WO1993012234A1 - Antiviral reagents based on rna-binding proteins - Google Patents

Antiviral reagents based on rna-binding proteins Download PDF

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Publication number
WO1993012234A1
WO1993012234A1 PCT/US1992/010770 US9210770W WO9312234A1 WO 1993012234 A1 WO1993012234 A1 WO 1993012234A1 US 9210770 W US9210770 W US 9210770W WO 9312234 A1 WO9312234 A1 WO 9312234A1
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polypeptide
rna
seq
hiv
cleavage
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PCT/US1992/010770
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French (fr)
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Sumedha D. Jayasena
Brian H. Johnston
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Sri International
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Priority claimed from US07/808,452 external-priority patent/US6063612A/en
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Publication of WO1993012234A1 publication Critical patent/WO1993012234A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6839—Triple helix formation or other higher order conformations in hybridisation assays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3511—Conjugate intercalating or cleaving agent
    • C—CHEMISTRY; METALLURGY
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to RNA-binding proteins modified to contain nucleic acid cleaving moieties, in particular, viral trans-acting pro ⁇ teins can be used as specific antiviral reagents.
  • the invention further includes methods for genera ⁇ ting said antiviral reagents, methods of cleaving viral nucleic acid, and methods of inactivating viral nucleic acid in cells.
  • phenanthroline attached to an oligonucleotide or polypeptide will bind cupric ion and this complex can be used to cleave DNA.
  • a reducing agent the bound cupric ion is reduced to cuprous ion, which reduces molecular oxygen to produce hydrogen peroxide.
  • the H-jO- reacts with the cuprous complex to form a copper-oxo species that is directly responsible for cleavage.
  • Chen et al. (1987) used this approach to convert the E. coli trp repressor to a site-specific deoxyribonuclease.
  • Copper-phenanthrolene has also been tethered to oligonucleotides to induce sequence-specific cleavage of single-stranded and double-stranded DNA (Francois et al., 1989).
  • An alternative but chemically analogous system utilizes EDTA-chelated iron tethered to an oligonucleotide to cleave DNA.
  • the present invention describes a polypeptide having site-specific RNA binding, where the polypeptide is modified to contain a moiety capable of cleaving an RNA backbone, in particular, viral polypeptides having site- specific viral RNA-binding.
  • exemplary of such polypeptides are the polypeptides presented as SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:22.
  • a number of cleaving moieties are useful in the practice of the present invention including the following: phenanthroline Cu(II) , Zn(II) , Fe(II)-EDTA, Cu(II)-bipyridine, and Cu(II)-terpyridine.
  • the RNA-binding polypeptide can be either the HIV TAT or REV proteins, or polypeptides derived therefrom.
  • a preferred embodiment of the present invention is the polypeptide having the sequence presented as SEQ ID N0:1, which further contains an end-terminal cysteine residue, and where the cleaving moiety is phenanthroline Cu(II) .
  • Another preferred embodiment is the polypeptide having the sequence presented as SEQ ID NO:2 where the cleaving moiety is phenanthroline Cu(II) .
  • a further embodiment of the polypeptide cleaving reagents of the present invention is the generation of fusion polypeptides containing the RNA-binding polypeptide coding sequence fused in frame to a non-specific nuclease.
  • the non-specific nuclease may be covalently bound to the RNA-binding polypeptide.
  • One preferred embodiment of this aspect of the present invention is the polypeptide having the sequence presented as SEQ ID NO:2 coupled to Staphylococcal non ⁇ specific nuclease.
  • the present invention also includes a method of cleaving a target RNA. The method involves contacting an RNA molecule containing a cognate RNA-binding site with the RNA-binding polypeptide cleaving reagent. Both chemical and nuclease cleaving reagents are useful in this aspect of the present invention.
  • the RNA molecule is an HIV RNA and the RNA-binding polypeptide is an HIV- encoded RNA-binding protein, such as TAT, REV, or polypeptides derived therefrom.
  • the RNA-binding polypeptide reagent is supplied at a concentration effective to produce cleavage of the target RNA molecule.
  • Cleavage reactions may further include the addition of a reducing agent, such as mercaptopropionic acid, N-acetyl cysteine, or ascorbate.
  • the cleavage reactions include the addition of the polypeptide reagent, CuS0 4 and mecaptopropionic acid.
  • the invention further includes a method of inhibiting expression of viral antigens in infected cells.
  • the method in ⁇ volves exposing the infected cells to the above described polypeptide cleaving reagent where the polypeptide reagent binds, site-specifically, to the RNA target and is able to be taken up by the infected cells.
  • the cells are exposed to a concentration of the polypeptide reagent which is effective to produce reduction in (i) viral antigen expression or (ii) viral transcription in the infected cells.
  • the cells may be exposed to the polypeptide reagent in the presence of a reducing agent, such as N-acetyl cysteine and ascorbate.
  • the viral RNA is HIV RNA and the polypeptide reagent is based on the HIV-encoded TAT or REV proteins, or polypeptides derived therefrom.
  • the polypeptide reagent includes a non-specific nuclease, expression of the polypeptide reagent in infected and/or un-infected cells provides a useful gene therapy to fight viral disease.
  • the cleaving agents of the present invention are oligonucleotides having nuclease resistant backbones to which a moiety capable of cleaving RNA backbones has been attached.
  • Figure 1 illustrates the sequence and structure of a -variety of TAR elements.
  • Figure 1A shows the TAR element of HIV-1 (also presented as SEQ ID NO:3) .
  • Figure IB shows a TAR element of HIV-2 (also presented as SEQ ID NO:4) .
  • Figure 1C shows a truncated HIV-1 TAR element designated ⁇ TAR (also presented as SEQ ID NO:5).
  • Figure ID illustrates the binding of a TAT-peptide, having an attached cleaving moiety, to a TAR element- containing target RNA.
