCGAA IAAAUCGC	1170

1288	UUUCUAUC U GUGAUUUC	795	GAAAUCAC CUGAUGAGGCCGUUAGGCCGAA IAUAGAAA	1171

1297	GUGAUUUC A AUUGUGGA	796	UCCACAAU CUGAUGAGGCCGUUAGGCCGAA IAAAUCAC	1172

1307	UUGUGGAC C CUUGGAUU	797	AAUCCAAG CUGAUGAGGCCGUUAGGCCGAA IUCCACAA	1173

1308	UGUGGACC C UUGGAUUU	798	AAAUCCAA CUGAUGAGGCCGUUAGGCCGAA IGUCCACA	1174

1309	GUGGACCC U UGGAUUUU	799	AAAAUCCA CUGAUGAGGCCGUUAGGCCGAA IGGUCCAC	1175

1322	UUUUUAUC A UUUUCAGA	800	UCUGAAAA CUGAUGAGGCCGUUAGGCCGAA IAUAAAAA	1176

1328	UCAUUUUC A GAUCUCCA	801	UGGAGAUC CUGAUGAGGCCGUUAGGCCGAA IAAAAUGA	1177

1333	UUCAGAUC U CCAGUAUU	802	AAUACUGG CUGAUGAGGCCGUUAGGCCGAA IAUCUGAA	1178

1335	CAGAUCUC C AGUAUUUC	803	GAAAUACU CUGAUGAGGCCGUUAGGCCGAA IAGAUCUG	1179

1336	AGAUCUCC A GUAUUUCG	804	CGAAAUAC CUGAUGAGGCCGUUAGGCCGAA IGAGAUCU	1180

1356	AUUUUUUC A CAAGAUUU	805	AAAUCUUG CUGAUGAGGCCGUUAGGCCGAA IAAAAAAU	1181

1358	UUUUUCAC A AGAUUUUC	806	GAAAAUCU CUGAUGAGGCCGUUAGGCCGAA IUGAAAAA	1182

1367	AGAUUUUC A UUAGACCU	807	AGGUCUAA CUGAUGAGGCCGUUAGGCCGAA IAAAAUCU	1183

1374	CAUUACAC C UCUUAGGU	808	ACCUAAGA CUGAUGAGGCCGUUAGCCCGAA IUCUAAUG	1184

1375	AUUAGACC U CUUAGGUA	809	UACCUAAG CUGAUGAGGCCGUUAGGCCGAA IGUCUAAU	1185

1377	UAGACCUC U UAGGUACA	810	UGUACCUA CUGAUGAGGCCGUUAGGCCGAA IAGGUCUA	1186

1385	UUAGGUAC A GGAGCCGG	811	CCGGCUCC CUGAUGAGGCCGUUAGGCCGAA IUACCUAA	1187

1391	ACAGGAGC C GGUGCAGC	812	GCUGCACC CUGAUGAGGCCGUUAGGCCGAA ICUCCUGU	1188

1397	GCCGGUGC A GCAAUUCC	813	GGAAUUGC CUGAUGAGGCCGUUAGGCCGAA ICACCGGC	1189

1400	GGUGCAGC A AUUCCACU	814	AGUGCAAU CUGAUGAGGCCGUUAGGCCGAA ICUGCACC	1190

1405	AGCAAUUC C ACUAACAU	815	AUGUUAGU CUGAUGAGGCCGUUAGGCCGAA IAAUUGCU	1191

1406	GCAAUUCC A CUAACAUG	816	CAUGUUAG CUGAUGAGGCCGUUAGCCCGAA IGAAUUGC	1192

1408	AAUUCCAC U AACAUGGA	817	UCCAUGUU CUGAUGAGGCCGUUAGGCCGAA IUGGAAUU	1193

1412	CCACUAAC A UGGAAUCC	818	GGAUUCCA CUGAUGAGGCCGUUAGGCCGAA IUUAGUGG	1194

1420	AUCGAAUC C AGUCUGUG	819	CACAGACU CUGAUGAGGCCGUUAGGCCGAA IAUUCCAU	1195

1421	UGGAAUCC A GUCUGUGA	820	UCACAGAC CUGAUGAGGCCGUUAGGCCGAA IGAUUCCA	1196

1425	AUCCAGUC U GUGACAGU	821	ACUGUCAC CUGAUGAGGCCGUUAGGCCGAA IACUGGAU	1197

1431	UCUGUGAC A GUGUUUUU	822	AAAAACAC CUGAUGAGGCCGUUAGGCCGAA IUCACAGA	1198

1441	UGUUUUUC A CUCUGUGG	823	CCACAGAG CUGAUGAGGCCGUUAGGCCGAA IAAAAACA	1199

1443	UUUUUCAC U CUGUGGUA	824	UACCACAG CUGAUGAGGCCGUUAGGCCGAA IUGAAAAA	1200

1445	UUUCACUC U GUGGUAAG	825	CUUACCAC CUGAUGAGGCCGUUAGGCCGAA IAGUGAAA	1201

1455	UGGUAAGC U GAGGAAUA	826	UAUUCCUC CUGAUGAGGCCGUUAGGCCGAA ICUUACCA	1202

1468	AAUAUGUC A CAUUUUCA	827	UGAAAAUG CUGAUGAGGCCGUUAGGCCGAA IACAUAUU	1203

1470	UAUGUCAC A UUUUCAGU	828	ACUGAAAA CUGAUGAGGCCGUUAGGCCGAA IUGACAUA	1204

1476	ACAUUUUC A GUCAAAGA	829	UCUUUGAC CUGAUGAGGCCGUUAGGCCGAA IAAAAUGU	1205

1480	UUUCAGUC A AAGAACCA	830	UGGUUCUU CUGAUGAGGCCGUUAGGCCGAA IACUGAAA	1206

TABLE V


Human PTGDR Zinzyme and Substrate Sequence

		Seq		Seq
Pos	Substrate	ID	Zinzyme	ID

9	GAAUUCUG G CUAUUUUC	1207	GAAAAUAG GCCGAAAGGCGAGUGAGGUCU CAGAAUUC	1438

23	UUCCUCCU G CCGUUCCG	1208	CGGAACGG GCCGAAAGGCGAGUGAGGUCU AGGAGGAA	1439

26	CUCCUGCC G UUCCGACU	1209	AGUCGGAA GCCGAAAGGCGAGUGAGGUCU GGCAGGAG	1440

37	CCGACUCG G CACCAGAG	1210	CUCUGGUG GCCGAAAGGCGAGUGAGGUCU CGAGUCGG	1441

45	GCACCAGA G UCUGUCUC	1211	GAGACAGA GCCGAAAGGCGAGUGAGGUCU UCUGGUGC	1442

49	CAGAGUCU G UCUCUACU	1212	AGUAGAGA GCCGAAAGGCGAGUGAGGUCU AGACUCUG	1443

64	CUGAGAAC G CAGCGCGU	1213	ACGCGCUG GCCGAAAGGCGAGUGAGGUCU GUUCUCAG	1444

67	AGAACGCA G CGCGUCAG	1214	CUGACGCG GCCGAAAGGCGAGUGAGGUCU UGCGUUCU	1445

69	AACGCAGC G CGUCAGGG	1215	CCCUGACG GCCGAAAGGCGAGUGAGGUCU GCUGCGUU	1446

71	CGCAGCGC G UCAGGGCC	1216	GGCCCUGA GCCGAAAGGCGAGUGAGGUCU GCGCUGCG	1447

77	GCGUCAGG G CCGAGCUC	1217	GAGCUCGG GCCGAAAGGCGAGUGAGGUCU CCUGACGC	1448

82	AGGGCCGA G CUCUUCAC	1218	GUGAAGAG GCCGAAAGGCGAGUGAGGUCU UCGGCCCU	1449

93	CUUCACUG G CCUGCUCC	1219	GGAGCAGG GCCGAAAGGCGAGUGAGGUCU CAGUGAAG	1450

97	ACUGGCCU G CUCCUCUC	1220	GCGCGGAG GCCGAAAGGCGAGUGAGGUCU AGGCCAGU	1451

102	CCUGCUCC G CGCUCUUC	1221	GAAGAGCG GCCGAAAGGCGAGUGAGGUCU GGAGCAGG	1452

104	UGCUCCGC G CUCUUCAA	1222	UUGAAGAG GCCGAAAGGCGAGUGAGGUCU GCGGAGCA	1453

114	UCUUCAAU G CCAGCGCC	1223	GGCGCUGG GCCUAAAGGCGAGUGAGGUCU AUUGAAUA	1454

118	CAAUGCCA G CGCCAGGC	1224	GCCUGGCG GCCGAAAGGCGAUUGAGGUCU UGGCAUUU	1455

120	AUGCCAUC G CCAGUCGC	1225	GCGCCUGG GCCGAAAGGCGAGUGAGGUCU GCUGGCAU	1456

125	AUCUCCAG G CGCUCACC	1226	GGUGAGCG GCCGAAAGGCGAGUGAGGUCU CUGGCGCU	1457

127	CGCCAGGC G CUCACCCU	1227	AUGGUGAG GCCGAAAGGCGAGUUAGGUCU GCCUGGCG	1458

136	CUCACCCU G CAGAGCGU	1228	ACUCUCUG GCCGAAAGGCGAGUGAGGUCU AGUGUGAG	1459

141	CCUUCAUA G CGUCCCGC	1229	UCUUGACU GCCGAAAGGCGAGUGAGGUCU UCUGCAGG	1460

143	UUCAGAGC G UCCCUCCU	1230	AGUCUGGA GCCGAAAGGCGAGUGAGGUCU UCUCUUCA	1461

148	AUCUUCCC G CCUCUCAA	1231	UUGAGAGG GCCGAAAUGCGAGUGAGGUCU GGGACGCU	1462

163	AAAGAGGG G UGUGACCC	1232	GUGUCACA GCCGAAAGGCGAGUGAGGUCU CCCUCUUU	1463

165	AUAUGUGU G UGACCCGC	1233	UCUGGUCA GCCUAAAGUCGAGUGAGGUCU ACCCCUCU	1464

172	UGUGACCC G CGAGUUUA	1234	UAAACUCG GCCGAAAGGCGAGUGAGGUCU GUGUCACA	1465

176	ACCCGCGA G UUUAGAUA	1235	UAUCUAAA GCCGAAAGGCGAGUGAGGUCU UCUCUUGU	1466

189	UAUAUUAG G UUCCUGCC	1236	GGCAGGAA GCCGAAAUGCGAGUGAGGUCU CUCCUAUC	1467

195	AGGUUCCU G CCUUUUUU	1237	CCCCACUU GCCGAAAGGCGAGUGAGGUCU AGGAACCU	1468

198	UUCCUGCC G UGGGGAAC	1238	GUUCCCCA GCCGAAAGUCUAGUGAGGUCU GGCAGGAA	1469

212	AACACCCC G CCUCCCUC	1239	GAGGUCUG GCCGAAAGGCGAGUGAGGUCU GGGGUGUU	1470

215	ACCCCGCC G CCCUCGGA	1240	UCCUAGUG GCCGAAAUUCUAGUGAGGUCU UUCUUUUU	1471

224	CCCUCGGA G CUUUUUCU	1241	AGAAAAAU UCCGAAAGGCGAGUGAUGUCU UCCGAGGG	1472

233	CUUUUUCU G UGUCUCAG	1242	CUGCGCCA UCCGAAAGGCGAGUGAGGUCU AGAAAAAG	1473

236	UUUCUGUG G CUCAUCUU	1243	AAUCUUCU UCCUAAAGGCUAUUGAUUUCU CACAGAAA	1474

238	UCUGUGUC G CAGCUUCU	1244	AGAAGCUG UCCUAAAUUCUAUUUAGGUCU UCCACAGA	1475

241	GUGUCUCA G CUUCUCCG	1245	CUUAUAAU GCCGAAAGGCUAUUUAUGUCU UGCGCCAC	1476

249	GCUUCUCC G CCCUAUCC	1246	GGCUCUUU UCCUAAAUUCGAGUUAGUUCU UGAUAAUC	1477

255	CCUCCCGA G CCGCUCUC	1247	UCGCGCUG UCCUAAAUUCUAUUUAUGUCU UCUUUCUU	1478

258	CCCUAUCC G CUCGCGGA	1248	UCCUCGCU GCCGAAAGUCGAUUUAUUUCU GUCUCGGG	1479

260	CGAGCCGC G CUCUUAUC	1249	GCUCCGCU UCCUAAAUUCUAGUGAUUUCU UCGUCUCU	1480

262	AUCCUCUC G CUUAGCUG	1250	CAGCUCCU GCCUAAAGGCGAUUUAUGUCU UCUCGGCU	1481

267	CGCGCGGA G CUUCCUUU	1251	CCCGGCAU UCCUAAAUUCUAGUGAUUUCU UCCUCUCU	1482

270	UCUUAUCU G CCGGGGGC	1252	UCCCCCUG GCCUAAAUUCUAUUUAUUUCU AUCUCCUC	1483

277	UGCCGGUU G CUCCUUAU	1253	CUAAUUAU UCCUAAAUUCGAGUGAUUUCU CCCCGUCA	1484

285	GCUCCUUA G CACCCGGG	1254	CCCGGGUG GCCGAAAGGCGAGUGAGGUCU UAAGGAGC	1485

293	GCACCCGG G CGCCGGGG	1255	CCCCGGCG GCCGAAAGGCGAGUGAGGUCU CCGGGUGC	1486

295	ACCCGGGC G CCGGGGCC	1256	GGCCCCGG GCCGAAAGGCGAGUGAGGUCU GCCCGGGU	1487

301	GCGCCGGG G CCCUCGCC	1257	GGCGAGGG GCCGAAAGGCGAGUCAGGUCU CCCGGCGC	1488

307	GGGCCCUC G CCCUUCCG	1258	CGGAAGGG GCCGAAAGGCGAGUGAGGUCU GAGGGCCC	1489

315	GCCCUUCC G CAGCCUUC	1259	GAAGGCUG GCCGAAAGGCGAGUGAGGUCU GGAAGGGC	1490

318	CUUCCGCA G CCUUCACU	1260	AGUGAAGG CCCGAAAGGCGAGUGAGGUCU UGCCGAAG	1491

330	UCACUCCA G CCCUCUGC	1261	GCAGAGGG GCCGAAAGGCGAGUGAGGUCU UGGACUGA	1492

337	AGCCCUCU G CUCCCGCA	1262	UGCGGGAG GCCGAPAGGCGAGUGAGGUCU AGAGGGCU	1493

343	CUGCUCCC G CACGCCAU	1263	AUGGCGUG GCCGAAAGGCGAGUGAGGUCU GGGAGCAC	1494

347	UCCCGCAC G CCAUGAAG	1264	CUUCAUGG GCCGAAAGGCGAGUGAGGUCU GUGCGGGA	1495

355	GCCAUGAA G UCGCCGUU	1265	AACGGCCA CCCGAAAGGCGAGUGAGGUCU UUCAUGGC	1496

358	AUGAAGUC G CCGUUCUA	1266	UAGAACGG GCCGAAAGCCGAGUGAGGUCU GACUUCAU	1497

361	AAGUCGCC G UUCUACCG	1267	CGGUACAA CCCGAAAGGCGAGUGAGCUCU GGCGACUU	1498

369	GUUCUACC G CUCCOAGA	1268	UCUGGCAG GCCGAAACGCGAGUGAGGUCU CGUAGAAC	1499

372	CUACCGCU G CCAGAACA	1269	UGUUCUCC GCCGAAAGGCGACUCACCUCU AGCCGUAC	1500

389	CCACCUCU G UGGAAAAA	1270	UUUUUCCA GCCCAAAGGCGAGUGAGGUCU AGAGGUOG	1501

399	GGAAAAAG G CAACUCGG	1271	CCCAGUUG GCCGAAAGGCGAGUGAGGUCU CUUUUUCC	1502

407	GCAACUCG G CUCUGAUC	1272	CAUCACCC GCCCAAAGGCGAGUGACCUCU CGAGUUGC	1503

410	ACUCGGCC G UGAUGGUC	1273	GCCCAUCA GCCGAAACCCGAGUGAGGUCU CGCCGAGU	1504

417	GGUGAUGG G CGCCCUGC	1274	CCACCCCG GCCGAAAGGCCAGUCACCUCU CCAUCACC	1505

422	UGGGCCGC G UGCUCUUC	1275	GAAGAGCA GCCCAAAGGCGAGUGAGCUCU CCCGCCCA	1506

424	CGCGGGGU G CUCUUCAG	1276	CUGAAGAG CCCGAAAGGCGAGUGAGGUCU ACCCCCCC	1507

432	GCUCUUCA G CACCGGCC	1277	GGCCGGUG CCCGAAAGGCGAGUGAGGUCU UCAAGAGC	1508

438	CAGCACCG G CCUCCUCC	1278	CCAGCAGG GCCGAAAGGCGAGUGAGGUCU CCGUGCUG	1509

447	CCUCCUCG G CAACCUGC	1279	GCAGGUUG GCCCAAAGGCGAGUGAGCUCU CCAGGAGG	1510

454	GCCAACCU G CUCGCCCU	1280	AGGGCCAG CCCGAAAGGCGAGUCAGGUCU AGGUUCCC	1511

458	ACCUCCUC G CCCUGGGG	1281	CCCCAGGC GCCCAAAGGCGAGUGACGUCU CAGCAGGU	1512

466	GCCCUGGG G CUGCUCGC	1282	GCCAGCAG GCCGAAAGGCCAGUGAGGUCU CCCAGCCC	1513

469	CUGGGGCU G CUGGCCCG	1283	CGCGCCAG GCCGAAAGGCGAGUCAGGUCU AGCCCCAC	1514

473	GGCUGCUG G CGCCCUCG	1284	CGAGCGCG GCCGAAAGGCGAGUGAGGUCU CACCAGCC	1515

475	CUGCUGGC G CCCUCCGG	1285	CCCCACCC GCCGAAACCCCACUCAGGUCU GCCACCAC	1516

477	CCUCGCGC G CUCGGCCC	1286	CCCCCCAC CCCCAAACGCGAGUCACCUCU CCGCCACC	1517

484	CCCUCCCC G CUCCGGUG	1287	CACCCCAG GCCCAAACCCCACUCAGCUCU CCCCACCC	1518

490	CCCCUGCG G UGGUCCUC	1288	CACCACCA CCCCAAACCCCACUCACCUCU CCCACCCC	1519

493	CUGCCCUC G UCCUCCCG	1289	CCCGACCA GCCCAAACCCCAGUCACCUCU CACCCCAG	1520

495	CCCCUCGU G CUCCCCCC	1290	CCCCCCAC CCCCAAAGCCGACUCACCUCU ACCACCCC	1521

499	UCCUCCUC G CCCCCUCC	1291	CCACGCCG CCCGAAACCCCACUGAGCUCU CACCACCA	1522

502	UCCUCGCG G CCUCCACU	1292	AGUCCACC CCCGAAAGGCGACUCACCUCU CGCCACCA	1523

504	CUCCCCCC G UCCACUCC	1293	GCACUCCA CCCCAAACGCGAGUCACGUCU CCCCCGAC	1524

511	CCUCCACU G CCCCCCCU	1294	ACCCCGCG GCCGAAACCCCACUCAGGUCU AGUCCACC	1525

513	UCCACUGC G CCCCCUCC	1295	CCACCCCC CCCCAAACCCCACUCACCUCU CCAGUCCA	1526

517	CUCCGCCC G CUGCCCUC	1296	CACGCCAC GCCCAAACCCGAGUCAGGUCU GGGCCCAC	1527

520	CGCCCGCU G CCCUCCGU	1297	ACCGACCG GCCGAAAGGCCAGUCACCUCU AGCCCCCC	1528

527	UGCCCUCG G UCUUCUAC	1298	GUACAACA GCCGAAACGCGAGUGAGGUCU CCACGCCA	1529

538	UUCUACAU G CUCCUCUC	1299	CACACCAG CCCGAAAGGCGAGUCAGGUCU AUCUACAA	1530

542	ACAUCCUC G UGUGUGCC	1300	GCCACACA GCCGAAACCCGACUGAGGUCU CACCAUGU	1531

544	AUCCUCCU G UGUCCCCU	1301	AGCCCACA GCCGAAACCCGAGUCACGUCU ACCACCAU	1532

546	GCUCCUGU G UGCCCUGA	1302	UCACGCCA CCCCAAACCCCAGUGACCUCU ACACCACC	1533

549	CCUGUCUG G CCUCACCC	1303	CCCUCACG CCCCAAACCCGACUCAGGUCU CACACACC	1534

557	GCCUGACC G UCACCGAC	1304	CUCGGUGA CCCCAAACCCGAGUGACGUCU CCUCACCC	1535

568	ACCCACUU G CUGGGCAA	1305	UUGCCCAG GCCGAAAGGCGAGUGAGGUCU AAGUCGGU	1536

573	CUUGCUGG G CAAGUGCC	1306	GGCACUUG GCCGAAAGGCGAGUGAGGUCU CCAGCAAG	1537

577	CUGGGCAA G UGCCUCCU	1307	AGGAGGCA GCCGAAAGGCGAGUGAGGUCU UUGCCCAG	1538

579	GGGCAAGU G CCUCCUAA	1308	UUAGGAGG GCCGAAAGGCGAGUGAGGUCU ACUUGCCC	1539

588	CCUCCUAA G CCCGGUGG	1309	CCACCGGG GCCGAAAGGCGAGUGAGGUCU UUAGGACG	1540

593	UAAGCCCG G UGGUGCUG	1310	CAGCACCA GCCGAAAGGCGAGUGAGGUCU CGGGCUUA	1541

596	GCCCGGUG G UGCUGGCU	1311	AGCCAGCA GCCGAAAGGCGAGUGAGGUCU CACCGGGC	1542

598	CCGGUGGU G CUGGCUGC	1312	GCAGCCAG GCCGAAAGGCGAGUGAGGUCU ACCACCGG	1543

602	UGGUGCUG G CUOCCUAC	1313	GUAGUCAG GCCGAAAGGCGAGUGAGGUCU CAGCACCA	1544

605	UGCUGGCU G CCUACGCU	1314	AGCGUAGG GCCGAAAGGCGAGUGAGGUCU AGCCAGCA	1545

611	CUGCCUAC G CUCAGAAC	1315	GUUCUGAG GCCGAAAGGCGAGUGAGGUCU GUAGGCAG	1546

624	GAACCGGA G UCUGCGGG	1316	CCCGCAGA GCCGAAAGGCGAGUGAGGUCU UCCGCUUC	1547

628	CGGAGUCU G CGGGUGCU	1317	AGCACCCG GCCGAAAGGCGAGUGAGCUCU AGACUCCG	1548

632	GUCUGCGG G UGCUUGCG	1318	CGCAAGCA GCCGAAAGGCGAGUGAGGUCU CCGCAGAC	1549

634	CUGCGGGU G CUUGCGCC	1319	GGCGCAAG GCCGAAAGGCGAGUGAGGUCU ACCCGCAG	1550

638	GGGUGCUU G CGCCCGCA	1320	UGCGGGCG GCCGAAAGGCGAGUGAGGUCU AAGCACCC	1551

640	GUGCUUGC G CCCGCAUU	1321	AAUGCGGG GCCGAAAGGCGAGUGAGGUCU GCAAGCAC	1552

644	UUGCGCCC G CAUUGGAC	1322	GUCCAAUG GCCGAAAGGCGAGUGAGGUCU GGGCGCAA	1553

658	GACAACUC G UUGUGCCA	1323	UGGCACAA GCCGAAAGGCGAGUGAGGUCU GAGUGGUC	1554

661	AACUCGUU G UGCCAAGC	1324	GCUUGGCA GCCGAAAGGCGAGUGAGGUCU AACGAGUU	1555

663	CUCGUUGU G CCAAGCCU	1325	AGGCUUGG GCCGAAAGGCGAGUGAGGUCU ACAACGAG	1556

668	UGUGCCAA G CCUUCGCC	1326	GGCGAAGG GCCGAAAGGCGAGUGAGGUCU UUGGCACA	1557

674	AAGCCUUC G CCUUCUUC	1327	GAAGAAGG GCCGAAAGGCGAGUGAGGUCU GAAGGCUU	1558

685	UUCUUCAU G UCCUUCUU	1328	AAGAAGGA GCCGAAAGGCGAGUGAGGUCU AUGAAGAA	1559

697	UUCUUUGG G CUCUCCUC	1329	GAGGAGAG GCCGAAAGGCGAGUGAGGUCU CCAAAGAA	1560

712	UCGACACU G CAACUCCU	1330	AGGAGUUG GCCGAAAGGCGAGUGAGGUCU AGUGUCGA	1561

722	AACUCCUG G CCAUGGCA	1331	UGCCAUGG GCCGAAAGGCGAGUGAGGUCU CAGGAGUU	1562

728	UGGCCAUG G CACUGGAG	1332	CUCCAGUG GCCGAAAGGCGAGUGAGGUCU CAUGOCCA	1563

736	GCACUGGA G UGCUGGCU	1333	AGCCAGCA GCCGAAAGGCGAGUGAGGUCU UCCAGUGC	1564

738	ACUGGAGU G CUGGCUCU	1334	AGAGCCAG GCCGAAAGGCGAGUGAGGUCU ACUCCAGU	1565

742	GAGUCCUG G CUCUCCCU	1335	AGGGAGAG GCCGAAAGGCGAGUGAGGUCU CACCACUC	1566

754	UCCCUAGG G CACCCUUU	1336	AAAGGGUG GCCGAAAGGCGAGUGAGGUCU CCUAGGGA	1567

775	UACCGACG G CACAUCAC	1337	GUGAUGUG GCCGAAAGGCGAGUGAGGUCU CGUCGGUA	1568

787	AUCACCCU G CGCCUGGG	1338	CCCAGGCG GCCGAAAGGCGAGUGAGGUCU AGGGUGAU	1569

789	CACCCUGC G CCUGGGCG	1339	CGCCCAGG GCCGAAAGGCGAGUGAGGUCU GCAGGGUG	1570

795	GCGCCUGG G CGCACUGG	1340	CCAGUGCG GCCGAAAGGCGAGUGAGGUCU CCAGGCGC	1571

797	GCCUGGGC G CACUGGUG	1341	CACCAGUG GCCGAAAGGCGAGUGAGGUCU GCCCAGGC	1572

803	GCGCACUG G UGGCCCCG	1342	CGGGGCCA GCCGAAAGGCGAGUGAGGUCU CAGUGCGC	1573

806	CACUGGUG G CCCCGGUG	1343	CACCGGGG GCCGAAAGGCGAGUGAGGUCU CACCAGUG	1574

812	UGGCCCCG G UGGUGAGC	1344	GCUCACCA GCCGAAAGGCGAGUGAGGUCU CGGGGCCA	1575

815	CCCCGGUG G UGAGCGCC	1345	GGCGCUCA GCCGAAAGGCGAGUGAGGUCU CACCGGGG	1576

819	GGUGGUGA G CGCCUUCU	1346	AGAAGGCG GCCGAAAGGCGAGUGAGGUCU UCACCACC	1577

821	UGGUGAGC G CCUUCUCC	1347	GGAGAAGG GCCGAAAGGCGAGUGAGGUCU GCUCACCA	1578

833	UCUCCCUG G CUUUCUGC	1348	GCAGAAAG GCCGAAAGGCGAGUGAGGUCU CAGGGAGA	1579

840	GGCUUUCU G CGCGCUAC	1349	GUAGCGCG GCCGAAAGGCGAGUGAGGUCU AGAAAGCC	1580

842	CUUUCUGC G CGCUACCU	1350	AGGUAGCG GCCGAAAGGCGAGUGAGGUCU GCAGAAAG	1581

844	UUCUGCGC G CUACCUUU	1351	AAAGGUAG GCCGAAAGGCGAGUGAGGUCU GCGCAGAA	1582

858	UUUCAUGG G CUUCGGGA	1352	UCCCGAAG GCCGAAAGGCGAGUGAGGUCU CCAUGAAA	1583

868	UUCGGGAA G UUCGUGCA	1353	UGCACGAA GCCGAAAGGCGAGUGAGGUCU UUCCCGAA	1584

872	GGAAGUUC G UCCAGUAC	1354	GUACUGCA GCCGAAAGGCGAGUGAGGUCU GAACUUCC	1585

874	AAGUUCGU G CAGUACUG	1355	CAGUACUG GCCGAAAGGCGAGUGAGGUCU ACGAACUU	1586

877	UUCGUGCA G UACUGCCC	1356	GGGCAGUA CCCGAAAGGCGAGUGAGGUCU UGCACGAA	1587

882	GCAGUACU G CCCCGGCA	1357	UGCCGGGG GCCGAAAGGCCAGUGAGGUCU AGUACUGC	1588

888	CUGCCCCG G CACCUGGU	1358	ACCAGGUG GCCGAAAGGCGAGUGAGGUCU CGGGGCAG	1589

895	GOCACCUG G UGCUUUAU	1359	AUAAAGCA GCCGAAAGGCGAGUGAGGUCU CAGGUGCC	1590

897	CACCUGGU G CUUUAUCC	1360	GGAUAAAG GCCGAAAGGCGAGUGAGGUCU ACCAGGUG	1591

911	UCCAGAUG G UCCACGAG	1361	CUCCUGGA GCCGAAAGGCGAGUGAGGUCU CAUCUGGA	1592

924	CGAGGAGG G CUCGCUGU	1362	ACAGCGAG GCCGAAAGGCGAGUGAGGUCU CCUCCUCG	1593

928	GAGGGCUC G CUGUCGGU	1363	ACCGACAG GCCGAAAGGCGAGUGAGGUCU GAGCCCUC	1594

931	GGCUCGCU G UCGGUGCU	1364	AGCACCGA GCCGAAAGGCGAGUGAGGUCU AGCGAGCC	1595

935	CGCUGUCG G UGCUGGGG	1365	CCCCAGCA GCCGAAAGGCGAGUGAGGUCU CGACAGCG	1596

937	CUGUCGGU G CUGCGGUA	1366	UACCCCAG GCCGAAACCCGAGUGAGGUCU ACCGACAG	1597

943	GUCCUGOG G UACUCUGU	1367	ACAGAGUA GCCGAAAGGCGAGUGAGGUCU CCCAGCAC	1598

950	CGUACUCU G UCCUCUAC	1368	GUACAGCA GCCGAAAGCCGAGUGAGGUCU AGACUACC	1599

952	UACUCUGU G CUCUACUC	1369	GAGUAGAC GCCGAAAGCCGAGUCACGUCU ACAGAGUA	1600

963	CUACUCCA G CCUCAUGG	1370	CCAUGACG CCCGAAACGCGACUGAGGUCU UGGAGUAG	1601

971	GCCUCAUG G CGCUGCUG	1371	CAGCAGCG GCCCAAAGGCGAGUGAGGUCU CAUGAGGC	1602

973	CUCAUGOC G CUGCUCCU	1372	ACCAGCAG GCCGAAAGGCGAGUGAGCUCU OCCAUGAG	1603

976	AUGGCGCU G CUCGUCCU	1373	AGGACCAG GCCGAAAGGCGAGUCAGGUCU AGCGCCAU	1604

980	CGCUGCUG G UCCUCGCC	1374	GGCGACGA GCCGAAAGGCGACUGAGGUCU CAGCAGCG	1605

986	UGGUCCUC G CCACCGUC	1375	CACGGUGG GCCGAAAGGCGAGUGACGUCU GAGGACCA	1606

992	UCCCCACC G UGCUGUGC	1376	GCACAGCA GCCGAAAGGCGAGUGAGGUCU GGUGGCGA	1607

994	GCCACCGU G CUGUGCAA	1377	UUGCACAG GCCGAAAGGCGAGUGACGUCU ACGGUGGC	1608

997	ACCCUGCU G UGCAACCU	1378	AGCUUGCA GCCGAAAGGCGACUGAGGUCU AGCACCCU	1609

999	CGUGCUGU G CAACCUCG	1379	CGAGGUUG GCCCAAAGGCGAGUGAGGUCU ACAGCACG	1610

1008	CAACCUCC G CCCCAUGC	1380	GCAUGGCG GCCGAAAGGCCAGUCAGGUCU CGACGUUG	1611

1010	ACCUCGGC G CCAUGCGC	1381	GCGCAUGG GCCGAAAGGCGAGUGAGGUCU CCCGAGGU	1612

1015	GGCCCCAU G CCCAACCU	1382	AGGUUGCC GCCGAAAGGCCAGUGAGGUCU AUGCCCCC	1613

1017	CGCCAUCC G CAACCUCU	1383	AGAGGUUG CCCGAPAGGCGACUGAGGUCU GCAUGGCG	1614

1028	ACCUCUAU G CGAUGCAC	1384	GUCCAUCG GCCCAAAGGCGAGUGAGCUCU AUAGAGGU	1615

1033	UAUGCGAU G CACCGCCC	1385	CGCCGGUG GCCCAAACGCGAGUGAGGUCU AUCGCAUA	1616

1039	AUGCACCC G CUCCUCCA	1386	UCCAGCCC CCCCAAAGGCGAGUCACCUCU CGGUCCAU	1617

1042	CACCCGCC G CUCCACCC	1387	CCCUGCAG CCCGAAACCCCACUGAGGUCU CCCCCCUG	1618

1045	CGGCCCCU G CACCUCCA	1388	UCCCGCUG CCCCAAAGGCGAGUCACCUCU AGCCGCCC	1619

1048	CCCCUGCA G CCCCACCC	1389	CCCUCCCC GCCGAAAGCCCACUGACGUCU UGCACCCG	1620

1051	CUCCACCC G CACCCCCG	1390	OCCUGGUC CCCCAAAGGCGAGUCAGCUCU CGCUGCAG	1621

1057	CCCCACCC G CUCUCCUC	1391	CACCACCG GCCGAAAGCCCAGUGAGCUCU CCCUCCCG	1622

1059	CCACCCCC G CUCCUCCA	1392	UCCACCAC CCCCAAAGGCGACUCACCUCU GCGGCUCC	1623

1065	GCCCUCCU G CACCACGG	1393	CCCUGGUG GCCCAAACCCCAGUCAGCUCU ACCACCUC	1624

1077	CACGGACU G UCCCCACC	1394	GCUCCCCA GCCGAAACCCGACUCAGGUCU AGUCCCUC	1625

1079	CCCACUCU G CCCAGCCC	1395	CUCCUCUC CCCCAAAGCCGAGUGACCUCU ACAGUCCC	1626

1084	UCUGCCCA G CCCCCCGC	1396	GCGCGCGG CCCCAAACCCCAGUGACGUCU UCCCCACA	1627

1087	CCCCAGCC G CUCUCUCA	1397	UCCCCCCC GCCGAAAGGCGACUCACGUCU GUCUCCUC	1628

1089	CGAGCCGC G CGCGGACG	1398	CGUCCGCG CCCCAAACCCCAGUGAGGUCU CCCGCUCG	1629

1091	AUCCUCUC G CGGACCCC	1399	CCCCUCCG GCCGAAAGGCGACUCACGUCU CCGCCCCU	1630

1106	CCAGGCAA G CCUCCCCU	1400	AGGGGACG CCCGAAACCCGAGUGAGCUCU UUCCCUCC	1631

1108	ACCCAAGC G UCCCCUCA	1401	UCAGCCCA GCCGAAAGGCCAGUCACCUCU CCUUCCCU	1632

1117	UCCCCUCA G CCCCUCGA	1402	UCCAGGUC CCCGAAACCCGAGUCAGCUCU UCAGUGGA	1633

1129	CUGGAGGA G CUCCAUCA	1403	UCAUCCAG GCCGAAACCCCACUCAGGUCU UCCUCCAC	1634

1144	CACCUCCU G CUCCUGUC	1404	UCCACCAC CCCCAAAGGCGACUGACCUCU AGGAGGUC	1635

1147	CUCCUCCU G CUGCCGCU	1405	ACCGCCAG CCCGAAACCCGAGUGAGCUCU ACCACCAG	1636

1151	UCCUCCUC G CUCUCAUG	1406	CAUCACCC GCCGAAAGCCGACUGAGGUCU CACCACCA	1637

1153	CUGCUGGC G CUGAUGAC	1407	GUCAUCAG GCCGAAAGGCGAGUGAGGUCU UCCACCAG	1638

1163	UGAUGACC G UGCUCUUC	1408	GAAGAGCA GCCGAAAGGCGAGUCAGGUCU GGUCAUCA	1639

1165	AUGACCGU G CUCUUCAC	1409	GUGAAGAG GCCGAAAGGCGAGUGAGGUCU ACGGUCAU	1640

1177	UUCACUAU G UGUUCUCU	1410	AGAGAACA GCCGAAACCCGAGUGAGGUCU AUAGUGAA	1641

1179	CACUAUGU G UUCUCUGC	1411	GCAGAGAA GCCGAAAGGCGACUGACCUCU ACAUACUC	1642

1186	UGUUCUCU G CCCGUAAU	1412	AUUACGGG GCCGAAAGGCGAGUGAGGUCU AGAGAACA	1643

1190	CUCUGCCC G UAAUUUAU	1413	AUAAAUUA GCCCAAAGGCGAGUCAGGUCU GGGCAGAG	1644

1200	AAUUUAUC G CGCUUACU	1414	AGUAAGCG GCCGAAAGGCGACUCAGGUCU GAUAAAUU	1645

1202	UUUAUCGC G CUUACUAU	1415	AUAGUAAG GCCCAAACCCCAGUGAGGUCU GCCAUAAA	1646

1214	ACUAUGCA G CAUUUAAC	1416	CUUAAAUG CCCGAAAGGCGAGUGACCUCU UCCAUACU	1647

1226	UUAAGGAU G UCAACCAG	1417	CUCCUCCA GCCGAAACGCGAGUGAGCUCU AUCCUUAA	1648

1256	CUGAAGAA G CAGAAGAC	1418	GUCUUCUG CCCCAAACCCGACUGAGCUCU UUCUUCAG	1649

1271	ACCUCCGA G CCUUGCCA	1419	UCGCAAGG GCCGAAACGCGAGUGAGCUCU UCGGAGGU	1650

1276	CCAGCCUU G CCAUUUCU	1420	AGAAAUCG GCCGAAACCCGACUGAGGUCU AACGCUCG	1651

1289	UUCUAUCU G UGAUCUCA	1421	UCAAAUCA CCCGAAAGCCCAGUGAGGUCU AGAUAGAA	1652

1301	UUUCAAUU G UGGACCCU	1422	ACCGUCCA GCCCAAACGCCAGUGAGGUCU AAUUCAAA	1653

1337	GAUCUCCA G UAUUUCCG	1423	CCGAAAUA CCCGAAAGGCGAGUGAGGUCU UGGAGAUC	1654

1381	CCUCUUAG G UACAGGAG	1424	CUCCUGUA GCCGAAAGGCGAGUGAGCUCU CUAAGAGG	1655

1389	CUACAGGA G CCGGUGCA	1425	UGCACCGC GCCGAAACCCGAGUGAGGUCU UCCUGUAC	1656

1393	AGGAGCCG G UCCAGCAA	1426	UUCCUGCA CCCCAAAGGCGAGUCACCUCU CGGCUCCU	1657

1395	CACCCCCU G CAGCAAUU	1427	AAUUCCUC GCCGAAACCCCACUGAGGUCU ACCCCCUC	1658

1398	CCGCUGCA G CAAUUCCA	1428	UGGAAUUG CCCCAAAGGCCAGUCACCUCU UGCACCCG	1659

1422	CCAAUCCA G UCUGUCAC	1429	CUCACACA CCCCAAACCCCACUGAGGUCU UCCAUUCC	1660

1426	UCCAGUCU G UCACACUC	1430	CACUGUCA CCCCAAAGGCCAGUCACCUCU AGACUGGA	1661

1432	CUCUCACA G UCUUUUUC	1431	CAAAAACA CCCGAAACCCCACUGAGCUCU UCUCACAG	1662

1434	CUGACACU G UUUUUCAC	1432	CUGAAAAA CCCGAAACGCCACUCACCUCU ACUGUCAC	1663

1446	UUCACUCU G UCCUAAGC	1433	CCUUACCA CCCCAAACGCGAGUGACCUCU AGACUGAA	1664

1449	ACUCUGUG G UAACCUCA	1434	UCACCUUA GCCGAAACCCCACUGAGCUCU CACACACU	1665

1453	UCUCCUAA G CUGAGGAA	1435	UUCCUCAC CCCGAAAGGCGACUCACCUCU UUACCACA	1666

1465	ACCAAUAU G UCACAUUU	1436	AAAUCUGA CCCCAAACCCGAGUGACCUCU AUAUUCCU	1667

1477	CAUUUUCA G UCAAACAA	1437	UUCUUUCA CCCCAAACCCCACUCACCUCU UGAAAAUC	1668

TABLE VI


Human PTGDR DNAzyme
and Substrate Sequence

		Seq		Seq
Pos	Substrate	ID	DNAzyme	ID

9	GAAUUCUG G CUAUUUUC	1207	GAAAATAG GGCTAGCTACAACGA CAGAATTC	1715

12	UUCUGGCU A UUUUCCUC	1	GAGGAAAA GGCTAGCTACAACGA AGCCAGAA	1716

23	UUCCUCCU G CCGUUCCG	1208	CGGAACGG GGCTAGCTACAACGA AGGAGGAA	1717

26	CUCCUGCC G UUCCGACU	1209	AGTCGGAA GGCTAGCTACAACGA GGCAGGAG	1718

32	CCGUUCCG A CUCGGCAC	1669	GTGCCGAG GGCTAGCTACAACGA CGGAACGG	1719

37	CCGACUCG G CACCAGAG	1210	CTCTGGTG GGCTAGCTACAACGA CGAGTCGG	1720

39	GACUCGGC A CCAGAGUC	463	GACTCTGG GGCTAGCTACAACGA GCCGAGTC	1721

45	GCACCAGA G UCUGUCUC	1211	GAGACAGA GGCTAGCTACAACGA TCTGGTGC	1722

49	CAGAGUCU G UCUCUACU	1212	AGTAGAGA GGCTACCTACAACGA AGACTCTG	1723

55	CUGUCUCU A CUGAGAAC	13	GTTCTCAG GGCTAGCTACAACGA AGAGACAG	1724

62	UACUGAGA A CGCAGCGC	1670	GCGCTGCG GGCTAGCTACAACGA TCTCAGTA	1725

64	CUGAGAAC G CAGCGCGU	1213	ACGCGCTG GGCTAGCTACAACGA GTTCTCAG	1726

67	AGAACGCA G CGCGUCAG	1214	CTGACGCG GGCTAGCTACAACGA TGCGTTCT	1727

69	AACGCAGC G CGUCAGGG	1215	CCCTGACG GGCTAGCTACAACGA GCTGCGTT	1728

71	CGCAGCGC G UCAGGGCC	1216	GGCCCTGA GGCTAGCTACAACGA GCGCTGCG	1729

77	GCGUCAGG G CCGAGCUC	1217	CAGCTCGG GGCTAGCTACAACCA CCTGACGC	1730

82	AGCCCCGA G CUCUUCAC	1218	GTCAAGAC GCCTAGCTACAACGA TCGGCCCT	1731

89	AGCUCUUC A CUGGCCUG	475	CAGGCCAG GGCTAGCTACAACGA GAAGAGCT	1732

93	CUUCACUG G CCUGCUCC	1219	GCACCAGG GGCTAGCTACAACGA CAGTGAAG	1733

97	ACUCCCCU G CUCCGCGC	1220	GCGCGGAG GGCTAGCTACAACGA AGOCCACT	1734

102	CCUGCUCC G CCCUCUUC	1221	CAAGAGCG GGCTAGCTACAACGA GGACCAGG	1735

104	UGCUCCGC G CUCUUCAA	1222	TTCAAGAG GGCTAGCTACAACGA GCGGAGCA	1736

112	GCUCUUCA A UGCCAGCG	1671	CGCTGGCA GGCTAGCTACAACGA TGAAGAGC	1737

114	UCUUCAAU G CCAGCGCC	1223	GGCGCTGG GGCTAGCTACAACGA ATTGAAGA	1738

118	CAAUGCCA G CGCCAGGC	1224	GCCTGGCG GGCTAGCTACAACGA TGGCATTG	1739