  • Figure 2A presents the primary coding sequence of the HIV-1 TAT protein. This sequence is also presented as SEQ ID N0:1. The region in bold represents the nuclear targeting domain.
  • the underlined polypeptide a proteolytic product of wild type TAT protein, binds specifically to TAR- element-containing RNA (Weeks et al. , 1990) .
  • Figure 2B presents the sequence of the TAT-derived polypeptide, TAT24C (SEQ ID NO:2), which is derived from the underlined sequence of Figure 2A.
  • Figure 2C illustrates a phenanthroline moiety attached to a cysteine residue of a polypeptide.
  • Figures 3A-D show the results of gel shift mobility assays using modified and unmodified polypeptides and different RNA substrates.
  • Figure 4 presents the sequence of the HIV-1 encoded REV protein. This sequence is also presented as SEQ ID NO:6.
  • Figure 5 illustrates the chemistry of the at- tachment of a phenanthroline moiety to a cysteine- containing polypeptide.
  • Figure 6 illustrates the use of 2-iminothiolane for attachment of cleaving moieties to amino groups of polypeptides.
  • Figure 7 shows one scheme for attachment of iminodiacetic acid to polypeptides for Zn(II) binding.
  • Figures 8A and 8B show the results of cleavage assays using a variety of cleaving agents and an HIV-1 TAR-element containing substrate.
  • the drawing of the RNA substrate indicates cleavage sites (arrows) of TAT-based cleaving agents.
  • Figure 8C shows the results of cleavage assays using a variety of cleaving agents and a truncated HIV-1 TAR-element-containing substrate.
  • the drawing of the RNA substrate indicates cleavage sites (arrows) of TAT-based cleaving agents.
  • Figure 9A shows the results of cleavage assays using a variety of cleaving agents and an HIV-2 TAR-element containing substrate.
  • the drawing of the RNA substrate indicates cleavage sites (arrows) of TAT-based cleaving agents.
  • Figure 9B shows the cleavage products resulting from using tRNA as a substrate RNA.
  • Figure 10 provides an overview of a method for targeting and inactivation of HIV mRNA using oligonucleotides which contain cleaving moieties.
  • Figure 11 shows, in bold, potential triplex target sites within the HIV-1-LTR region: A (SEQ ID NO:8), B (SEQ ID NO:9), and C (SEQ ID NO:10).
  • Figure 12 shows oligonucleotides [A 1 (SEQ ID N0:11) and 2 (SEQ ID NO:12), B 1 (SEQ ID NO:13) and 2 (SEQ ID NO:14), and C 1 (SEQ ID NO:15) and 2 (SEQ ID NO:16)] designed to target the sequences presented in Figure 11 A, B, and C, respectively.
  • Next to each of these oligonucleotides is the general pattern of base triplets expected when triplexes are formed.
  • Oligonucleotides K (SEQ ID N0:17), L (SEQ ID N0:18), and M (SEQ ID N0:19) are the control oligonucleotides.
  • Figure 13 shows a schematic representation of triplex formation at an mRNA target site (bold) using a linear complementary oligonucleotide where the end loop contains basic oligonucleotides (-X- ) .
  • the RNA sequence is presented as SEQ ID NO:21 and the oligonucleotide is presented as SEQ ID NO:20, where N is an basic residue.
  • Figure 14 shows a schematic representation of the HIV-1 REV response element (RRE) ; this sequence is also presented as SEQ ID NO:23.
  • SEQ ID NO:24 presents the sequence of a truncated RRE binding site which corresponds to Stem II in the figure.
  • RNA-specific binding proteins can be adapted to perform site-specific cleavage of a target RNA when the target RNA contains the cognate binding site to which the RNA-binding protein binds.
  • One important application of these protein-based cleaving agents is the inactivation of mammalian viruses; in particular, RNA binding proteins modified to accomplish site specific cleavage can be used for inactivating RNA viruses, including the human immunodeficiency viruses (HIV) .
  • HIV human immunodeficiency viruses
  • RNA target site specific recognition RNA target site specific recognition
  • known or identifiable recognition sequence the ability to get the protein or polypeptide into cells containing the target RNA
  • a relatively small protein binding domain is preferable
  • fragments containing the protein binding domain should compete well for binding at the RNA target site with the native protein from which they are derived.
  • RNA-binding proteins Two examples of RNA-binding proteins useful in the practice of the present invention are the TAT and REV proteins encoded by HIV-l.
  • TAT consists of 86 amino acids and is a potent transactivator of long terminal repeat (LTR)-directed viral gene expression and is essential for viral replication (Dayton et al., 1986; Fisher et al., 1986).
  • LTR long terminal repeat
  • the amino acid sequence of the TAT protein is presented as SEQ ID NO:l.
  • TAT-induced transactivation requires the presence of the TAR (transactivation response) element, located at the untranslated 5' end of the viral mRNA element.
  • the RNA sequence of the TAR element is presented as SEQ ID NO:3.
  • the TAR element is capable of forming a stable stem-loop structure (Muesing et al., 1987) in the native viral RNA ( Figure 1) .
  • a 3 nucleotide (nt) bulge on the stem of TAR has been demonstrated to be essential for specific and high-affinity binding of the TAT protein to the TAR element (Roy et al., 1990; Cordingley et al . , 1990; Dingwall et al., 1989; Weeks et al., 1990).
  • the ability of (i) purified TAT protein and (ii) polypeptide fragments derived from TAT, which contain the nuclear targeting domain, to bind in vitro to RNA containing the TAR element make TAT a useful model for the RNA-cleaving reagents of the present invention.
  • the integrity of the stem and the initial U22 of the bulge are important or TAT protein binding (Roy et al. , 1990b) .
  • TAT protein binding Rost al. , 1990b
  • other sequences that do not affect the binding of the TAT protein to the TAR site are needed for trans-activation of tran ⁇ scription.
  • One such region is the sequence at the loop, which is required for the binding of cellular factors that may interact with the TAT protein to mediate transactivation (Gatignol et al. , 1989; Gaynor et al. , 1989; Marciniak et al. , 1990a; Gatignol et al., 1991).
  • the important components of the protein/RNA interaction are (i) the portion of the protein involved in binding to the specific target sequence, and (ii) the RNA target sequence required for binding.
  • TAT protein both full- length TAT (86 amino acids) and a truncated TAT protein (the N-terminal 72 amino acids) have been expressed as fusion proteins in Escherichia coli (Weeks et al., 1990).
  • the TAT proteins were linked to the E . coli catabolite activator protein via a protease recognition site.
  • polypeptide fragments were recovered from each fused protein which resulted from an unexpected cleavage between Gly48 and Arg49 ( Figure 2) , at the start of the lysine/arginine-rich RNA-binding region.
  • the fragments resulting from this cleavage, TRF38 and TRF24 corn-prise the regions extending from Arg49 to residue 86 and Arg49 to residue 72, respectively.
  • Both of these polypeptide fragments specifically bound RNA containing the TAR site (TAR-RNA) .
  • a synthetic 14-residue polypeptide spanning the basic region (amino acid residues 48-61 of the TAT protein) bound TAR-RNA as well.
  • TRF38 Up to three copies of TRF38 could bind to the wild- type TAR (wt-TAR) site, the first with an apparent dissociation constant of 5 nM and a second copy with an apparent dissociation constant of 20 nM. Only one copy of the TRF38 polypeptide could bind to a truncated version of TAR consisting of the minimal TAT binding site: the minimal site consists of 26 bases containing the upper part of the wt-TAR ( Figure 1C; Muesing et al., 1987). Similar binding characteristics were found for TRF24.
  • TAT24C A polypeptide similar to TRF24, designated TAT24C, was chemically synthesized (Example 1A) .
  • TAT24C consists of amino acid residues 49-72 of the TAT protein (Figure 2A) and an additional cysteine residue at the C-terminus ( Figure 2B, SEQ ID NO:2).
  • the TAT24C polypeptide was purified by HPLC and reacted with 5-iodoacetamido-l,10- phenanthroline to attach a 1,10-phenanthroline moiety to the cysteine residue (peptide designated TAT24C-phen, see below) .
  • RNA substrates are prepared as described in Example 2.
  • Several TAR-containing RNAs were synthesized to use as substrates in binding assays to test the binding activity of the modified polypeptide
  • TAT24C-phen (Example 2) .
  • the predicted secondary structures of the target RNAs are shown in Figure 1C.
  • the RNA substrate designated HIV-1 TAR is the 57-nt RNA stem-loop structure found in native HIV-1 mRNA (Sharp et al. , 1989).
  • the RNA substrate designated ⁇ TAR is a truncated RNA containing the minimum TAT binding site (nt 17-43) (Weeks et al., 1990).
  • HIV-1 the etiologically associated virus of AIDS, another retrovirus, termed type 2 (HIV-2) has been reported (Clavel et al., 1986). HIV-2 also possess a functional TAT gene (Arya et al.
  • HIV-2 TAT responsive element Two subelements responsible for the TAT-mediated transactivation in the HIV-2 TAT responsive element (TAR) have been identified and contain two stem-loop struc ⁇ tures confined to +1 - +103 nucleotides (Arya et al., 1985).
  • the sequence of HIV-2 TAR RNA used in the present study is from +13 - 90 nucleotides and contains both stem-loop structures.
  • FIG. 3 shows a photograph of a representative gel mobility shift assay.
  • discrete bands having retarded mobility for samples containing either phenanthroline- odified or un ⁇ modified polypeptides demonstrate the binding of both polypeptides to each of the three RNAs con ⁇ taining TAR elements ( Figure 3A-C) .
  • the attach- ment of the extra cysteine and the phenanthroline moiety at the C-terminus of the TAT24 polypeptide does not substantially affect binding to the TAR site.
  • Essentially no retardation of tRNA was observed when it was incubated with high levels of the TAT24C polypeptide ( Figure 3D) , indicating relatively specific binding to TAR-site-containing RNA substrates.
  • TAT protein As well as fragments of TAT containing the nuclear targeting region, are rapidly taken up by cells.
  • the TAT protein taken up by cells in this fashion specifically activates HIV-1 LTR-linked gene expression (Green et al., 1988; Frankel et al., 1988).
  • the entry of polypeptides containing (i) the nuclear targeting region of TAT and (ii) nucleic acid cleaving agents can be evaluated as described below.
  • REV protein is a regulatory factor essential for viral replication; it is required for the production of viral structural proteins. It appears to exert its effect at the level of splicing and perhaps transport of viral mRNA into the cytoplasm (Malim et al., 1989a, 1989b; Felber et al., 1989); further REV appears to increases the stability of unspliced HIV mRNA (Felber et al., 1989).
  • the REV protein consists of 116 amino acids ( Figure 4, SEQ ID NO:6), encoded by two exons.
  • arginine-rich domain acts as the nuclear tar ⁇ geting domain (Malim et al., 1989b). Mutational analysis has demonstrated that some C-terminal deletion mutants of the REV protein are non ⁇ functional, in terms of normal REV functions, but are trans-dominant as illustrated by competitive inhibition of wild-type REV functions.
  • REV response element The action of REV requires the presence of a target sequence termed the REV response element
  • RRE Figure 14, SEQ ID NO:23
  • Malim et al. , 1989a, 1989b RRE
  • RRE has been mapped to a 234-nucleotide region capable of forming four stem-loop structures and one branched stem-loop structure
  • REV offers another potential means of targeting a cleaving agent specifically to HIV RNA and has a potential advantage over the TAT protein in that REV has more potential binding sites.