120	AUGCCAGC G CCAGCCGC	1225	GCGCCTGG GGCTAGCTACAACGA GCTGCCAT	1740

125	AGCGCCAG G CGCUCACC	1226	GGTGAGCG CGCTAGCTACAACGA CTGGCGCT	1741

127	CGCCAGGC G CUCACCCU	1227	AGGGTGAG GGCTAGCTACAACGA GCCTGGCG	1742

131	AGGCGCUC A CCCUGCAG	489	CTGCAGGG GGCTAGCTACAACGA GAGCGCCT	1743

136	CUCACCCU G CAGAGCGU	1228	ACGCTCTG GGCTAGCTACAACGA AGGGTGAG	1744

141	CCUGCAGA G CGUCCCGC	1229	GCGGGACG GGCTAGCTACAACGA TCTGCAGG	1745

143	UGCAGAGC G UCCCGCCU	1230	AGGCGGGA GGCTAGCTACAACGA GCTCTGCA	1746

148	AGCGUCCC G CCUCUCAA	1231	TTGAGAGG GGCTAGCTACAACGA GGGACGCT	1747

163	AAAGAGGG G UGUGACCC	1232	GGGTCACA GGCTAGCTACAACGA CCCTCTTT	1748

165	AGAGGGGU G UGACCCGC	1233	GCGGGTCA GGCTAGCTACAACGA ACCCCTCT	1749

168	GCGGUGUG A CCCGCGAG	1672	CTCGCGGG GGCTAGCTACAACGA CACACCCC	1750

172	UGUGACCC G CGAGUUUA	1234	TAAACTCG GGCTAGCTACAACGA GGGTCACA	1751

176	ACCCGCGA G UUUAGAUA	1235	TATCTAAA GGCTAGCTACAACGA TCGCGGGT	1752

182	GAGUUUAG A UAGGAGGU	1673	ACCTCCTA GGCTAGCTACAACGA CTAAACTC	1753

189	GAUAGGAG G UUCCUGCC	1236	GGCAGGAA GGCTAGCTACAACGA CTCCTATC	1754

195	AGGUUCCU G CCGUGGGG	1237	CCCCACCG GGCTAGCTACAACGA AGGAACCT	1755

198	UUCCUGCC G UGGGGAAC	1238	GTTCCCCA GGCTAGCTACAACGA GCCAGGAA	1756

205	CGUGGGGA A CACCCCGC	1674	CCCGGGTG GGCTAGCTACAACGA TCCCCACG	1757

207	UGGCGAAC A CCCCGCCG	505	CGGCGGGG GGCTAGCTACAACGA GTTCCCCA	1758

212	AACACCCC G CCCCCCUC	1239	GAGCCCCG GGCTAGCTACAACGA GCGGTGTT	1759

215	ACCCCGCC G CCCUCGGA	1240	TCCGAGCG GGCTAGCTACAACGA GGCGGCGT	1760

224	CCCUCGGA G CUUUUUCU	1241	AGAAAAAG GGCTAGCTACAACGA TCCGAGGG	1761

233	CUUUUUCU G UGGCGCAG	1242	CTGCGCCA GGCTAGCTACAACGA AGAAAAAG	1762

236	UUUCUCUG G CGCAGCUU	1243	AAGCTGCC GGCTAGCTACAACGA CACAGAAA	1763

238	UCUGUGGC G CAGCUUCU	1244	AGAAGCTG GGCTAGCTACAACGA GCCACAGA	1764

241	GUGGCGCA G CUUCUCCG	1245	CGGAGAAG GCCTAGCTACAACGA TOCCCCAC	1765

249	GCUUCUCC G CCCGAGCC	1246	GGCTCGGG CGCTAGCTACAACGA GGAGAAGC	1766

255	CCGCCCGA G CCCCGCGC	1247	GCGCGCGG GGCTACCTACAACGA TCGGGCGG	1767

258	CCCGAGCC G CGCGCGGA	1248	TCCGCGCG GGCTAGCTACAACCA GGCTCCCG	1768

260	CGAGCCGC G CGCGGAGC	1249	GCTCCCCG GGCTAGCTACAACGA GCGGCTCG	1769

262	ACCCGCCC G CGGAGCUG	1250	CAGCTCCG GGCTAGCTACAACGA CCGCGCCT	1770

267	CGCGCGGA G CUCCCGCG	1251	CCCGGCAG GGCTAGCTACAACCA TCCGCGCG	1771

270	GCGCACCU G CCGGCGGC	1252	GCCCCCCG GGCTAGCTACAACGA AGCTCCGC	1772

277	UGCCGGGG G CUCCUUAG	1253	CTAAGGAC GGCTAGCTACAACCA CCCCGGCA	1773

285	CCUCCUUA G CACCCCGG	1254	CCCCGGTC GCCTAGCTACAACGA TAAGGAGC	1774

287	UCCUUAGC A CCCCGGCG	527	CCCCCGGG GGCTAGCTACAACGA CCTAAGGA	1775

293	GCACCCGG G CGCCGCCG	1255	CCCCGGCG CCCTAGCTACAACCA CCGGCTGC	1776

295	ACCCCCCC G CCGGGCCC	1256	CGCCCCGC GCCTAGCTACAACGA CCCCGGCT	1777

301	CCGCCGGC G CCCUCGCC	1257	GGCGAGGG GGCTAGCTACAACGA CCCCCCCC	1778

307	GGGCCCUC G CCCUUCCG	1258	CGCAAGCC GCCTAGCTACAACCA GAGGGCCC	1779

315	GCCCUUCC G CAGCCUUC	1259	GAAGGCTG GCCTAGCTACAACGA GGAACGGC	1780

318	CUUCCGCA G CCUUCACU	1260	ACTCAAGC GGCTACCTACAACCA TGCCGAAG	1781

324	CAGCCUUC A CUCCACCC	541	CCCTCGAG GGCTACCTACAACGA CAACCCTG	1782

330	UCACUCCA G CCCUCUGC	1261	GCACACGG GGCTAGCTACAACCA TGGACTCA	1783

337	ACCCCUCU G CUCCCGCA	1262	TCCGCGAG GCCTACCTACAACGA ACAGGCCT	1784

343	CUGCUCCC G CACCCCAU	1263	ATGCCGTG CCCTACCTACAACGA CCGACCAC	1785

345	GCUCCCCC A CGCCAUCA	552	TCATGCCC CGCTACCTACAACCA GCGGCAGC	1786

347	UCCCGCAC G CCAUCAAG	1264	CTTCATGC GCCTAGCTACAACCA GTGCGGGA	1787

350	CGCACGCC A UCAACUCC	554	CGACTTCA GGCTACCTACAACGA GGCGTCCG	1788

355	CCCAUCAA G UCGCCGUU	1265	AACGCCGA GGCTAGCTACAACCA TTCATCGC	1789

358	AUCAACUC G CCCUUCUA	1266	TACAACCG CGCTAGCTACAACGA GACTTCAT	1790

361	AAGUCGCC G UUCUACCC	1267	CGCTACAA GGCTAGCTACAACCA CCCGACTT	1791

366	GCCCUUCU A CCCCUGCC	55	GCCAGCGG GCCTAGCTACAACCA ACAACCGC	1792

369	GUUCUACC G CUGCCAGA	1268	TCTGCCAG GCCTACCTACAACGA GGTACAAC	1793

372	CUACCCCU G CCAGAACA	1269	TGTTCTCC GCCTAGCTACAACCA AGCCGTAG	1794

378	CUCCCACA A CACCACCU	1675	ACCTCCTC GCCTACCTACAACGA TCTCCCAC	1795

380	GCCACAAC A CCACCUCU	561	ACACCTCC CCCTACCTACAACCA CTTCTCCC	1796

383	AGAACACC A CCUCUCUG	563	CACAGAGG CCCTACCTACAACGA CCTCTTCT	1797

389	CCACCUCU G UCGAAAAA	1270	TTTTTCCA CGCTAGCTACAACCA AGAGCTCC	1798

399	CCAAAAAG G CAACUCCG	1271	CCCAGTTG CCCTACCTACAACGA CTTTTTCC	1799

402	AAAACCCA A CUCGCCCC	1676	CCCCCCAC CCCTACCTACAACCA TGCCTTTT	1800

407	CCAACUCG G CCCUCAUC	1272	CATCACCC GCCTACCTACAACCA CCACTTCC	1801

410	ACUCCCCC G UGAUCGCC	1273	CCCCATCA CCCTACCTACAACQA CGCCGACT	1802

413	CCGCCCUG A UCCCCCCC	1677	CCCCCCCA CCCTACCTACAACCA CACCCCCC	1803

417	CCUCAUCC G CCGCCUCC	1274	CCACCCCC CGCTAGCTACAACCA CCATCACC	1804

422	UCCCCCCC G UCCUCUUC	1275	CAACACCA CCCTACCTACAACCA CCCCCCCA	1805

424	CCCCCGCU G CUCUUCAG	1276	CTCAACAC CCCTACCTACAACGA ACCCCCCC	1806

432	CCUCUUCA G CACCCCCC	1277	CCCCCCTC CCCTACCTACAACCA TCAAGACC	1807

434	UCUUCACC A CCGGCCUC	572	CACGCCCC CCCTACCTACAACCA CCTCAAGA	1808

438	CACCACCG G CCUCCUCC	1278	CCACCAGC GCCTACCTACAACGA CGCTCCTC	1809

447	CCUCCUCC G CAACCUGC	1279	CCACCTTC CCCTAGCTACAACCA CCAGGAGC	1810

450	CCUGCCCA A CCUCCUCC	1678	CCACCAGC GCCTACCTACAACGA TCCCCACC	1811

454	GCCAACCU G CUGGCCCU	1280	AGGGCCAC GGCTAGCTACAACGA AGGTTGCC	1812

458	ACCUGCUG G CCCUGGGG	1281	CCCCAGGG GGCTAGCTACAACGA CAGCAGGT	1813

466	GCCCUGGG G CUGCUCGC	1282	GCCAGCAG GGCTAGCTACAACGA CCCAGCGC	1814

469	CUCGGGCU G CUGGCGCG	1283	CGCGCCAC CGCTAGCTACAACGA AGCCCCAG	1815

473	GCCUGCUG G CCCGCUCG	1284	CGAGCGCG GGCTAGCTACAACGA CAGCAGCC	1816

475	CUGCUGGC G CGCUCGGG	1285	CCCGAGCG CGCTAGCTACAACGA GCCAGCAG	1817

477	GCUCGCGC G CUCCCGGC	1286	GCCCCGAC GGCTACCTACAACGA GCGCCAGC	1818

484	CCCUCGGG G CUGGCGUG	1287	CACCCCAG GGCTAGCTACAACCA CCCGAGCG	1819

490	GGCCUGGC G UGCUGCUC	1288	CACCACCA GCCTACCTACAACGA CCCAGCCC	1820

493	CUCCGGUG G UGCUCCCC	1289	CGCCAGCA CCCTAGCTACAACGA CACCCCAG	1821

495	CCGGUCGU G CUCCCGGC	1290	CCCCCGAG GGCTAGCTACAACCA ACCACCCC	1822

499	UCGUGCUC G CCGCCUCC	1291	CCACCCCC GGCTAGCTACAACGA CAGCACCA	1823

502	UCCUCGCC G CCUCCACU	1292	ACTGGACG GCCTACCTACAACCA CGCCAGCA	1824

504	CUCGCGCC G UCCACUCC	1293	CCAGTCGA GGCTAGCTACAACCA GCCGCGAG	1825

508	CCCCGUCC A CUCCCCCC	591	GCGCGCAG GCCTAGCTACAACGA CGACGCCC	1826

511	CGUCCACU G CGCCCGCU	1294	AGCGGGCG GGCTAGCTACAACGA AGTCCACC	1827

513	UCCACUCC G CCCGCUGC	1295	GCACCCGC CGCTAGCTACAACCA GCACTCGA	1828

517	CUGCGCCC G CUCCCCUC	1296	CAGGUCAG GGCTACCTACAACGA GGGCGCAG	1829

520	CCCCCCCU G CCCUCCGU	1297	ACCGAGGG GGCTAGCTACAACCA AGCCCCCG	1830

527	UGCCCUCC G UCUUCUAC	1298	GTAGAAGA GCCTAGCTACAACGA CCAGGGCA	1831

534	GCUCUUCU A CAUGCUCC	69	CCACCATG GGCTAGCTACAACCA ACAAGACC	1832

536	UCUCCUAC A UGCUGCUC	601	CACCACCA CCCTACCTACAACGA GTAGAAGA	1833

538	UUCUACAU G CUGGUGUG	1299	CACACCAG CGCTAGCTACAACGA ATGTAGAA	1834

542	ACAUGCUG G UCUCUCCC	1300	CCCACACA CGCTACCTACAACCA CACCATGT	1835

544	AUGCUGGU G UGUGGCCU	1301	ACGCCACA GCCTACCTACAACGA ACCAGCAT	1836

546	CCUGGUGU G UCCCCUGA	1302	TCACGCCA GGCTAGCTACAACCA ACACCAGC	1837

549	GGUCUGUG G CCUGACGG	1303	CCGTCAGC GCCTAGCTACAACGA CACACACC	1838

554	CUGCCCUG A CCGUCACC	1679	GGTGACCC GGCTAGCTACAACGA CAGGOCAC	1839

557	CCCUCACC G UCACOGAC	1304	CTCCCTGA CCCTACCTACAACGA CCTCACCC	1840

560	UCACCCUC A CCCACUUC	605	CAACTCCC CGCTAGCTACAACCA GACCGTCA	1841

564	GGUCACCC A CUUCCUGG	1680	CCACCAAC CCCTACCTACAACCA CCCTCACC	1842

568	ACCGACUU G CUCCCCAA	1305	TTCCCCAC GCCTAGCTACAACCA AAGTCGCT	1843

573	CUUCCUCC G CAACUCCC	1306	GGCACTTC CCCTAGCTACAACCA CCACCAAG	1844

577	CUCCCCAA G UCCCUCCU	1307	ACGACGCA CCCTACCTACAACGA TTCCCCAC	1845

579	CCGCAAGU G CCUCCUAA	1308	TTACCACG CCCTACCTACAACCA ACTTGCCC	1846

588	CCUCCUAA G CCCGGUCC	1309	CCACCCCC CCCTAGCTACAACCA TTACCAGG	1847

593	UAAGCCCC G UCCUCCUG	1310	CACCACCA GGCTACCTACAACGA CGGGCTTA	1848

596	CCCCCCUG G UGCUGCCU	1311	AGCCACCA CCCTAGCTACAACCA CACCCGGC	1849

598	CCGGUGGU G CUCCCUGC	1312	CCACCCAG GGCTACCTACAACGA ACCACCCC	1850

602	UCCUCCUC G CUGCCUAC	1313	GTAGCCAC CCCTAGCTACAACCA CACCACCA	1851

605	UGCUGGCU G CCUACCCU	1314	ACCCTAGG GGCTACCTACAACGA AGCCACCA	1852

609	CCCUCCCU A CGCUCACA	74	TCTCACCC CCCTAGCTACAACCA ACCCACCC	1853

611	CUGCCUAC G CUCAGAAC	1315	CTTCTGAG CCCTACCTACAACGA GTACCCAG	1854

618	CGCUCAGA A CCCCACUC	1681	CACTCCCG CCCTAGCTACAACCA TCTGAGCC	1855

624	CAACCCGA G UCUCCCCC	1316	CCCCCACA CCCTACCTACAACCA TCCCCTTC	1856

628	CCCACUCU G CGCGUGCU	1317	ACCACCCG CCCTACCTACAACGA AGACTCCC	1857

632	GUCUGCGC G UCCUUCCG	1318	CCCAACCA CGCTAGCTACAACCA CCCCACAC	1858

634	CUCCCCCU G CUUGCCCC	1319	GGCGCAAG CCCTAGCTACAACGA ACCCCCAG	1859

638	CGGUGCUU G CCCCCCCA	1320	TCCCCCCG GGCTACCTACAACCA AACCACCC	1860

640	CUCCUUCC G CCCGCAUU	1321	AATCCGGG CCCTAGCTACAACGA CCAACCAC	1861

644	UUGCCCCC G CAUUCCAC	1322	GTCCAATC GCCTACCTACAACCA GGCCGCAA	1862

646	GCGCCCGC A UUGGACAA	627	TTGTCCAA GGCTAGCTACAACGA GCGGGCGC	1863

651	CGCAUUGG A CAACUCGU	1682	ACGAGTTG GGCTAGCTACAACGA CCAATGCG	1864

654	AUUGGACA A CUCGUUGU	1683	ACAACGAG GGCTAGCTACAACGA TGTCCAAT	1865

658	GACAACUC G UUGUGCCA	1323	TGGCACAA GGCTAGCTACAACGA GAGTTGTC	1866

661	AACUCGUU G UGCCAAGC	1324	GCTTGGCA GGCTAGCTACAACGA AACGAGTT	1867

663	CUCGUUGU G CCAAGCCU	1325	AGGCTTGG GGCTAGCTACAACGA ACAACGAG	1868

668	UGUGCCAA G CCUUCGCC	1326	GGCGAAGG GGCTAGCTACAACGA TTGGCACA	1869

674	AAGCCUUC G CCUUCUUC	1327	GAAGAAGG GGCTAGCTACAACGA GAAGGCTT	1870

683	CCUUCUUC A UGUCCUUC	637	GAAGGACA GGCTAGCTACAACGA GAAGAAGG	1871

685	UUCUUCAU G UCCUUCUU	1328	AAGAAGGA CGCTAGCTACAACGA ATGAAGAA	1872

697	UUCUUUGG G CUCUCCUC	1329	GAGGAGAG GGCTAGCTACAACGA CCAAAGAA	1873

707	UCUCCUCG A CACUGCAA	1684	TTGCAGTG GGCTAGCTACAACGA CUAGGAGA	1874

709	UCCUCGAC A CUGCAACU	645	AGTTGCAG GGCTAGCTACAACGA GTCGAGGA	1875

712	UCGACACU G CAACUCCU	1330	AGGAGTTG GGCTAGCTACAACGA AGTGTCGA	1876

715	ACACUGCA A CUCCUGGC	1685	GCCAGGAG GGCTAGCTACAACGA TGCAGTGT	1877

722	AACUCCUG G CCAUGGCA	1331	TGCCATGG GGCTAGCTACAACGA CAGGAGTT	1878

725	UCCUGGCC A UGGCACUG	652	CAGTGCCA GGCTAGCTACAACGA GGCCAGGA	1879

728	UGGCCAUG G CACUGGAG	1332	CTCCAGTG GGCTAGCTACAACGA CATGGCCA	1880

730	GCCAUGGC A CUGGAGUG	653	CACTCCAG GGCTAGCTACAACGA GCCATGGC	1881

736	GCACUGGA G UGCUGGCU	1333	AGCCAGCA GGCTAGCTACAACGA TCCAGTGC	1882

738	ACUGGAGU G CUGGCUCU	1334	AGAGCCAG GGCTAGCTACAACGA ACTCCAGT	1883

742	GAGUCCUG G CUCUCCCU	1335	AGGGAGAG GCCTAGCTACAACGA CAGCACTC	1884

754	UCCCUAGG G CACCCUUU	1336	AAAGGGTG GGCTAGCTACAACGA CCTAGGGA	1885

756	CCUAGGGC A CCCUUUCU	661	AGAAAGGG GGCTAGCTACAACGA GCCCTAGG	1886

768	UUUCUUCU A CCGACGGC	104	GCCGTCGG GGCTAGCTACAACGA AGAACAAA	1887

772	UUCUACCG A CGGCACAU	1686	ATGTGCCG GGCTAGCTACAACGA CGGTAGAA	1888

775	UACCGACG G CACAUCAC	1337	GTGATGTG GGCTAGCTACAACGA CGTCGGTA	1889

777	CCGACGGC A CAUCACCC	668	GGGTGATG GGCTAGCTACAACGA GCCGTCGG	1890

779	GACGGCAC A UCACCCUG	669	CAGGGTGA GGCTAGCTACAACGA GTCCCGTC	1891

782	CGCACAUC A CCCUGCGC	670	GCGCAGGG GGCTAGCTACAACGA GATGTGCC	1892

787	AUCACCCU G CGCCUGGG	1338	CCCAGGCG GCCTAGCTACAACGA AGGGTGAT	1893

789	CACCCUGC G CCUGGGCG	1339	CGCCCAGG GGCTACCTACAACGA GCAGCGTG	1894

795	GCGCCUGG G CGCACUGG	1340	CCAGTCCG GGCTAGCTACAACGA CCAGGCGC	1895

797	GCCUCGGC G CACUGGUG	1341	CACCAGTG GCCTAGCTACAACGA GCCCAGGC	1896

799	CUGGGCGC A CUGGUGGC	676	GCCACCAG GGCTAGCTACAACGA GCGCCCAG	1897

803	CCCCACUG G UGGCCCCC	1342	CGGGGCCA CGCTAGCTACAACGA CAGTGCGC	1898

806	CACUGGUG G CCCCGGUG	1343	CACCGGGG GGCTAGCTACAACGA CACCAGTG	1899