  • RNA-binding proteins and their cognate binding sites can be characterized as has been de ⁇ scribed above for the TAT protein and TAR site. Truncated versions of any such protein and/or binding site can be evaluated for protein/-peptide binding using, for example, the gel mobility shift assay or standard filter binding assays (Sauer et al. ; Radding et al.). Further, cellular uptake can be evaluated. If the protein is not adequately taken up by cells at concentrations useful to provide intracellular catalytic function, i.e., cleavage, then alternative methods of cellular uptake, such as targeted liposomal delivery where the liposomes carry target cell surface recognition moieties, can be employed to get polypeptide fragments into target cells.
  • a number of chemical moieties are capable of cleaving nucleic acid substrates including phenan ⁇ throline (Chen et al., 1986, 1987; Francois et al., 1989; Ebright et al., 1990), Fe(II)-EDTA (Dreyer et al., 1985; Dervan, 1986; Moser et al., 1987; Maher et al., 1989; Sluka et al. , 1987), Cu(II)-bipyridine, Cu(II)-terpyridine, and Zn(II) (Modak et al., 1991; Eichhorn et al., 1971; Ikenaga et al., 1974; Breslow et al., 1989).
  • These chemical cleaving moieties can be employed in the present invention as exemplified below with reference to the TAT24 C-phen protein.
  • Nucleic acid cleaving moieties are attached to the TAT derived polypeptides as described in Example IB. The chemically synthesized
  • HPLC-purified polypeptides are reacted with 5-iodoacetamido-l,10-phenanthroline (phenanthroline moiety) to obtain polypeptides containing the phenanthroline moiety uniquely attached to the side chain of the cysteine residue.
  • Figure 5 illustrates the attachment of a phenanthroline moiety to cysteine-containing polypeptides.
  • TAT24-C polypeptide (SEQ ID NO:2), consisting of that 24-residue domain plus a single cysteine residue at the C-terminus, was chemically synthesized (Example 1A) .
  • a phenanthroline moiety was then attached to the sulfhydryl group of the cysteine to obtain TAT24C-phen (Example IB) .
  • the cleaving agent can also be attached at residues other than cysteines.
  • phenanthroline can be attached to the side chain- amino groups of lysines and arginines, as well as to the amino group at the N-terminus, by reacting the protein first with 2-iminothiolane hydro ⁇ chloride, followed by coupling with 5-iodoace- tamido-l,10-phenanthroline (Chen et al. , 1987). This coupling is illustrated in Figure 6 and described in Example IB. Because of the higher loading of cleaving moieties per protein/peptide molecules, such protein/peptide molecules are expected to be efficient reagents for cleavage and are expected to be resistant to in vivo degradation.
  • nucleic acid cleaving agents can also be used in the methods of the present invention, including Fe(II)-EDTA,
  • the intracellular reduction potential can be affected using N-acetyl cysteine, which increases the intracellular glutathione level (Roederer et al. , 1990; Kalebic et al. , 1991), in order to assist in keeping the metal atom of the cleaving agent in the reduced state.
  • N-acetyl cysteine increases the intracellular glutathione level (Roederer et al. , 1990; Kalebic et al. , 1991)
  • RNA degradation is known to be induced by divalent metal ions, especially Zn(II) , in the absence of a reducing agent.
  • the reduced hydrolytic cleavage compared to reaction in the presence of a reducing agent can be at least partly of set by having more than one chelating molecule attached to the polypeptide.
  • One technique for tethering Zn(II) to a protein is via coordination by iminodiacetic acid (Aldrich, Milwaukee WI) : one possible scheme for such attachment is shown in Figure 7. Briefly, iminodiacetic acid is converted to its diethyl ester to protect carboxylic function ⁇ alities. The resulting product is condensed with iodoacetic acid in the presence of dicyclohexyl carbodiimide (DCC) to obtain compound 3 ( Figure 7) . Compound 3 is then reacted with, for example, a polypeptide containing a cysteine residue.
  • DCC dicyclohexyl carbodiimide
  • the polypeptides are screened for specific binding to their cognate nucleic acid binding site, the polypeptides carrying nucleic acid cleaving moieties are next evaluated for their ability to cleave the target nucleic acid.
  • a non-specific enzymatic nuclease can be attached to a sequence specific RNA binding protein to form a sequence-specific ribonuclease.
  • Staphylococcal nuclease a non-specific nuclease that attacks both RNA and DNA, has been converted to site- specific DNA endonuclease by attaching the protein to an oligonucleotide (Corey et al., 1987; Pei et al., 1990).
  • hybrid fusion pro ⁇ teins are generated between the RNA sequence- specific protein and the coding sequence of Staphylococcal nuclease.
  • the hybrid proteins can be expressed using any number of standard expres ⁇ sion systems (e.g., "CLONTECH” commercially available vectors) .
  • CLONTECH commercially available vectors
  • the hybrid protein is expressed in E. coli using the OmpA-derived expression system (plasmid pONFl) already adopted for the secretion of staphylococcal nuclease (Takahara et al. , 1985) .
  • the signal peptide required for the secretion of o pA protein is fused to the nuclease gene to obtain large amounts of secreted nuclease: the nuclease is then released from the signal polypeptide by appropriate processing.
  • DNA sequences encoding the TAT, REV, or derivative RNA-binding poly ⁇ peptides are cloned adjacent to and in-frame with the staphylococcal nuclease gene in plasmid pONFl.
  • the RNA-binding protein coding sequence can be generated by any number of methods including polymerase chain reaction amplification (Mullis et al. ; Mullis) and standard cloning technology (Ausubel et al.
  • RNA-binding protein/- peptide coding sequence is performed by standard cloning methods (Ausubel et al. ; Maniatis et al.; Sambrook, et al.) .
  • the resulting hybrid proteins are then expressed in E. coli and purified (Takahara et al., 1985).
  • hybrid proteins/peptide nucleases to bind their cognate nucleic acid substrate is evaluated as described above using, for example, the gel mobility shift or filter binding assays.
  • hybrid protein/peptide nucleases are screened for specific binding to their cognate nucleic acid binding site, they are next evaluated for their ability to cleave the target nucleic acid.
  • the chemical nuclease activity of Cu(II)-com- plexed l,10-phenanthroline derives from an oxida- tive attack on the sugar ring by a copper-oxo species generated in the presence of a reducing agent (Sigman et al., 1990).
  • RNA target molecules containing the binding sites were 5'-end- labeled, purified on denaturing polyacryl- amide gels, and annealed for use as substrates for cleavage (Example 2) .
  • a typical cleavage reaction contained 10 3 cpm of end-labeled RNA and 40-100 ng of polypeptide.
  • Example 4A describes RNA cleavage reactions utilizing the TAT24C-phen polypeptide. The target RNA was incubated with TAT24C-phen at 25"C for 10 min before the cleavage was initiated by adding CuS0 4 and mercaptopropionic acid.
  • Cu(II) is different from their reactivity toward the polypeptide.
  • the effect of the TAT24C-phen was examined relative to the HIV-2 TAR site (Example 4C) .
  • the primary cleavage site found on the HIV-2 TAR is somewhat unexpected ( Figure 9, lanes 1 and 2).
  • the cleavage site of the HIV-2 target was anticipated to be predominantly at the loop close to the TAT binding site (loop 1, Figure 9A) .
  • the TAT24C-phen polypeptide appears to cleave the HIV-2 TAR-RNA at a site located towards the 5' end of its binding site ( Figure 9A) .
  • the primary cleavage site in the HIV-2 TAR site is not in the loop and does not appear to have unpaired bases (as implied by the SI nuclease cleavage reactions the results of which are shown in lane 4, Figure 9A) .
  • the primary site of cleavage in the HIV-2 substrate is likely the consequence of the tertiary structure of the HIV-2 TAR RNA.
  • the HIV-2 target consists of two stem- loop structures: the HIV-2 TAR RNA sequence may have a complex tertiary structure reduces the otherwise favorable interaction of the polypeptide-bound cleaving moiety with the loop.
  • the TAT24C-phen polypeptide does, however, cleave both HIV-2 target loops.
  • the cleavage of the loop 1 is ex ⁇ pected and the cleavage of the remote second loop may be the result of the two loops being in a spatially close orientation within the overall secondary structure of the HIV-2 substrate.
  • cleavage sites of the RNA tar ⁇ gets lie on either side of the bulge where the TAT protein is known to bind (Roy et al., 1990;
  • HIV-2 TAR In contrast to cleavage of HIV-1 TAR, for which the primary site is at the loop adjacent to the TAT binding site, cleavage of HIV-2 TAR takes place mainly at the stem, roughly midway between the two loops ( Figure 9A, lanes 1 and 2) .
  • HIV-2 TAR has two 2-nt bulges, both of which have the consensus TAT binding motif (Weeks et al. , 1990; Green- et al., 1988; Frankel et al. , 1988; Milligan et al., 1987; Arya et al. , 1988; Weeks et al., 1991; Murakawa et al. , 1989).
  • RNA target mole ⁇ cules using modified binding polypeptides, i.e., proteins known to have binding sites in a selected RNA target molecule.
  • modified binding polypeptides i.e., proteins known to have binding sites in a selected RNA target molecule.
  • REV-derived polypeptides encompassing the nuclear targeting domain are synthesized and attached to cleaving agents as described for reagents based on the TAT protein.
  • a synthetic peptide spanning the basic domain of the REV protein (SEQ ID NO:22) has been shown to bind specifically to the RRE target (Kje s et al., 1991) ; in vitro the same peptide inhibits the splicing of mRNA containing RRE.
  • target RNA molecules can be cleaved using polypeptides modi ⁇ fied with iminodiacetic acid in the presence of ZnCl 2 .
  • RNA binding proteins or polypeptides which are derived from these proteins, depends on seve ⁇ ral factors: (a) the binding affinity and speci- ficity of the reagent; (b) the spatial positioning of the cleaving moiety; (c) the nature of the cleaving reagent; and (d) reaction conditions.
  • the specificity and the binding affinity of a binding polypeptide to its cognate binding site in an RNA target molecule may potentially be increased by increasing the length of the polypeptide, for example: (i) by extending the polypeptide length from the N-terminus, (ii) the C-terminus, or (iii) both ends of the basic nuclear targeting domain of a transactivator.
  • TAT24C-phen In the case of the TAT and REV proteins the maximum number of residues needed to maximize specific binding is not expected to be more than 50 residues, because both trans-activators are relatively small, on the order of 100 residues, and both have functional domains besides the RNA binding domain.
  • Two other approaches to increa ⁇ sing the binding specificity of TAT24C-phen to the target TAR site are as follows: (i) evaluating in vitro generated mutations (Ausubel et al.) for mutations that increase the binding specificity of the protein/peptide to the cognate binding site (Sauer et al.); (ii) chemical alterations of the polypeptides which can effect a general improve ⁇ ment of RNA binding, such as replacing Asp and Glu with Asn and Gin.
  • chemical modifi ⁇ cation can be used to convert Asp and Glu to esters or amides to increase the net positive charge of the polypeptide.
  • Chemical modification reactions that occur in solution can be performed on the polypeptide bound to the target RNA where possible, so that sites critical for binding are protected against alteration (Galas et al. ; Siebenlist et al.).
  • the spatial positioning of the cleaving moi ⁇ ety may be adjusted. Because of the three-dimen ⁇ sional folding of polypeptides, the position of the cleaving agent within the polypeptide molecule can be crucial for the cleavage. In the cases of TAT and REV proteins the three dimensional struc ⁇ ture of the proteins is not known. Accordingly, favorable locations for the placement of the cleaving moiety can be empirically determined; the single cysteine residue can be placed at several positions, including the N-terminus, C-terminus, and internal positions of the polypeptide which do not affect RNA-binding ability.