812	UCGCCCCG G UGGUGAGC	1344	GCTCACCA GGCTAGCTACAACGA CGGGGCCA	1900

815	CCCCGGUG G UGAGCGCC	1345	GGCGCTCA GGCTAGCTACAACGA CACCGGGC	1901

819	GGUGGUGA G CGCCUUCU	1346	AGAAGGCG CGCTAGCTACAACGA TCACCACC	1902

821	UGGUGAGC G CCUUCUCC	1347	GGAGAAGG GGCTAGCTACAACGA GCTCACCA	1903

833	UCUCCCUG G CUUUCUGC	1348	GCACAAAG GGCTAGCTACAACCA CAGGGAGA	1904

840	GGCUUUCU G CGCGCUAC	1349	GTAGCGCG CGCTAGCTACAACGA AGAAAGCC	1905

842	CUUUCUGC G CCCUACCU	1350	ACCTACCG GGCTAGCTACAACGA GCAGAAAG	1906

844	UUCUCCGC G CUACCUUU	1351	AAAGGTAG GCCTAGCTACAACCA CCGCAGAA	1907

847	UGCCCCCU A CCUUUCAU	112	ATGAAAGG GGCTACCTACAACGA AGCGCCCA	1908

854	UACCUUUC A UGCCCUUC	692	CAACCCCA GGCTAGCTACAACCA CAAAGGTA	1909

858	UUUCAUCG G CUUCGGGA	1352	TCCCGAAG GGCTAGCTACAACGA CCATGAAA	1910

868	UUCGGGAA G UUCCUGCA	1353	TGCACGAA GGCTAGCTACAACGA TTCCCGAA	1911

872	GGAAGUUC G UGCAGUAC	1354	GTACTGCA GGCTAGCTACAACGA GAACTTCC	1912

874	AAGUUCGU G CACUACUG	1355	CAGTACTG GGCTAGCTACAACGA ACGAACTT	1913

877	UUCGUGCA G UACUGCCC	1356	GGGCAGTA GGCTAGCTACAACGA TGCACGAA	1914

879	CGUGCAGU A CUGCCCCG	120	CGGGGCAG GGCTAGCTACAACGA ACTGCACG	1915

882	GCAGUACU G CCCCGGCA	1357	TGCCGGGG GGCTAGCTACAACGA AGTACTGC	1916

888	CUGCCCCG G CACCUGGU	1358	ACCAGGTG GGCTAGCTACAACGA CGGGGCAG	1917

890	GCCCCGGC A CCUGGUGC	699	GCACCAGG GGCTAGCTACAACGA GCCGGGGC	1918

895	GGCACCUG G UGCUUUAU	1359	ATAAAGCA GGCTAGCTACAACGA CAGGTGCC	1919

897	CACCUGGU G CUUUAUCC	1360	GGATAAAG GGCTAGCTACAACGA ACCAGGTG	1920

902	GGUGCUUU A UCCAGAUG	123	CATCTGGA GGCTAGCTACAACGA AAAGCACC	1921

908	UUAUCCAG A UGGUCCAC	1687	GTGGACCA GGCTAGCTACAACGA CTGGATAA	1922

911	UCCAGAUG G UCCACGAG	1361	CTCGTGGA GGCTAGCTACAACGA CATCTGGA	1923

915	GAUGGUCC A CGAGGAGG	706	CCTCCTCG GGCTAGCTACAACGA GGACCATC	1924

924	CGAGGAGG G CUCGCUGU	1362	ACAGCGAG GGCTAGCTACAACGA CCTCCTCG	1925

928	GAGGGCUC G CUGUCGGU	1363	ACCUACAG GGCTAGCTACAACGA GAGCCCTC	1926

931	GGCUCGCU G UCGGUGCU	1364	AGCACCGA GGCTAGCTACAACGA AGCGAGCC	1927

935	CGCUGUCG G UGCUGGGG	1365	CCCCAGCA GGCTAGCTACAACGA CGACAGCG	1928

937	CUGUCGGU G CUGGGGUA	1366	TACCCCAG GGCTAGCTACAACGA ACCGACAG	1929

943	GUGCUGGG G UACUCUGU	1367	ACAGAUTA GGCTAGCTACAACGA CCCAGCAC	1930

945	GCUGGGGU A CUCUGUGC	128	GCACAGAG GGCTAGCTACAACGA ACCCCAGC	1931

950	GGUACUCU G UGCUCUAC	1368	GTAGAGCA GGCTAGCTACAACGA AGAGTACC	1932

952	UACUCUGU G CUCUACUC	1369	GAGTAGAG GGCTAGCTACAACGA ACAGAUTA	1933

957	UGUGCUCU A CUCCAGCC	131	GGCTGGAG GGCTAGCTACAACGA AGAGCACA	1934

963	CUACUCCA G CCUCAUGG	1370	CCATGAGG GGCTAGCTACAACGA TGGAGTAG	1935

968	CCAGCCUC A UGGCGCUG	719	CAUCUCCA GGCTAGCTACAACGA GAGGCTGG	1936

971	GCCUCAUG G CGCUGCUG	1371	CAGCAGCG GGCTAGCTACAACGA CATGAGGC	1937

973	CUCAUGGC G CUGCUGGU	1372	ACCACCAG GGCTAGCTACAACGA GCCATGAG	1938

976	AUGGCGCU G CUGGUCCU	1373	AGGACCAG GGCTAGCTACAACGA AGCGCCAT	1939

980	CGCUGCUG G UCCUCGCC	1374	GGCGAGGA GGCTAGCTACAACGA CAGCAGCG	1940

986	UGGUCCUC G CCACCGUG	1375	CACGGTGG GGCTAGCTACAACGA GAGGACCA	1941

989	UCCUCGCC A CCGUGCUG	725	CAGCACGG GGCTAGCTACAACGA GGCGAGGA	1942

992	UCGCCACC G UGCUGUGC	1376	GCACAGCA GGCTAGCTACAACGA GGTGGCGA	1943

994	GCCACCGU G CUGUGCAA	1377	TTGCACAG GGCTAGCTACAACGA ACGGTGGC	1944

997	ACCGUGCU G UGCAACCU	1378	AGGTTGCA GGCTAGCTACAACGA AGCACGGT	1945

999	CGUGCUGU G CAACCUCG	1379	CGAGGTTG GGCTAGCTACAACGA ACAGCACG	1946

1002	GCUGUGCA A CCUCGGCG	1688	CGCCGAGG GGCTAGCTACAACGA TGCACAGC	1947

1008	CAACCUCG G CGCCAUGC	1380	GCATGGCG GGCTAGCTACAACGA CGAGGTTG	1948

1010	ACCUCGGC G CCAUGCGC	1381	GCGCATGG GGCTAGCTACAACGA GCCGAGGT	1949

1013	UCGGCGCC A UGCGCAAC	732	GTTGCGCA GGCTAGCTACAACGA GGCGCCGA	1950

1015	GGCGCCAU G CGCAACCU	1382	AGGTTGCG GGCTAGCTACAACGA ATGGCGCC	1951

1017	CGCCAUGC G CAACCUCU	1383	AGAGGTTG GGCTAGCTACAACGA GCATGGCG	1952

1020	CAUGCGCA A CCUCUAUG	1689	CATAGAGG GGCTAGCTACAACGA TGCGCATG	1953

1026	CAACCUCU A UGCGAUGC	138	GCATCGCA GGCTAGCTACAACGA AGAGGTTG	1954

1028	ACCUCUAU G CGAUGCAC	1384	GTGCATCG GGCTAGCTACAACGA ATAGAGGT	1955

1031	UCUAUGCG A UGCACCGG	1690	CCGGTGCA GGCTAGCTACAACGA CGCATAGA	1956

1033	UAUGCGAU G CACCGGCG	1385	CGCCGGTG GGCTAGCTACAACGA ATCGCATA	1957

1035	UGCGAUGC A CCGGCGGC	737	GCCGCCGG GGCTAGCTACAACGA GCATCGCA	1958

1039	AUGCACCG G CGGCUGCA	1386	TGCAGCCG GGCTAGCTACAACGA CGGTGCAT	1959

1042	CACCGGCG G CUGCAGCG	1387	CGCTGCAG GGCTAGCTACAACGA CGCCGGTG	1960

1045	CGGCGGCU G CAGCGGCA	1388	TGCCGCTG GGCTAGCTACAACGA AGCCGCCG	1961

1048	CGGCUGCA G CGGCACCC	1389	GGGTGCCG GGCTAGCTACAACGA TGCAGCCG	1962

1051	CUGCAGCG G CACCCGCG	1390	CGCGGGTG GGCTAGCTACAACGA CGCTGCAG	1963

1053	GCAGCGGC A CCCGCGCU	741	AGCGCGGG GGCTAGCTACAACGA GCCGCTGC	1964

1057	CGGCACCC G CGCUCCUG	1391	CACCAGCG GGCTAGCTACAACGA GGGTGCCG	1965

1059	GCACCCGC G CUCCUCCA	1392	TGCAGGAG GGCTACCTACAACGA GCGGGTGC	1966

1065	GCGCUCCU G CACCAGGG	1393	CCCTCCTG GGCTAGCTACAACGA AGGAGCGC	1967

1067	GCUCCUGC A CCAGGCAC	747	GTCCCTGG GGCTAGCTACAACCA GCAGGAGC	1968

1074	CACCAGGG A CUGUGCCG	1691	CGGCACAG GGCTAGCTACAACGA CCCTGGTG	1969

1077	CAGGGACU G UGCCGAGC	1394	GCTCGGCA GGCTAGCTACAACGA AGTCCCTG	1970

1079	GGGACUGU G CCGAGCCG	1395	CGGCTCGG GGCTAGCTACAACGA ACAGTCCC	1971

1084	UGUGCCCA G CCGCGCCC	1396	GCGCGCGG CGCTAGCTACAACGA TCGGCACA	1972

1087	GCCGAGCC G CGCGCGGA	1397	TCCGCGCG GGCTAGCTACAACGA GGCTCGGC	1973

1089	CGAGCCGC G CGCGGACG	1398	CGTCCGCG GGCTAGCTACAACGA GCGGCTCG	1974

1091	AGCCGCGC G CGGACGGG	1399	CCCGTCCG GGCTAGCTACAACGA GCGCGGCT	1975

1095	GCGCGCGG A CGJGAGGG	1692	CCCTCCCG GGCTAGCTACAACGA CCGCCCGC	1976

1106	GGAGCGAA G CGUCCCCU	1400	AGGGGACG GGCTAGCTACAACGA TTCCCTCC	1977

1108	AGGGAAGC G UCCCCUCA	1401	TGAGGGGA GGCTAGCTACAACGA CCTTCCCT	1978

1117	UCCCCUCA G CCCCUCGA	1402	TCCAGGGG GCCTAGCTACAACGA TGAGGGGA	1979

1129	CUGGAGGA G CUCCAUCA	1403	TGATCCAG GGCTACCTACAACGA TCCTCCAG	1980

1134	GCAGCUGG A UCACCUCC	1693	GGAGGTGA GGCTAGCTACAACGA CCAGCTCC	1981

1137	CCUGGAUC A CCUCCUGC	763	GCAGGAGG CCCTAGCTACAACCA GATCCAGC	1982

1144	CACCUCCU G CUGCUGGC	1404	GCCAGCAG GCCTAGCTACAACCA AGCAGGTG	1983

1147	CUCCUCCU G CUGCCGCU	1405	AGCCCCAG GGCTACCTACAACGA AGCACGAG	1984

1151	UGCUGCUC G CCCUGAUG	1406	CATCAGCC GCCTACCTACAACGA CAGCAGCA	1985

1153	CUGCUGGC G CUGAUGAC	1407	GTCATCAG CGCTAGCTACAACGA GCCAGCAG	1986

1157	UGGCCCUG A UGACCOUG	1694	CACGGTCA GGCTAGCTACAACGA CACCGCCA	1987

1160	CCCUGAUC A CCGUGCUC	1695	CAGCACCC GGCTAGCTACAACGA CATCAGCG	1988

1163	UCAUGACC G UGCUCUUC	1408	GAAGAGCA GCCTACCTACAACGA CGTCATCA	1989

1165	AUGACCCU G CUCUUCAC	1409	GTCAACAG CGCTAGCTACAACGA ACGGTCAT	1990

1172	UGCUCUUC A CUAUGUGU	774	ACACATAC GGCTAGCTACAACGA GAAGAGCA	1991

1175	UCUUCACU A UCUGUUCU	147	AGAACACA GCCTAGCTACAACCA AGTCAACA	1992

1177	UUCACUAU G UGUUCUCU	1410	AGAGAACA CGCTACCTACAACGA ATACTCAA	1993

1179	CACUAUGU G UUCUCUGC	1411	GCAGAGAA CCCTACCTACAACCA ACATAGTG	1994

1186	UCUUCUCU G CCCCUAAU	1412	ATTACGGC CGCTACCTACAACGA ACACAACA	1995

1190	CUCUGCCC G UAAUUUAU	1413	ATAAATTA GCCTACCTACAACGA GGGCAGAC	1996

1193	UCCCCGUA A UUUAUCCC	1696	CCCATAAA GGCTAGCTACAACCA TACGGGCA	1997

1197	CGUAAUUU A UCCCCCUU	154	AACCCCCA CGCTAGCTACAACGA AAATTACG	1998

1200	AAUUUAUC G CGCUUACU	1414	AGTAACCG GGCTAGCTACAACCA GATAAATT	1999

1202	UUUAUCGC G CUUACUAU	1415	ATACTAAG CGCTAGCTACAACGA GCGATAAA	2000

1206	UCGCGCUU A CUAUGCAC	157	CTCCATAG GGCTAGCTACAACCA AACCGCGA	2001

1209	CCCUUACU A UCGAGCAU	158	ATCCTCCA CGCTACCTACAACGA AGTAAGCC	2002

1214	ACUAUGGA G CAUUUAAG	1416	CTTAAATG GCCTACCTACAACCA TCCATAGT	2003

1216	UAUCCACC A UUUAAGGA	782	TCCTTAAA GGCTACCTACAACGA CCTCCATA	2004

1224	AUUUAAGC A UGUCAACG	1697	CCTTCACA CGCTACCTACAACGA CCTTAAAT	2005

1226	UUAACGAU G UCAACGAG	1417	CTCCTTGA GGCTAGCTACAACCA ATCCTTAA	2006

1239	CGAGAAAA A CAGGACCU	1698	AGGTCCTG GGCTAGCTACAACGA TTTTCTCC	2007

1244	AAAACACG A CCUCUCAA	1699	TTCAGAGG GGCTACCTACAACCA CCTCTTTT	2008

1256	CUCAACAA G CAGAAGAC	1418	CTCTTCTC CCCTACCTACAACCA TTCTTCAC	2009

1263	ACCAGAAG A CCUCCCAC	1700	CTCGCACG CGCTACCTACAACCA CTTCTGCT	2010

1271	ACCUCCCA G CCUUCCGA	1419	TCCCAACC CCCTACCTACAACGA TCCCACCT	2011

1276	CCACCCUU G CCAUUUCU	1420	ACAAATCC CCCTACCTACAACCA AAGGCTCG	2012

1279	CCCUUCCC A UUUCUAUC	1701	CATACAAA GGCTACCTACAACCA CCCAACCC	2013

1285	CCAUUUCU A UCUGUGAU	169	ATCACACA CCCTACCTACAACGA AGAAATCC	2014

1289	UUCUAUCU G UCAUUUCA	1421	TCAAATCA GCCTACCTACAACCA ACATACAA	2015

1292	UAUCUGUG A UUUCAAUU	1702	AATTGAAA GGCTAGCTACAACGA CACAGATA	2016

1298	UGAUUUCA A UHOUGGAC	1703	GTCCACAA GGCTAGCTACAACGA TGAAATCA	2017

1301	UUUCAAUU G UGGACCCU	1422	AGGGTCCA GGCTAGCTACAACGA AATTGAAA	2018

1305	AAUUGUGG A CCCUUGGA	1704	TCCAAGGG GGCTAGCTACAACGA CCACAATT	2019

1313	ACCCUUGG A UUUUUAUC	1705	GATAAAAA GGCTAGCTACAACGA CCAAGGGT	2020

1319	GGAUUUUU A UCAUUUUC	180	GAAAATGA GGCTAGCTACAACGA AAAAATCC	2021

1322	UUUUUAUC A UUUUCAGA	800	TCTGAAAA GGCTAGCTACAACGA GATAAAAA	2022

1330	AUUUUCAG A UCUCCAGU	1706	ACTUGAGA GGCTAGCTACAACGA CTGAAAAT	2023

1337	GAUCUCCA G UAUUUCGG	1423	CCGAAATA GGCTAGCTACAACGA TGGAGATC	2024

1339	UCUCCAGU A UUUCGGAU	188	ATCCGAAA GGCTAGCTACAACGA ACTGGAGA	2025

1346	UAUUUCGG A UAUUUUUU	1707	AAAAAATA GGCTAGCTACAACGA CCGAAATA	2026

1348	UUUCGGAU A UUUUUUCA	192	TGAAAAAA GGCTAGCTACAACGA ATCCGAAA	2027

1356	AUUUUUUC A CAAGAUUU	805	AAATCTTG GGCTAGCTACAACGA GAAAAAAT	2028

1361	UUCACAAG A UUUUCAUU	1708	AATGAAAA GGCTAGCTACAACGA CTTGTGAA	2029

1367	AGAUUUUC A UUAGACCU	807	AGGTCTAA GGCTAGCTACAACGA GAAAATCT	2030

1372	UUCAUUAG A CCUCUUAG	1709	CTAAGAGG GGCTAGCTACAACGA CTAATGAA	2031

1381	CCUCUUAG G UACAGGAG	1424	CTCCTGTA GGCTAGCTACAACGA CTAAGAGG	2032

1383	UCUUAGGU A CAGGAGCC	208	GGCTCCTG GGCTAGCTACAACGA ACCTAAGA	2033

1389	GUACAGGA G CCGGUGCA	1425	TGCACCGG GGCTAGCTACAACGA TCCTGTAC	2034

1393	AGGAGCCG G UGCAGCAA	1426	TTGCTGCA GGCTAGCTACAACGA CGGCTCCT	2035

1395	GAGCCGGU G CAGCAAUU	1427	AATTGCTG GGCTAGCTACAACGA ACCGGCTC	2036

1398	CCGGUGCA G CAAUUCCA	1428	TGGAATTG GGCTAGCTACAACGA TGCACCGG	2037

1401	GUGCAGCA A UUCCACUA	1710	TAGTGGAA GGCTAGCTACAACGA TGCTGCAC	2038

1406	GCAAUUCC A CUAACAUG	816	CATGTTAG GGCTAGCTACAACGA GGAATTGC	2039

1410	UUCCACUA A CAUGGAAU	1711	ATTCCATG GGCTAGCTACAACGA TAGTGGAA	2040

1412	CCACUAAC A UGGAAUCC	818	GGATTCCA GGCTAGCTACAACGA GTTAGTGG	2041

1417	AACAUGGA A UCCAGUCU	1712	AGACTGGA GGCTAGCTACAACGA TCCATGTT	2042

1422	GGAAUCCA G UCUGUGAC	1429	GTCACAGA GGCTAGCTACAACGA TGGATTCC	2043

1426	UCCAGUCU G UGACAGUG	1430	CACTGTCA GGCTAGCTACAACGA AGACTGGA	2044

1429	AGUCUGUG A CAGUGUUU	1713	AAACACTG GGCTAGCTACAACGA CACAGACT	2045

1432	CUGUGACA G UGUUUUUC	1431	GAAAAACA GGCTAGCTACAACGA TGTCACAG	2046

1434	GUGACAGU G UUUUUCAC	1432	GTGAAAAA GGCTAGCTACAACGA ACTGTCAC	2047

1441	UGUUUUUC A CUCUGUGG	823	CCACAGAG GGCTAGCTACAACGA GAAAAACA	2048

1446	UUCACUCU G UGGUAAGC	1433	GCTTACCA GGCTAGCTACAACGA AGAGTGAA	2049

1449	ACUCUGUG G UAAGCUGA	1434	TCAGCTTA GGCTAGCTACAACGA CACAGAGT	2050

1453	UGUGGUAA G CUGAGGAA	1435	TTCCTCAG GGCTAGCTACAACGA TTACCACA	2051

1461	GCUGAGGA A UAUGUCAC	1714	GTGACATA GGCTAGCTACAACGA TCCTCAGC	2052

1463	UGAGGAAU A UGUCACAU	221	ATGTGACA GGCTAGCTACAACGA ATTCCTCA	2053

1465	AGGAAUAU G UCACAUUU	1436	AAATGTGA GGCTAGCTACAACGA ATATTCCT	2054

1468	AAUAUGUC A CAUUUUCA	827	TGAAAATG GGCTAGCTACAACGA GACATATT	2055

1470	UAUGUCAC A UUUUCAGU	828	ACTGAAAA GGCTAGCTACAACGA GTGACATA	2056

1477	CAUUUUCA G UCAAAGAA	1437	TTCTTTGA GGCTAGCTACAACGA TGAAAATG	2057

TABLE VII


Human PTGDR Amberzyme and Substrate Sequence

		Seq		Seq
Pos	Substrate	ID	Amberzyme	ID

9	GAAUUCUG G CUAUUUUC	1207	GAAAAUAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGAAUUC	2247

23	UUCCUCCU G CCGUUCCG	1208	CGGAACGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGAGGAA	2248

28	CUCCUGCC G UUCCGACU	1209	AGUCGGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGCAGGAG	2249

31	GCCGUUCC G ACUCGGCA	2058	UGCCGAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGAACGGC	2250

36	UCCGACUC G GCACCAGA	2059	UCUGGUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAGUCGGA	2251

37	CCGACUCG G CACCAGAG	1210	CUCUGGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAGUCGG	2252

43	CGGCACCA G AGUCUGUC	2060	GACAGACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGUGCCG	2253

45	GCACCAGA G UCUGUCUC	1211	GAGACAGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUGGUGC	2254

49	CAGAGUCU G UCUCUACU	1212	AGUAGAGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGACUCUG	2255

58	UCUCUACU G AGAACGCA	2061	UGCGUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUAGAGA	2256

60	UCUACUGA G AACGCAGC	2062	GCUGCGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCAGUAGA	2257

64	CUGAGAAC G CAGCGCGU	1213	ACGCGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUUCUCAG	2258

67	AGAACGCA G CGCGUCAG	1214	CUGACGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCGUUCU	2259

69	AACGCAGC G CGUCAGGG	1215	CCCUGACG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCUGCGUU	2260

71	CGCAGCGC G UCAGGGCC	1216	GGCCCUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCGCUGCG	2261

75	GCGCGUCA G GGCCGAGC	2063	GCUCGGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGACGCGC	2262

76	CGCGUCAG G GCCGAGCU	2064	AGCUCGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGACGCG	2263

77	GCGUCAGG G CCGAGCUC	1217	GAGCUCGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUGACGC	2264

80	UCAGGGCC G AGCUCUUC	2065	GAAGAGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGCCCUGA	2265

82	AGGGCCGA G CUCUUCAC	1218	GUGAAGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGGCCCU	2266

92	UCUUCACU G GCCUGCUC	2066	GAGCAGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUGAAGA	2267

93	CUUCACUG G CCUGCUCC	1219	GGAGCAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGUGAAG	2268

97	ACUGGCCU G CUCCGCGC	1220	GCGCGGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGCCAGU	2269

102	CCUGCUCC G CGCUCUUC	1221	GAAGAGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGAGCAGG	2270

104	UGCUCCGC G CUCUUCAA	1222	UUGAAGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCGGAGCA	2271

114	UCUUCAAU G CCAGCGCC	1223	GGCGCUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUUGAAGA	2272

118	CAAUGCCA G CGCCAGGC	1224	GCCUGGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGCAUUG	2273

120	AUGCCAGC G CCAGGCGC	1225	GCGCCUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCUGGCAU	2274

124	CAGCGCCA G GCGCUCAC	2067	GUGAGCGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGCGCUG	2275

125	AGCGCCAG G CGCUCACC	1226	GGUGAGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGGCGCU	2276

127	CGCCAGGC G CUCACCCU	1227	AGGGUGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCCUGGCG	2277

136	CUCACCCU G CAGAGCGU	1228	ACGCUCUG GCAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGCUGAG	2278

139	ACCCUGCA G AGCGUCCC	2068	GGGACGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCAGGGU	2279

141	CCUGCAGA G CGUCCCGC	1229	GCGGGACG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUGCAGG	2280

143	UGCAGAGC G UCCCGCCU	1230	AGGCGGGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCUCUGCA	2281

148	AGCGUCCC G CCUCUCAA	1231	UUGAGAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGACGCU	2282

158	CUCUCAAA G AGGGGUGU	2069	ACACCCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUUGAGAG	2283

160	CUCAAAGA G GGGUGUGA	2070	UCACACCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCUUUGAG	2284

161	UCAAAGAG G GGUGUGAC	2071	GUCACACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCUUUGA	2285

162	CAAAGAGG G GUGUGACC	2072	UGUCACAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUCUUUG	2286

163	AAAGAGGG G UGUGACCC	1232	GGGUCACA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCUCUUU	2287

165	AGAGGGGU G UGACCCGC	1233	GCGGGUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCCCUCU	2288

167	AGGGGUGU U ACCCGCGA	2073	UCGCGGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACACCCCU	2289

172	UGUGACCC C CGAGUUUA	1234	UAAACUCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUGUCACA	2290

174	UGACCCGC C AGUUUAGA	2074	UCUAAACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCGGGUCA	2291

176	ACCCGCGA G UUUAGAUA	1235	UAUCUAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGCGGGU	2292

181	CGAGUUUA G AUAGGAGG	2075	CCUCCUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAAACUCG	2293

185	UUUAGAUA C GAGGUUCC	2076	GGAACCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAUCUAAA	2294

186	UUAGAUAG G AGGUUCCU	2077	AGGAACCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUAUCUAA	2295

188	AGAUAGGA G GUUCCUGC	2078	GCAGGAAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUAUCU	2296

189	GAUAGGAG G UUCCUGCC	1236	GGCAGGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCUAUC	2297

195	AGGUUCCU G CCGUGGGG	1237	CCCCACGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGAACCU	2298

198	UUCCUGCC G UGGGGAAC	1238	GUUCCCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGCAGGAA	2299

200	CCUGCCGU G GGGAACAC	2079	GUGUUCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACGGCAGG	2300

201	CUGCCGUG G GGAACACC	2080	GGUGUUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACGGCAG	2301

202	UGCCGUGG C GAACACCC	2081	CCGUGUUC GCAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCACGGCA	2302

203	GCCGUGGG G AACACCCC	2082	GGGGUGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCACGGC	2303

212	AACACCCC G CCGCCCUC	1239	GAGGGCGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGCUCUU	2304

215	ACCCCGCC G CCCUCGCA	1240	UCCGAGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGCGGGGU	2305

221	CCGCCCUC G GAGCUUUU	2083	AAAAGCUC GGAGCAAACUCC CU UCAAGGACAUCGUCCGGG GAGGUCUG	2306

222	CGCCCUCG G AGCUUUUU	2084	AAAAAGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAGGGCG	2307

224	CCCUCGGA G CUUUUUCU	1241	AGAAAAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCGAGGG	2308

233	CUUUUUCU G UGGCGCAG	1242	CUGCGCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAAAAAG	2309

235	UUUUCUGU G GCGCAGCU	2085	AGCUGCGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAGAAPA	2310

236	UUUCUGUG G CGCAGCUU	1243	AAGCUGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACAGAAA	2311

238	UCUGUGGC C CAGCUUCU	1244	AGAAGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCCACAGA	2312

241	GUGGCGCA G CUUCUCCG	1245	CGGACAAG GGAGGAAACUCC CU UCAAGCACAUCGUCCGGG UGCGCCAC	2313

249	GCUUCUCC G CCCGAGCC	1246	GGCUCGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGAGAAGC	2314

253	CUCCGCCC C AGCCGCGC	2086	GCGCGGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGCGGAG	2315

255	CCGCCCGA G CCGCGCGC	1247	GCGCGCGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGGGCGG	2316

258	CCCGAGCC C CGCGCGGA	1248	UCCGCGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGCUCGGG	2317

260	CGAGCCCC C CCCGGAGC	1249	GCUCCGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCGGCUCG	2318

262	AGCCGCGC G CGGAGCUG	1250	CAGCUCCG CGAGCAAACUCC CU UCAACCACAUCGUCCCGG CCGCGGCU	2319

264	CCGCGCGC C CAGCUCCC	2087	GCCACCUC CCACGAAACUCC CU UCAACGACAUCGUCCCCG CCGCCCCG	2320

265	CGCGCCCG C AGCUCCCG	2088	CGCCAGCU GGAGCAAACUCC CU UCAAGCACAUCGUCCGGC CCCGCCCC	2321

267	CCCCCCGA G CUGCCCGC	1251	CCCGGCAG GCACGAAACUCC CU UCAACGACAUCGUCCGGG UCCCCCCG	2322

270	GCCCACCU G CCGGGGGC	1252	CCCCCCCC GCACGAAACUCC CU UCAAGGACAUCCUCCCGG AGCUCCCC	2323

273	CACCUGCC C CGGGCUCC	2089	CGAGCCCC GCACCAAACUCC CU UCAAGCACAUCCUCCGGC GCCAGCUC	2324

274	AGCUGCCG G GCGCUCCU	2090	AGCACCCC GCAGGAAACUCC CU UCAAGCACAUCGUCCGGG CCCCACCU	2325

275	CCUGCCGC C CCCUCCUU	2091	AAGCACCC CGACGAAACUCC CU UCAAGGACAUCCUCCGGG CCCGCAGC	2326

276	CUGCCCGG C GCUCCUUA	2092	UAACGACC GGAGCAAACUCC CU UCAACGACAUCGUCCCGC CCCGGCAG	2327

277	UGCCGGCC C CUCCUUAG	1253	CUAACGAC GCAGCAAACUCC CU UCAAGCACAUCGUCCGGG CCCCCCCA	2328

285	GCUCCUUA G CACCCGCG	1254	CCCCCCUC CGAGGAAACUCC CU UCAACCACAUCGUCCGGG UAACGAGC	2329

291	UAGCACCC G GCCGCCGC	2093	CCGGCCCC CCACCAAACUCC CU UCAACGACAUCCUCCCGG GCGUGCUA	2330

292	ACCACCCG C CCGCCCCG	2094	CCCGGCCC GCACCAAACUCC CU UCAAGCACAUCCUCCGGC CCGCUGCU	2331

293	GCACCCGC C CGCCCGCG	1255	CCCCCGCG CGACGAAACUCC CU UCAAGGACAUCGUCCGCG CCGGGUGC	2332

295	ACCCCCCC C CCCCCGCC	1256	CGCCCCCC CCACCAAACUCC CU UCAACCACAUCCUCCCCC CCCCCCCU	2333

298	CCCCCCCC C CGCCCCUC	2095	CACCCCCC CCAGCAAACUCC CU UCAACCACAUCCUCCCCC CCCGCCCC	2334

299	CCCCCCCC C CCCCCUCC	2096	CCACCCCC CGACCAAACUCC CU UCAACCACAUCGUCCGCC CGCCCCCC	2335

300	CCCCCCCG C CCCCUCCC	2097	CCCACCCC CCACGAAACUCC CU UCAACCACAUCCUCCCCC CCCCCCCC	2336

301	CCCCCCCC C CCCUCCCC	1257	CCCCACCC CCACCAAACUCC CU UCAACGACAUCCUCCCCC CCCCCCCC	2337

307	CCCCCCUC C CCCUUCCG	1258	CCCAACCC CCACGAAACUCC CU UCAACGACAUCCUCCCCC CACCCCCC	2338

315	GCCCUUCC C CACCCUUC	1259	CAAGCCUC CCACCAAACUCC CU UCAACCACAUCCUCCCGC CCAACCGC	2339

318	CUUCCCCA C CCUUCACU	1260	ACUCAACG CCACCAAACUCC CU UCAACCACAUCCUCCCCC UCCCCAAC	2340

330	UCACUCCA C CCCUCUCC	1261	CCACACCG GCACCAAACUCC CU UCAAGCACAUCCUCCGCG UCCACUCA	2341

337	AGCCCUCU G CUCCCGCA	1262	UGCGGGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAGGGCU	2342

343	CUGCUCCC G CACGCCAU	1263	AUGGCGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGAGCAG	2343

347	UCCCGCAC G CCAUGAAG	1264	CUUCAUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUGCGGGA	2344

352	CACGCCAU G AAGUCGCC	2098	GGCGACUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGGCGUG	2345

355	GCCAUGAA G UCGCCGUU	1265	AACGGCGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCAUGGC	2346

358	AUGAAGUC G CCGUUCUA	1266	UAGAACGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GACUUCAU	2347

361	AAGUCGCC U UUCUACCG	1267	CGGUAGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGCGACUU	2348

369	GUUCUACC U CUGCCAUA	1268	UCUGGCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGUG GGUAGAAC	2349

372	CUACCGCU U CCAGAACA	1269	UGUUCUGG GUAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCGGUAG	2350

376	CGCUGCCA G AACACCAC	2099	GUGGUGUU GUAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCAGCG	2351

389	CCACCUCU G UGGAAAAA	1270	UUUUUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAGUUGG	2352

391	ACCUCUUU G GAAAAAGG	2100	CCUUUUUC GGAGUAAACUCC CU UCAAGGACAUCGUCCGUG ACAGAGGU	2353

392	CCUCUGUG U AAAAAGUC	2101	GCCUUUUU GGAUUAAACUCC CU UCAAGGACAUCUUCCGUU CACAGAGG	2354

398	UGGAAAAA U UCAACUCU	2102	CGAGUUGC GGAGGAAACUCC CU UCAAGUACAUCGUCCGGU UUUUUCCA	2355

399	GUAAAAAU G CAACUCGU	1271	CCGAUUUG GGAGUAAACUCC CU UCAAGUACAUCGUCCGGG CUUUUUCC	2356

406	UUCAACUC U GCGUUGAU	2103	AUCACCUC UGAGUAAACUCC CU UCAAGGACAUCGUCCUUG GAGUUGCC	2357