  • cleaving reagent in order to improve the efficiency of cleavage reactions using RNA binding proteins or polypeptides derived from these proteins the nature of the cleaving reagent can be modified as described above, using chemical or enzymatic cleaving moieties.
  • reaction conditions can be modified to improve cleavage of RNA substrates by, for example, increasing reduction potential in the reaction mixture or intracellularly by, for example, adding N-acetyl cysteine (or perhaps ascorbate) to the system.
  • In vitro reaction conditions can also be modified by altering temperature, ionic conditions, the amount and type of reducing agent, and pH.
  • the cleavage assay will be used to assess the effects of the above factors on the efficiency of the cleavage reaction.
  • Single variables will be modified to evaluate efficacy. For example, to assess the cleavage induced by different peptides, an identical concentration of the different pep ⁇ tides are used in reaction mixtures containing the other reagents at fixed concentrations. After the cleavage reaction has been carried out for a specified period, digested end-labeled RNAs (e.g., HIV-1 TAR) will be resolved on sequencing gels, the gels will be autoradiographed, and bands corresponding to starting material (intact RNA) and cleavage products will be excised.
  • digested end-labeled RNAs e.g., HIV-1 TAR
  • the radioactive counts present in the excised bands will be determined by scintillation counting.
  • the relative concentrations of cleavage product to starting material is then determined ( (cpm prod /cpm Pro +c P in i ntact ) 10 °) •
  • Densitometry scanning can also be used to evaluate efficiency of cleavage reac ⁇ tions by using films which have not been overex ⁇ posed.
  • RNA molecules contain both single- and double-stranded regions that can offer targets for oligonucleotide binding (Zamecnik et al., 1986; Rittner et al., 1991).
  • RNA oligonucleotide agents must con ⁇ tinuously bind to the target molecules in such a way as to inactivate them.
  • a clea ⁇ vage agent is attached to RNA oligonucleotides, the oligonucleotides only need to bind the target RNA long enough to cleave it in order to achieve permanent inactivation.
  • a chosen cleaving group (see above for chemical and enzyma- tic cleaving groups) is attached to oligonucleo ⁇ tides which are resistant to cleavage by endo ⁇ genous nucleases.
  • backbones include deoxy- ribose or ribose sugar moieties connected by methyl phosphonate or phosphorothioate linkages (Miller et al., 1985).
  • RNA-binding oligonucleotides have sequences of the following two types: (i) sequences designed to form a du ⁇ plex with putatively single-stranded regions of a target RNA, and (ii) sequences designed to form triplexes with homopurine regions of the DNA which encodes the RNA-target, for example, a DNA pro- virus.
  • one mRNA target site is the TAR region, because base pairing at this site by a complementary oligonucleotide is expected to block formation of the stem-loop structure required for binding and transactivation by TAT.
  • An inter-molecular duplex is potentially more stable than the intra-molecular stem-loop duplex due to the absence of unpaired bases. Further, such an inter-molecular duplex may be able to displace the stem-loop structure by pairing initially with the loop or with single-stranded regions adjacent to the stem, particularly in view of the observation that alteration of non-essential sequences adjacent to TAR create competing secondary structures which inhibit TAR function (Berkhout et al., 1989).
  • a major advantage of targeting the DNA pro- virus associated with an RNA virus is that typically only one, or a few copies, of integrated, transcriptionally active DNA are present per cell in contrast to many copies of mRNA which may be present in an infected cell (Soma et al., 1988).
  • Homopurine-homopyrimidine regions of duplex DNA can bind single-stranded oligonucleotides having the same sequence as either the homopurine or the homopyrimidine strand of the target DNA but with the reverse polarity (Dervan, 1986) , forming purine-purine-pyrimidine or pyrimidine-purine-pyrimidine triplexes, respectively.
  • the purine-purine-pyrimidine triplexes typically require a divalent cation such as Mg ++ or Zn ++ for their stability but are relatively independent of pH (Lyamichev et al., 1991) .
  • the pyrimidine-purine-pyrimidine triplexes require divalent cation but are favored by slightly acid pH.
  • triple helix approach for targeting DNA to inhibit expression has had limited use due to the requirement for homopurine target sequences.
  • Triplex formation at an oligopurine*oligopyrimi ⁇ dine tract can be induced by a single strand consisting of either only pyrimidines or only purines. Sequence-specific recognition by the oligopyrimidine strand relies on the formation of PyPuPy (C+ «GC and T ⁇ T) base triplets (Moser et al., 1987). In this case, the oligopyrimidine strand is parallel to the purine tract of the duplex.
  • oligopurine strand lies anti-parallel to the purine tract of the duplex, and sequence-specific recognition in this case is brought about by Pu «PuPy (G «GC and A*AT) base triplets (Kohwi et al., 1988; Beal et al.,
  • a sequence of 15-18 purines is required to achieve sufficient specificity, and this requirement limits the triplex approach in controlling the expression of a particular gene. Although long homopurine stretches do occur in viral genomes, finding such a sequence within a gene vital to the virus can be difficult.
  • triplex formation can occur at tandem oligopurineOligopyrimidine sequences using normal DNA, without any unnatural linkages or synthetic base analogues.
  • sequences utilize both types of base triplets, Pu'PuPy and PyPuPy, in forming a triplex.
  • this approach allows the formation of triplexes at base sequences made up of both purines and pyrimidines.
  • the incorporation of Pu «PuPy base triplets has the advantage that triplex formation does not demand low pH, which is usually the case when the C+ «GC base triplet is involved.
  • triplex- forming oligonucleotides are designed to interact with ho opurine-homopyrimidine sequences in the pro-virus.
  • three potential targeting sites are three potential targeting sites (sequences in bold, Figure 11A, 11B, 11C) for targeting with single-stranded oligonucleotides.
  • These cleaving-oligonucleotide reagents bind and cleave the DNA provirus as well as the mRNA of HIV, increasing the likelihood of preventing viral replication.