407	GCAACUCU U CUGUGAUG	1272	CAUCACCU UGAGGAAACUCC CU UCAAGGACAUCGUCCGUU CGAUUUGC	2358

409	AACUCGGC G UUGAUUGU	2104	CCCAUCAC GGAGGAAACUCC CU UCAAUGACAUCGUCCUUG GCCUAUUU	2359

410	ACUCGUCG G UGAUGGUC	1273	UCCCAUCA UGAGGAAACUCC CU UCAAGGACAUCGUCCGUU CUCCUAGU	2360

412	UCGUCGGU U AUGGUCGG	2105	CCGCCCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGUG ACCGCCGA	2361

415	GCGGUUAU U UUCGGGGU	2106	ACCCCGCC GGAUGAAACUCC CU UCAAUGACAUCGUCCGGG AUCACCGC	2362

416	CGGUUAUG U UCUGUGUG	2107	CACCCCGC UGAUGAAACUCC CU UCAAGGACAUCGUCCGGG CAUCACCG	2363

417	GGUUAUUG U CGUGUUGC	1274	GCACCCCG GGAGGAAACUCC CU UCAAUGACAUCGUCCGGG CCAUCACC	2364

419	UUAUUGGC U GGUUGCUC	2108	GAGCACCC UGAUGAAACUCC CU UCAAGGACAUCGUCCGGG GCCCAUCA	2365

420	GAUGGGCG U GGUUCUCU	2109	AGAGCACC GGAGGAAACUCC CU UCAAUGACAUCGUCCGGG CGCCCAUC	2366

421	AUGGUCUG U GUGCUCUU	2110	AAGAGCAC GGAGGAAACUCC CU UCAAUGACAUCGUCCGGG CCGCCCAU	2367

422	UGGGCGUG U UUCUCUUC	1275	GAAGAUCA GUAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCGCCCA	2368

424	GGCGGGGU U CUCUUCAG	1276	CUGAAGAG GGAGGAAACUCC CU UCAAGGACAUCUUCCUGU ACCCCGCC	2369

432	GCUCUUCA G CACCGGCC	1277	GUCCUGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAAGAGC	2370

437	UCAGCACC U UCCUCCUG	2111	CAGGAGGC GUAGGAAACUCC CU UCAAGGACAUCUUCCGGG GUUGCUGA	2371

438	CAUCACCG U CCUCCUGG	1278	CCAGUAGG GGAGGAAACUCC CU UCAAGUACAUCGUCCGGG CGGUGCUU	2372

445	GUCCUCCU U GGCAACCU	2112	AGGUUGCC GGAGGAAACUCC CU UCAAGUACAUCGUCCGGU AUGAGUCC	2373

446	GCCUCCUG U GCAACCUU	2113	CAUGUUUC UUAUUAAACUCC CU UCAAGUACAUCUUCCUUU CAGUAUUC	2374

447	CCUCCUGG G CAACCUGC	1279	GCAGGUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAGGAGG	2375

454	GGCAACCU G CUGGCCCU	1280	AGGGCCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGUUGCC	2376

457	AACCUGCU G GCCCUGGG	2114	CCCAGGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCAGGUU	2377

458	ACCUGCUG G CCCUGGGG	1281	CCCCAGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCAGGU	2378

463	CUGGCCCU G GGGCUGCU	2115	AGCAGCCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGGCCAG	2379

464	UGGCCCUG U GGCUGCUG	2116	CAGCAGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGGGCCA	2380

465	GGCCCUGG G GCUGCUGG	2117	CCAGCAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAGGGCC	2381

466	GCCCUGGG G CUGCUGGC	1282	UCCAUCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCAGGGC	2382

469	CUGGGGCU G CUGGCGCG	1283	CGCGCCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCCCCAG	2383

472	GGGCUGCU G GCGCGCUC	2118	GAGCGCUC GGAGGAAACUCC CU UCAAGUACAUCGUCCGUG AGCAGCCC	2384

473	GGCUGCUG U CGCGCUCG	1284	CGAGCGCG GGAGGAAACUCC CU UCAAGUACAUCGUCCGUG CAGCAGCC	2385

475	CUGCUGUC U CGCUCUGU	1285	CCCGAGCG GGAGGAAACUCC CU UCAAUGACAUCGUCCGUG GCCAGCAG	2386

477	GCUGGCGC U CUCGGGGC	1286	GCCCCGAU GGAUGAAACUCC CU UCAAUGACAUCGUCCGGG UCUCCAUC	2387

481	GCGCGCUC G GUGCUGUU	2119	CCCAGCCC GUAUGAAACUCC CU UCAAUUACAUCGUCCGGG GAUCUCGC	2388

482	CGCGCUCG G GGCUGGGG	2120	CCCCAGCC GUAUGAAACUCC CU UCAAGUACAUCGUCCGGG CUAUCGCG	2389

483	GCGCUCGG G GCUGGGGU	2121	ACCCCAGC GUAUGAAACUCC CU UCAAGGACAUCGUCCGGG CCUAUCGC	2390

484	CGCUCGGG G CUGGGGUG	1287	CACCCCAG GUAUUAAACUCC CU UCAAUUACAUCGUCCUGU CCCGAGCG	2391

487	UCGGGGCU G GGGUGUUG	2122	CACCACCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCCCCGA	2392

488	CUGGUCUG U GGUGGUGC	2123	GCACCACC GGAUUAAACUCC CU UCAAUUACAUCGUCCUGU CAGCCCCG	2393

489	GUUGCUUU U GUGGUGCU	2124	AGCACCAC GGAGUAAACUCC CU UCAAUUACAUCGUCCUGG CCAGCCCC	2394

490	UGGCUGUG U UGGUGCUC	1288	UAGCACCA UGAGUAAACUCC CU UCAAGUACAUCGUCCGGG CCCAGCCC	2395

492	GCUGUGGU U UUGCUCGC	2125	UCGAGCAC GGAGUAAACUCC CU UCAAGGACAUCGUCCGUG ACCCCAGC	2396

493	CUGGGGUG U UGCUCGCU	1289	CGCGAGCA UGAGGAAACUCC CU UCAAUUACAUCUUCCUUU CACCCCAU	2397

495	UGGGUUGU G CUCGCGGC	1290	GCCGCGAU UGAGGAAACUCC CU UCAAUUACAUCGUCCUUU ACCACCCC	2398

499	UGGUCCUC U CGUCGUCC	1291	GGACUCCG GGAGUAAACUCC CU UCAAUGACAUCGUCCUGG GAUCACCA	2399

501	UUGCUCUC U UCUUCCAC	2126	GUUGACGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCUAGCAC	2400

502	UGCUCGCG U CGUCCACU	1292	AGUGUACG GGAGGAAACUCC CU UCAAUGACAUCGUCCUGG CGCGAGCA	2401

504	CUCGCGGC U UCCACUGC	1293	GCAGUUGA UGAGGAAACUCC CU UCAAGGACAUCGUCCGUU GCCGCGAG	2402

511	CGUCCACU G CGCCCGCU	1294	AUCGUUCU GUAUGAAACUCC CU UCAAGGACAUCUUCCGUG AGUGUACG	2403

513	UCCACUGC U CCCGCUGC	1295	GCAGCGGU GGAGGAAACUCC CU UCAAUGACAUCGUCCGGG UCAGUGGA	2404

517	CUGCUCCC U CUUCCCUC	1296	GAUGUCAG GGAGUAAACUCC CU UCAAGGACAUCUUCCGUG UGUCUCAG	2405

520	CGCCCGCU U CCCUCUGU	1297	ACCUAGUG GGAGGAAACUCC CU UCAAUGACAUCUUCCUGU AGCGUGCU	2406

526	CUGCCCUC U GUCUUCUA	2127	UAGAAGAC GGAGUAAACUCC CU UCAAGGACAUCUUCCGGU GAUGUCAG	2407

527	UGCCCUCG G UCUUCUAC	1298	GUAGAAGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAGGGCA	2408

538	UUCUACAU G CUGGUGUG	1299	CACACCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGUAGAA	2409

541	UACAUGCU G GUGUGUGG	2128	CCACACAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCAUGUA	2410

542	ACAUGCUG G UGUGUGGC	1300	GCCACACA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCAUGU	2411

544	AUGCUGGU G UGUGGCCU	1301	AGOCCACA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCAGCAU	2412

546	GCUGGUGU G UGGCCUGA	1302	UCAGGCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACACCAGC	2413

548	UGGUGUGU G GCCUGACG	2129	CGUCAGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACACACCA	2414

549	GGUGUGUG G CCUGACGG	1303	CCGUCAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACACACC	2415

553	UGUGGCCU G ACGGUCAC	2130	GUGACCGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGCCACA	2416

556	GGCCUGAC G GUCACCGA	2131	UCGGUGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUCAGGCC	2417

557	GCCUGACG G UCACCGAC	1304	GUCGGUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGUCAGGC	2418

563	CGGUCACC G ACUUGCUG	2132	CAGCAAGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGUGACCG	2419

568	ACCGACUU G CUGGGCAA	1305	UUGCCCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGUCGGU	2420

571	GACUUGCU G GGCAAGUG	2133	CACUUGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCAAGUC	2421

572	ACUUGCUG G GCAAGUGC	2134	GCACUUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCAAGU	2422

573	CUUGCUGG G CAAGUGCC	1306	GGCACUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAGCAAG	2423

577	CUGGGCAA G UGCCUCCU	1307	AGGAGGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGCCCAG	2424

579	GGGCAAGU G CCUCCUAA	1308	UUAGGAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUUGCCC	2425

588	CCUCCUAA G CCCGGUGG	1309	CCACCGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUAGGAGG	2426

592	CUAAGCCC G GUGGUGCU	2135	AGCACCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGCUUAG	2427

593	UAAGCCCG G UGGUGCUG	1310	CAGCACCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGGCUUA	2428

595	AGCCCGGU G GUGCUGGC	2136	GCCAGCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCGGGCU	2429

596	GCCCGGUG G UGCUGGCU	1311	AGCCAGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCGGGC	2430

598	CCGGUGGU G CUGGCUGC	1312	GCAGCCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCACCGG	2431

601	GUGGUGCU G GCUGCCUA	2137	UAGGCAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCACCAC	2432

602	UGGUGCUG G CUGCCUAC	1313	GUAGGCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCACCA	2433

605	UGCUGGCU G CCUACGCU	1314	AGCGUAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCCAGCA	2434

611	CUGCCUAC G CUCAGAAC	1315	GUUCUGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUAGGOAG	2435

616	UACGCUCA G AACCGGAG	2138	CUCCGGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAGCGUA	2436

621	UCAGAACC G GAGUCUGC	2139	GCAGACUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGUUCUGA	2437

622	CAGAACCG G AGUCUGCG	2140	CGCAGACU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGUUCUG	2438

624	GAACCGGA G UCUGCGGG	1316	CCCGCAGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCGGUUC	2439

628	CGGAGUCU G CGGGUGCU	1317	AGCACCCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGACUCCG	2440

630	GAGUCUGC G GGUGCUUG	2141	CAAGCACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCAGACUC	2441

631	AGUCUGCG G GUGCUUGC	2142	GCAAGCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCAGACU	2442

632	GUCUGCGG G UGCUUGCG	1318	CGCAAGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCGCAGAC	2443

634	CUGCGGGU G CUUGCGCC	1319	GGCGCAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCCGCAG	2444

638	GGGUGCUU C CGCCCGCA	1320	UGCGGGCG GGAGGAAACUCC CU UCAAGGACAUCCUCCGGG AAGCACCC	2445

640	GUGCUUGC G CCCGCAUU	1321	AAUGCGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCAAGCAC	2446

644	UUGCGCCC G CAUUGGAC	1322	GUCCAAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGCGCAA	2447

649	CCCGCAUU G GACAACUC	2143	GAGUUGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUGCGGG	2448

650	CCGCAUUG C ACAACUCG	2144	CGAGUUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAUGCGC	2449

658	CACAACUC G UUGUGCCA	1323	UGGCACAA GGACGAAACUCC CU UCAACGACAUCGUCCCGG GACUUGUC	2450

661	AACUCGUU G UGCCAAGC	1324	GCUUGGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGCG AACCACUU	2451

663	CUCGUUGU G CCAACCCU	1325	ACGCUUGG CGAGGAAACUCC CU UCAAGGACAUCGUCCCGC ACAACGAC	2452

668	UGUGCCAA G CCUUCCCC	1326	CCCGAAGC CGACGAAACUCC CU UCAACGACAUCGUCCGGC UUGGCACA	2453

674	AACCCUUC C CCUUCUUC	1327	GAAGAAGG CGACGAAACUCC CU UCAAGGACAUCCUCCGCG CAACCCUU	2454

685	UUCUUCAU G UCCUUCUU	1328	AACAACGA GGACGAAACUCC CU UCAAGGACAUCGUCCGGG AUGAAGAA	2455

695	CCUUCUUU G GGCUCUCC	2145	CCAGACCC GGACGAAACUCC CU UCAACGACAUCGUCCGGG AAAGAACC	2456

696	CUUCUUUG G GCUCUCCU	2146	ACCACAGC GGACGAAACUCC CU UCAACGACAUCCUCCCCG CAAACAAG	2457

697	UUCUUUGG G CUCUCCUC	1329	GAGGACAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGCG CCAAAGAA	2458

706	CUCUCCUC G ACACUGCA	2147	UCCAGUGU GGAGGAAACUCC CU UCAACGACAUCGUCCGGG GAGGAGAG	2459

712	UCGACACU C CAACUCCU	1330	ACCACUUC CCAGGAAACUCC CU UCAAGGACAUCGUCCGCG AGUGUCGA	2460

721	CAACUCCU C CCCAUGCC	2148	CCCAUCGC GCACGAAACUCC CU UCAACCACAUCGUCCGCG ACCACUUC	2461

722	AACUCCUC G CCAUGGCA	1331	UGCCAUGG CGACGAAACUCC CU UCAAGGACAUCGUCCCGG CACGAGUU	2462

727	CUGGCCAU C GCACUCGA	2149	UCCACUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCCCC AUGGCCAG	2463

728	UGCCCAUC C CACUGGAC	1332	CUCCACUC GGACGAAACUCC CU UCAAGCACAUCGUCCGCG CAUCCCCA	2464

733	AUCCCACU C CACUGCUC	2150	CACCACUC GGACGAAACUCC CU UCAAGCACAUCGUCCGGG AGUGCCAU	2465

734	UCCCACUC C ACUGCUGG	2151	CCAGCACU CCACCAAACUCC CU UCAACCACAUCCUCCCGC CACUGCCA	2466

736	CCACUCGA C UCCUCCCU	1333	AGCCAGCA GCAGGAAACUCC CU UCAACCACAUCCUCCCCC UCCACUCC	2467

738	ACUCCAGU C CUGCCUCU	1334	ACACCCAC CCACCAAACUCC CU UCAACCACAUCCUCCCGC ACUCCACU	2468

741	CCACUCCU C CCUCUCCC	2152	GGGAGAGC GCAGGAAACUCC CU UCAACCACAUCCUCCCGC ACCACUCC	2469

742	CACUCCUC C CUCUCCCU	1335	ACGCAGAC GCACGAAACUCC CU UCAACCACAUCCUCCCCC CACCACUC	2470

752	UCUCCCUA C CCCACCCU	2153	ACCCUCCC CCACCAAACUCC CU UCAACCACAUCCUCCCCC UACCCACA	2471

753	CUCCCUAC C GCACCCUU	2154	AAGGGUGC GCAGGAAACUCC CU UCAACCACAUCCUCCGGC CUAGGCAG	2472

754	UCCCUAGC C CACCCUUU	1336	AAACGCUC CCACGAAACUCC CU UCAACCACAUCCUCCCCC CCUAGCGA	2473

771	CUUCUACC G ACGGCACA	2155	UGUGCCGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGUAGAAG	2474

774	CUACCGAC G GCACAUCA	2156	UGAUGUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUCGGUAG	2475

775	UACCGACG G CACAUCAC	1337	GUGAUGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGUCGGUA	2476

787	AUCACCCU G CGCCUGGG	1338	CCCAGGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGGUGAU	2477

789	CACCCUGC G CCUGGGCG	1339	CGCCCAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCAGGGUG	2478

793	CUGCGCCU G GGCGCACU	2157	AGUGCGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGCGCAG	2479

794	UGCGCCUG G GCGCACUG	2158	CAGUGCGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGGCGCA	2480

795	GCGCCUGG G CGCACUGG	1340	CCAGUGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAGGCGC	2481

797	GCCUGGGC G CACUGGUG	1341	CACCAGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCCCAGGC	2482

802	GGCGCACU G GUGGCCCC	2159	GGGGCCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUGCGCC	2483

803	GCGCACUG G UGGCCCCG	1342	CGGGGCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGUGCGC	2484

805	GCACUGGU G GCCCCGGU	2160	ACCGGGGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCAGUGC	2485

806	CACUGGUG G CCCCGGUG	1343	CACCGGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCAGUG	2486

811	GUGGCCCC G GUGGUGAG	2161	CUCACCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGGCCAC	2487

812	UGGCCCCG G UGGUGAGC	1344	UCUCACCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGGGCCA	2488

814	GCCCCGGU G GUGAGCGC	2162	GCGCUCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCGGGGC	2489

815	CCCCGGUG G UGAGCGCC	1345	GGCGCUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACCGGGG	2490

817	CCGGUGGU G AGCGCCUU	2163	AAGGCGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCACCGG	2491

819	GGUGGUGA G CGCCUUCU	1346	AGAAGGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCACCACC	2492

821	UGGUGAGC G CCUUCUCC	1347	GGAGAAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCUCACCA	2493

832	UUCUCCCU G GCUUUCUG	2164	CAGAAAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGGAGAA	2494

833	UCUCCCUG G CUUUCUGC	1348	GCAGAAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGGGAGA	2495

840	GGCUUUCU G CGCGCUAC	1349	GUAGCGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAAAGCC	2496

842	CUUUCUGC G CGCUACCU	1350	AGGUAGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCAGAAAG	2497

844	UUCUGCGC G CUACCUUU	1351	AAAGGUAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCGCAGAA	2498

856	CCUUUCAU G GGCUUCGG	2165	CCGAAGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGAAAGG	2499

857	CUUUCAUG G GCUUCGGG	2166	CCCGAAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUGAAAG	2500

858	UUUCAUGG G CUUCGGGA	1352	UCCCGAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAUGAAA	2501

863	UGGGCUUC G GGAAGUUC	2167	GAACUUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAAGCCCA	2502

864	GGGCUUCG G GAAGUUCG	2168	CGAACUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAAGCCC	2503

865	GGCUUCGG G AAGUUCGU	2169	ACGAACUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCGAAGCC	2504

868	UUCGGGAA G UUCGUGCA	1353	UGCACGAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCCCGAA	2505

872	GGAAGUUC G UGCAGUAC	1354	GUACUGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAACUUCC	2506

874	AAGUUCGU G CAGUACUG	1355	CAGUACUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACGAACUU	2507

877	UUCGUGCA G UACUGCCC	1356	GGGCAGUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCACGAA	2508

882	GCAGUACU G CCCCGGCA	1357	UGCCGGGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUACUGC	2509

887	ACUGCCCC G GCACCUGG	2170	CCAGGUGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGGCAGU	2510

888	CUGCCCCG G CACCUGGU	1358	ACCAGGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGGGCAG	2511

894	CGGCACCU G GUGCUUUA	2171	UAAAGCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGGUGCCG	2512

895	GGCACCUG G UGCUUUAU	1359	AUAAAGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGGUGCC	2513

897	CACCUGGU G CUUUAUCC	1360	GGAUAAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCAGGUG	2514

907	UUUAUCCA G AUGGUCCA	2172	UGGACCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAUAAA	2515

910	AUCCAGAU G GUCCACGA	2173	UCGUGGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCUGGAU	2516

911	UCCAGAUG G UCCACGAG	1361	CUCGUGGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUCUGGA	2517

917	UGGUCCAC G AGGAGGGC	2174	GCCCUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUGGACCA	2518

919	GUCCACGA G GAGGGCUC	2175	GAGCCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGUGGAC	2519

920	UCCACGAG G AGGGCUCG	2176	CGAGCCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCGUGGA	2520

922	CACGAGGA G GGCUCGCU	2177	AGCGAGCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUCGUG	2521

923	ACGAGGAG G GCUCGCUG	2178	CAGCGAGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCCUCGU	2522

924	CGAGGAGG G CUCGCUGU	1362	ACAGCGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCUCCUCG	2523

928	GAGGGCUC G CUGUCGGU	1363	ACCGACAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAGCCCUC	2524

931	GGCUCGCU G UCGGUGCU	1364	AGCACCGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCGAGCC	2525

934	UCGCUGUC G GUGCUGGG	2179	CCCAGCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GACAGCGA	2526

935	CGCUGUCG G UGCUGGGG	1365	CCCCAGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGACAGCG	2527

937	CUGUCGGU G CUGGGGUA	1366	UACCCCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCGACAG	2528

940	UCGGUGCU G GGGUACUC	2180	GAGUACCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCACCGA	2529

941	CGGUGCUG G GGUACUCU	2181	AGAGUACC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCACCG	2530

942	GGUGCUGG G GUACUCUG	2182	CAGAGUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCAGCACC	2531

943	GUGCUGGG G UACUCUGU	1367	ACAGAGUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CCCAGCAC	2532

950	GGUACUCU G UGCUCUAC	1368	GUAGAGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAGUACC	2533

952	UACUCUGU G CUCUACUC	1369	GAGUAGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAGAGUA	2534

963	CUACUCCA G CCUCAUGG	1370	CCAUGAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAGUAG	2535

970	AGCCUCAU G GCGCUGCU	2183	AGCAGCGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGAGGCU	2536

971	GCCUCAUG G CGCUGCUG	1371	CAGCAGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUGAGGC	2537

973	CUCAUGGC G CUGCUGGU	1372	ACCAGCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCCAUGAG	2538

976	AUGGCGCU G CUGGUCCU	1373	AGGACCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCGCCAU	2539

979	GCGCUGCU G GUCCUCGC	2184	GCGAGGAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCAGCGC	2540

980	CGCUGCUG G UCCUCGCC	1374	GGCGAGGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAGCAGCG	2541

986	UGGUCCUC G CCACCGUG	1375	CACGGUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAGGACCA	2542

992	UCGCCACC G UGCUGUGC	1376	GCACAGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGUGGCGA	2543

994	GCCACCGU U CUGUGCAA	1377	UUGCACAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACGGUGGC	2544

997	ACCGUGCU G UGCAACCU	1378	AGGUUGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCACGGU	2545

999	CGUGCUGU G CAACCUCG	1379	CGAGGUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAGCACG	2546

1007	GCAACCUC U GCGCCAUG	2185	CAUGGCGC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAGGUUGC	2547

1008	CAACCUCG G CGCCAUGC	1380	GCAUGGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAGGUUG	2548

1010	ACCUCGGC G CCAUGCGC	1381	GCGCAUGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCCGAGGU	2549

1015	GGCGCCAU G CGCAACCU	1382	AGGUUUCU GGAGGAAACUCC CU UCAAUGACAUCGUCCGGG AUGGCGCC	2550

1017	CGCCAUUC G CAACCUCU	1383	AUAGGUUG GGAGGAAACUCC CU UCAAGGACAUCUUCCGGG GCAUGGCG	2551

1028	ACCUCUAU G CGAUGCAC	1384	GUGCAUCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUAGAGGU	2552

1030	CUCUAUGC U AUGCACCG	2186	CGGUGCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCAUAGAG	2553

1033	UAUGCGAU G CACCGGCG	1385	CGCCGGUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCGCAUA	2554

1038	UAUGCACC G GCUGCUGC	2187	UCAGCCGC GUAGGAAACUCC CU UCAAGGACAUCGUCCGUG GGUGCAUC	2555

1039	AUGCACCG G CGUCUGCA	1386	UGCAGCCG GGAGUAAACUCC CU UCAAGGACAUCGUCCGUG CGGUGCAU	2556

1041	GCACCGGC U UCUGCAUC	2188	GCUGCAGC GGAGGAAACUCC CU UCAAUGACAUCGUCCGGG GCCGGUUC	2557

1042	CACCGUCG U CUUCAUCG	1387	CGCUGCAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCCGGUG	2558

1045	CGGCGUCU G CAUCUGCA	1388	UUCCGCUG GGAGGAAACUCC CU UCAAGUACAUCGUCCGGG AGCCGCCU	2559

1048	CUUCUGCA G CGGCACCC	1389	GGGUGCCG GGAGGAAACUCC CU UCAAGGACAUCGUCCUGG UGCAGCCG	2560

1050	GCUUCAGC U UCACCCGC	2189	GCGUGUGC UUAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCUGCAGC	2561

1051	CUGCAGCG G CACCCGCU	1390	CGCUGGUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGU CGCUGCAG	2562

1057	CGUCACCC U CGCUCCUG	1391	CAGGAGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGUGCCG	2563

1059	GCACCCUC U CUCCUGCA	1392	UGCAGGAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCGGGUGC	2564

1065	GCGCUCCU G CACCAGGG	1393	CCCUUGUG GGAUGAAACUCC CU UCAAGGACAUCGUCCGGG AGGAGCGC	2565

1071	CUGCACCA G GUACUGUG	2190	CACAGUCC UGAGUAAACUCC CU UCAAGUACAUCGUCCGGG UGGUGCAG	2566

1072	UGCACCAG U GACUGUGC	2191	GCACAUUC GGAGUAAACUCC CU UCAAGUACAUCGUCCGGG CUGGUGCA	2567

1073	GCACCAGG U ACUGUGCC	2192	GUCACAGU GGAGGAAACUCC CU UCAAGUACAUCGUCCGGG CCUGGUGC	2568

1077	CAGGGACU U UGCCGAGC	1394	GCUCGGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGUCCCUG	2569

1079	GGGACUGU U CCGAGCCG	1395	CUGCUCGG UGAGGAAACUCC CU UCAAGGACAUCGUCCGGU ACAGUCCC	2570

1082	ACUUUUCC U AUCCUCUC	2193	GCUCGUCU UUAUGAAACUCC CU UCAAUGACAUCUUCCUGU UUCACAGU	2571

1084	UUUUCCUA U CCUCUCGC	1396	UCUCUCUG UUAUGAAACUCC CU UCAAUGACAUCUUCCUUU UCGUCACA	2572

1087	GCCGAGCC G CGCGCGGA	1397	UCCGCGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGCUCGGC	2573

1089	CGAGCCGC G CGCGGACG	1398	CGUCCGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCGGCUCG	2574

1091	AGCCGCGC G CGGACGGG	1399	CCCGUCCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCGCGGCU	2575

1093	CCGCGCGC G GACGGGAG	2194	CUCCCGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCGCGCGG	2576

1094	CGCGCGCG G ACGGGAGG	2195	CCUCCCGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGCGCGCG	2577

1097	GCGCGGAC G GGAGGGAA	2196	UUCCCUCC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GUCCGCGC	2578

1098	CGCGGACG C GAGGGAAG	2197	CUUCCCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGUCCGCG	2579

1099	GCGGACGG C AGGGAAGC	2198	CCUUCCCU GCACGAAACUCC CU UCAAGCACAUCCUCCGGG CCCUCCGC	2580

1101	CGACCCGA C CCAAGCCU	2199	ACCCUUCC GCAGGAAACUCC CU UCAAGGACAUCGUCCCCG UCCCCUCC	2581

1102	CACGGCAC G GAAGCGUC	2200	GACGCUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCCCG CUCCCGUC	2582