  • All three target sites are located in the control region of the HIV-LTR (i.e., upstream of the transcription initiation site) and therefore do not interact with mRNA sequences to function as anti-sense mediators.
  • the potential target sites A, B, and C have different triplex forming motifs:
  • Site A consisting exclusively of purines, is targeted for triplex formation using oligonucleotides A-l and A-2 ( Figure 12) , which are capable of forming triplexes with Pu «PuPy and PyPuPy base triplets, respectively.
  • Site B consists of a tract of pyrimidine residues flanked by two purine tracts and is targeted with oligonucleotides B-1 and B-2 ( Figure 12) , having the correct polarity to bind with two strands of the target (see above) .
  • Site C has some pyrimidines scattered within a highly purine-rich sequence, and oligonu ⁇ cleotides C-l and C-2 ( Figure 12) are directed toward site C.
  • oligonucleotides K, L, and M correspond, respectively, to sites A, B, and C in the reverse polarity and are therefore blocked from triplex formation; these oligonucleotides are used as controls.
  • Test oligomers with and without phenanthroline are used to assess the effect of cleavage. Attachment of 1,10-phenanthroline to oligo ⁇ nucleotides is achieved as follows. During chemi ⁇ cal synthesis each oligonucleotide is synthesized with a thiol group at the 5' end by use of the "C6-thiol modifierTM 11 reagent from Clontech (Palo Alto, CA) according to the manufacturers instruc ⁇ tions.
  • the oligonucleotides are de-protected with NH 4 OH and treated with silver nitrate to expose the thiol group.
  • the oligonucleotide is immediately reacted with 5-iodoacetamido 1,10-phenanthroline as described above to covalently link 1,10-phenan ⁇ throline to polypeptides.
  • pHIV-lLTR-CAT is linearized with Hindlll, end-labeled with 32 P ⁇ 7 ⁇ ATP using polynucleotide kinase, and subjected to a second restriction digest to obtain a uniquely labeled DNA fragment containing the duplex target sequence.
  • this DNA fragment is mixed with an appropriate modified oligonucleotide in a buffer containing 10 mM Tris-HCl, 100 mM NaCl, 100 ⁇ M spermine, and 10 mM MgCl 2 .
  • the pH of the buffer is adjusted depending on the sequence of the target (a lower pH is used for the formation of C+ «GC base triplets) .
  • cleavage is initiated by adding CuSo 4 (to 10 ⁇ M) and mercaptopropionic acid (to 2.5 mM) .
  • Cleavage products are resolved on sequencing gels along with the products of sequencing reactions. This method allows the mapping of the site of triplex formation and the cleavage efficiency (detected by counting the radioactivity of excised gel bands) ; cleavage efficiency is used to quantitate the efficiency of triplex formation.
  • the CAT gene is transiently expressed under the direction of HIV-1 LTR in HeLa cells.
  • HeLa cells are trans- fected with pHIV-lLTR-CAT, using the DEAE-dextran technique (Queen et al., 1983). Twelve hours after transfection, the cells are incubated with an oligonucleotide, as described by Postel et al. (1991) , and itomycin C (SIGMA) is added to the medium to induce CAT expression.
  • SIGMA itomycin C
  • Cells are har ⁇ vested at 12 and 24 hr and CAT activities deter ⁇ mined as described by Gorman et al. (1982) and compared to controls, i.e., cells that have been exposed to control oligonucleotides (K, L, and M) and cells without oligonucleotide treatment.
  • oligonucleotides carrying phenanthroline are complexed with CuS0 4 before being introduced to the cell medium.
  • Ascorbic acid or mercaptopropionic acid
  • Ascorbic acid is supplied to the medium 12 hr after the oligonucleotide treatment and cells are harvested and assayed for CAT activity after another 24 hr.
  • the effect of oligonucleotides in the pre- sence of TAT protein is assayed using p-HIV-lLTR- CAT under stable expression conditions (described above) .
  • CAT-active clones are transfected with a TAT expression vector and these cells, which are transiently expressing TAT are used for oligonu- cleotide treatment followed by the measurement of CAT activity.
  • triplex helix methods An alternative to the above described triplex helix methods is to use an oligonucleotide-based approach where a single-stranded oligonucleotide is capable of forming a triplex with HIV mRNA by contributing two "strands" connected by a hairpin loop ( Figure 10; Figure 13) .
  • This triplex- directed anti-sense approach is expected to be more effective in arresting biological processes such as translation and reverse transcription than is the convention anti-sense approach where a DNA- RNA duplex is formed.
  • Triplex formation in this fashion is highly selective and of high affinity and may not be a substrate for enzymes such as helicases. The action of such helicases has been a potential problem in the conventional anti-sense approach (Bass et al. , 1987).
  • oligonucleotides For cleavage of target RNA substrates, oligonucleotides have a chemical cleaving group attached to one end ( Figure 10) and an inter- calator linked to the other end. Because de- protection procedures are different and indepen ⁇ dent from each other, derivatization at the two ends can be performed at two stages of oligonu ⁇ cleotide synthesis.
  • the cleaving reagents of the present inven ⁇ tion provide means for a method of cleaving RNA targets at specific sites. Such cleavage is useful for the analysis of RNA structure and function as well as diagnostic analyses.
  • One example of a diagnostic application is to isolate RNA from a cell infected with a particular RNA virus. Total or poly-A+ RNA (Ausubel et al.) is end labeled. The RNA is then isolated away from free label and the amount of incorporated label estimated, for example, by scintillation counting.
  • RNA cleaving agent such as an RNA-binding protein combined with a chemical cleaving moiety
  • the cleaving reagents of the present invention are particularly desirable for use with RNA virus tar ⁇ gets or their pro-viral DNA forms: for example, cleaving HIV genomic RNA or pro-viral DNA.