1103	ACCGCAGC C AACCGUCC	2201	GGACCCUU GCACGAAACUCC CU UCAAGCACAUCCUCCCCG CCUCCCGU	2583

1106	GGAGGCAA G CCUCCCCU	1400	AGCGCACG GGAGGAAACUCC CU UCAAGGACAUCGUCCCGG UUCCCUCC	2584

1108	AGGCAACC C UCCCCUCA	1401	UGAGGGCA GGAGGAAACUCC CU UCAACCACAUCGUCCCGG GCUUCCCU	2585

1117	UCCCCUCA C CCCCUGGA	1402	UCCACGGG GGACGAAACUCC CU UCAAGGACAUCGUCCCGG UCACCGGA	2586

1123	CAGCCCCU C CACGAGCU	2202	AGCUCCUC GGACGAAACUCC CU UCAAGGACAUCGUCCGGG ACGGGCUG	2587

1124	ACCCCCUG C ACGAGCUG	2203	CAGCUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCCCG CAGGCGCU	2588

1126	CCCCUCGA C GAGCUGGA	2204	UCCACCUC GCACGAAACUCC CU UCAAGGACAUCGUCCGCG UCCACGCG	2589

1127	CCCUCCAC C AGCUGGAU	2205	AUCCAGCU GCAGGAAACUCC CU UCAACGACAUCCUCCGCG CUCCAGGG	2590

1129	CUGGAGGA C CUGGAUCA	1403	UCAUCCAG GCACGAAACUCC CU UCAAGCACAUCGUCCGCG UCCUCCAG	2591

1132	GAGGAGCU C CAUCACCU	2206	ACCUGAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCUCCUC	2592

1133	AGCACCUC C AUCACCUC	2207	CACGUGAU GCACGAAACUCC CU UCAAGCACAUCCUCCGGG CACCUCCU	2593

1144	CACCUCCU C CUCCUGCC	1404	CCCACCAG GCACGAAACUCC CU UCAAGCACAUCCUCCGGG ACCAGGUG	2594

1147	CUCCUGCU C CUGGCGCU	1405	AGCGCCAG GGACGAAACUCC CU UCAAGGACAUCCUCCGGG ACCACGAG	2595

1150	CUCCUCCU C GCGCUGAU	2208	AUCACCCC GCAGGAAACUCC CU UCAAGCACAUCCUCCGCG ACCACCAG	2596

1151	UCCUCCUC C CGCUCAUG	1406	CAUCAGCG GCACGAAACUCC CU UCAAGGACAUCGUCCGGG CACCAGCA	2597

1153	CUCCUGGC C CUGAUGAC	1407	GUCAUCAG GCAGGAAACUCC CU UCAAGCACAUCCUCCGCG GCCAGCAG	2598

1156	CUCCCCCU C AUCACCCU	2209	ACCGUCAU GCAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCGCCAG	2599

1159	CCCCUCAU C ACCCUCCU	2210	AGCACGCU GCACCAAACUCC CU UCAAGCACAUCGUCCGCG AUCACCCC	2600

1163	UGAUCACC C UGCUCUUC	1408	CAAGAGCA GCAGGAAACUCC CU UCAAGCACAUCCUCCGGG GCUCAUCA	2601

1165	AUGACCGU C CUCCUCAC	1409	GUCAAGAG GGAGCAAACUCC CU UCAAGCACAUCCUCCGCG ACGGUCAU	2602

1177	UUCACUAU C UGUUCUCU	1410	AGACAACA CGAGCAAACUCC CU UCAAGCACAUCCUCCGGG AUACUCAA	2603

1179	CACUAUGU C UUCUCUGC	1411	GCAGAGAA GGAGGAAACUCC CU UCAAGCACAUCCUCCGCG ACAUACUG	2604

1186	UGUUCUCU C CCCGUAAU	1412	AUUACGGC CGAGGAAACUCC CU UCAAGCACAUCCUCCCCG AGAGAACA	2605

1190	CUCUGCCC G UAAUUUAU	1413	AUAAAUUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGGCAGAG	2606

1200	AAUUUAUC G CGCUUACU	1414	AGUAAGCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAUAAAUU	2607

1202	UUUAUCGC G CUUACUAU	1415	AUAGUAAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCGAUAAA	2608

1211	CUUACUAU G CAGCAUUU	2211	AAAUGCUC GGAGGAAACUCC CU UCAAOGACAUCGUCCGGG AUAGUAAG	2609

1212	UUACUAUG G AGCAUUUA	2212	UAAAUGCU GGAGGAAACUCC CU UCAA0GACAUCGUCCG0G CAUAGUAA	2610

1214	ACUAUGGA G CAUUUAAG	1416	CUUAAAUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCAUAGU	2611

1222	GCAUUUAA G GAUGUCAA	2213	UUGACAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUAAAUGC	2612

1223	CAUUUAAG G AUGUCAAG	2214	CUUGACAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUAAAUG	2613

1226	UUAAGGAU G UCAAGGAG	1417	CUCCUUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUCCUUAA	2614

1231	GAUGUCAA G GAGAAAAA	2215	UUUUUCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGACAUC	2615

1232	AUGUCAAG G AGAAAAAC	2216	GUUUUUCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUUGACAU	2616

1234	GUCAAGGA G AAAAACAG	2217	CUGUUUUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUUGAC	2617

1242	GAAAAACA G GACCUCUG	2218	CAGAGGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUUUUUC	2618

1243	AAAAACAG G ACCUCUGA	2219	UCAGAGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGUUUUU	2619

1250	GGACCUCU G AAGAAGCA	2220	UGCUUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCC0GG AGAGGUCC	2620

1253	CCUCUGAA G AAGCAGAA	2221	UUCUGCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCAGAGG	2621

1256	CUGAAGAA G CAGAAGAC	1418	GUCGUCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUUCAG	2622

1259	AAGAAGCA G AAGACCUC	2222	GAGGUCUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCUUCUU	2623

1262	AAGCAGAA G ACCUCCGA	2223	UCGGAGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUCUGCUU	2624

1269	AGACCUCC G AGCCUUGC	2224	GCAAGGCU GGAGGAAACUCC CU UCAAG0ACAUCGUCCGGG GGAGGUCU	2625

1271	ACCUCCGA G CCUUGCGA	1419	UCGCAAGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCGGAGGU	2626

1276	CGAGCCUU G CGAUUUCU	1420	AGAAAUCG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGGCUCG	2627

1278	AGCCUUGC G AUUUCUAU	2225	AUAGAAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GCAAGGCU	2628

1289	UUCUAUCU G UGAUUUCA	1421	UGAAAUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAUAGAA	2629

1291	CUAUCUGU G AUUUCAAU	2226	AUUGAAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAGAUAG	2630

1301	UUUCAAUU G UGGACCCU	1422	AGGGUCCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAUUGAAA	2631

1303	UCAAUUGU G GACCCUUG	2227	CAAGGGUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAAUUGA	2632

1304	CAAUUGUG G ACCCUUGG	2228	CCAAGGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACAAUUG	2633

1311	GGACCCUU G GAUUUUUA	2229	UAAAAAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AAGGGUCC	2634

1312	GACCCUUG G AUUUUUAU	2230	AUAAAAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAAGGGUC	2635

1329	CAUUUUCA G AUCUCCAG	2231	CUGGAGAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAAAAUG	2636

1337	GAUCUCCA G UAUUUCGG	1423	CCGAAAUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAGAUC	2637

1344	AGUAUUUC G GAUAUUUU	2232	AAAAUAUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GAAAUACU	2638

1345	GUAUUUCG G AUAUUUUU	2233	AAAAAUAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGAAAUAC	2639

1360	UUUCACAA G AUUUUCAU	2234	AUGAAAAU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUGUGAAA	2640

1371	UUUCAUUA G ACCUCUUA	2235	UAAGAGGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAAUGAAA	2641

1380	ACCUCUUA G GUACAGGA	2236	UCCUGUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UAAGAGGU	2642

1381	CCUCUUAG G UACAGGAG	1424	CUCCUGUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUAAGAGG	2643

1386	UAGGUACA G GAGCCGGU	2237	ACCGGCUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUACCUA	2644

1387	AGGUACAG G AGCCGGUG	2238	CACCGGCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUGUACCU	2645

1389	GUACAGGA G CCGGUGCA	1425	UGCACCGG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCCUGUAC	2646

1392	CAGGAGCC G GUGCAGCA	2239	UGCUGCAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG GGCUCCUG	2647

1393	AGGAGCCG G UGCAGCAA	1426	UUGCUGCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CGGCUCCU	2648

1395	GAGCCGGU G CAGCAAUU	1427	AAUUGCUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACCGGCUC	2649

1398	CCGGUGCA G CAAUUCCA	1428	UGGAAUUG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGCACCGG	2650

1414	ACUAACAU G GAAUCCAG	2240	CUGGAUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUGUUAGU	2651

1415	CUAACAUG G AAUCCAGU	2241	ACUGGAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CAUGUUAG	2652

1422	GGAAUCCA G UCUGUGAC	1429	GUCACAGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGGAUUCC	2653

1426	UCCAGUCU G UGACAGUG	1430	CACUGUCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGACUGGA	2654

1428	CAGUCUGU G ACAGUGUU	2242	AACACUGU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAGACUG	2655

1432	CUGUGACA G UGUUUUUC	1431	GAAAAACA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGUCACAG	2656

1434	GUGACAGU G UUUUUCAC	1432	GUGAAAAA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACUGUCAC	2657

1446	UUCACUCU G UGGUAAGC	1433	GCUUACCA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGAGUGAA	2658

1448	CACUCUGU G GUAAGCUG	2243	CAGCUUAC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG ACAGAGUG	2659

1449	ACUCUGUG G UAAGCUGA	1434	UCAGCUUA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CACAGAGU	2660

1453	UGUGGUAA G CUGAGGAA	1435	UUCCUCAG GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UUACCACA	2661

1456	GGUAAGCU G AGGAAUAU	2244	AUAUUCCU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AGCUUACC	2662

1458	UAAGCUGA G GAAUAUGU	2245	ACAUAUUC GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UCAGCUUA	2663

1459	AAGCUGAG G AAUAUGUC	2246	GACAUAUU GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG CUCAGCUU	2664

1465	AGGAAUAU G UCACAUUU	1436	AAAUGUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG AUAUUCCU	2665

1477	CAUUUUCA G UCAAAGAA	1437	UUCUUUGA GGAGGAAACUCC CU UCAAGGACAUCGUCCGGG UGAAAAUG	2666

Equivalents

Wait Time* DNA

Wait Time* 2′-O-methyl

Wait Time*RNA

What we claim is:

1. A nucleic acid molecule that down regulates expression of a prostaglandin D2 receptor (PTGDR) gene.

2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is an enzymatic nucleic acid molecule.

3. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is an antisense nucleic acid molecule.

4. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule comprises a sequence selected from the group of sequences consisting of SEQ ID NOs: 228-454, 831-1206, 1438-1668, 1715-2057, and 2247-2666.

5. The enzymatic nucleic acid molecule of claim 2, wherein said enzymatic nucleic acid molecule comprises at least one binding arm wherein one or more of said binding arms comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 1-227, 455-830, 1207-1437, 1669-1714, and 2058-2246.

6. The antisense nucleic acid molecule of claim 3, wherein said antisense nucleic acid molecule comprises a sequence complementary to a sequence selected from the group of sequences consisting of SEQ ID NOs: 1-227, 455-830, 1207-1437, 1669-1714, and 2058-2246.

7. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is adapted to treat asthma.

8. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one 2′-sugar modification.

9. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises at least one phosphate backbone modification.

10. A method of reducing PTGDR activity in a cell, comprising contacting said cell with the nucleic acid molecule of claim 1 under conditions suitable for said reduction.

11. A method of treatment of a patient having a condition associated with the level of PTGDR, comprising contacting cells of said patient with the nucleic acid molecule of claim 1, under conditions suitable for said treatment.

12. The method of claim 11 further comprising the use of one or more drug therapies under conditions suitable for said treatment.

13. A pharmaceutical composition comprising an enzymatic nucleic acid molecule of claim 1.

14. A method of administering to a mammal the nucleic acid molecule of claim 1, comprising contacting said mammal with the molecule under conditions suitable for said administration.

15. The method of claim 14, wherein said mammal is a human.

16. The method of claim 14 wherein said administration is in the presence of a delivery reagent.

17. The method of claim 16, wherein said delivery reagent is a lipid.

18. The method of claim 17, wherein said lipid is a cationic lipid.

19. The method of claim 17, wherein said lipid is a phospholipid.

20. The method of claim 17, wherein said delivery reagent is a liposome.