  • the cleaving reagents of the present inven ⁇ tion are also useful in a method of inhibiting expression of RNA viral (e.g., HIV) antigens in cells infected with the virus.
  • the infected cells are exposed to an RNA binding protein or polypeptide modified to contain a cleaving moiety (i.e., the reagent), at a re ⁇ agent concentration effective to produce reduction in viral antigen expression in the infected cells.
  • a cleaving moiety i.e., the reagent
  • both modified and unmodified polypeptides are assayed for their ability to enter the cell.
  • One method to evaluate cellular uptake is to label the poly ⁇ peptides with a fluorescent dye, such as fluores- cein isothiocyanate (FITC) (Pierce, Rockford, IL) at the single cysteine residue.
  • FITC fluores- cein isothiocyanate
  • the fluores ⁇ cent-labeled polypeptides are added to the cell culture medium and the cellular distribution analyzed by fluorescence microscopy.
  • fluorescent labeling is carried out at a single cysteine residue before reacting amino groups with 2-iminothilane for attachment of the cleaving moiety.
  • uptake of the re ⁇ agent polypeptide can be evaluated using radio ⁇ active label since any polypeptide can be easily made radioactive during synthesis (Chen et al. , 1986) .
  • Another alternative is to perform an immuno-fluorescence assay on fixed cells after incubation with the reagent using rabbit anti- peptide- antibodies and rhodamine-conjugated goat anti-rabbit antibodies (Malim et al., 1989).
  • RNA-binding protein can be applied to any protein or polypeptide under investigation, e.g., TAT or REV.
  • TAT covalently attached to the chemical cleaving group, 1,10-phenanthroline results in cleavage of target TAR sequences consistent with polypeptide binding to the 3-nt bulge.
  • RNA-cleaving protein/peptide reagents for example, the in vivo usefulness of the TAT24C-phen polypeptide is tested using a number of cell systems including the following:
  • Chloramphenicol acetyltransferase (CAT) assays HIV-1 LTR-directed CAT activity is mea ⁇ sured under transient expression as well as stable expression conditions.
  • HeLa cells will be transfected with an expression vector containing the entire U3 region and 78 base pairs of the R region of the HIV LTR (e.g., pHIV- 1LTR-CAT (S. Miller, SRI International, Menlo Park CA) ; or Gendelman et al., 1985).
  • the LTR region contains the enhancer, promoter and TAR elements. Transfection is performed using the DEAE-dextran technique (Queen et al. , 1983).
  • the cells are incubated with the polypeptide reagent, over a range of polypeptide concentrations. Mitomycin C is added to the medium to induce CAT expression. Since the HIV-1 LTR is under the influence of NF- kB, the expression of CAT activity can be induced by treating with either UV or mitomycin C (Nabel et al., 1987). After 12 and 24 hours the cells are harvested and CAT activities are determined as described by Gorman et al. (1982) . CAT activities are compared between (i) cell samples which were not treated with the polypeptide reagent, and (ii) cells samples which were treated with the poly ⁇ peptide reagent.
  • Cleavage of the target substrate by the polypeptide reagent is expected to result in a decrease of CAT activity.
  • Polypeptide re ⁇ agents containing phenanthrolene are complexed with CuS0 4 before addition to the cell samples. If the cellular reduction potential is not sufficient for the cleavage to occur, ascorbic acid (or mercaptopropionic acid) is added to the medium.
  • CAT expression system To assess the activity of polypeptide reagents in the presence of wild-type TAT protein, a stable CAT expression system is used. HeLa cells are cotransfected with a 1:5 ratio of pSV2neo (a mammalian integration plasmid which confers neomycin-resistance; Southern et al., 1982) and pHIV-lLTR-CAT plasmids using DEAE- dextran procedure. Cells are selected for G418 resistance, and individual colonies are picked, expanded, and tested for CAT expression. CAT- active clones are transfected with a wild-type TAT expression vector (e.g., pcDEBtat, S. Miller, SRI International; or pAR, available from the AIDS Re ⁇ search and Reference Program) . Cells now expressing TAT transiently are used for polypeptide treatment followed by the measurement of CAT activity.
  • pcDEBtat a mammalian integration plasmid which confers
  • the TAT24C-phen polypeptide reagent is added to the culture media over a range of concentra- tions.
  • the ability of TAT24C-phen to block the transactivation by endogenous TAT protein is determined by measuring chloramphenicol acetyl transferase (CAT) activity over time after the addition of the TAT24C-phen polypeptide.
  • CAT chloramphenicol acetyl transferase
  • HIV antigen levels including p24, asso ⁇ ciated with HIV-infected cells (e.g., by ELISA (Wang et al. , 1988, 1989; Crowe et al., 1990);
  • TAT24C-phen Blocking acute infection — The abil- ity of TAT24C-phen to prevent acute infection by HIV of the following cells will be assessed: PHA-stimulated human peripheral blood lymphocytes, MT4 and Jurkat cells (both CD4+ lymphocyte cell lines) , macrophages, and monocytes infected by monocytropic HIV isolates.
  • TAT24C-phen polypeptide is evaluated using, for example, killing of Jurkat cells. Also, mutagenicity is evaluated with a standard Ames test.
  • the intracellular reduction potential can be modulated using N-acetyl cysteine, which increases the intracellular glutathione level (Roederer et al., 1990; Kalebic et al., 1991).
  • N-acetyl cysteine increases the intracellular glutathione level
  • Such manipulation of the intracellular reduction potential assist in keeping, for example, a copper atom of a cleaving agent in the reduced state.
  • RNA cleaving reagents composed of an RNA- binding protein and a non-specific nuclease also have important in vivo applications. A specific RNA cleaving-hybrid nuclease can be evaluated as described above when the hybrid nuclease is taken up into cells.
  • CAT expression in Hela cells harboring target RNA-CAT fused genes are assayed in the presence and absence of hybrid- nuclease expressed from an independent promoter.
  • the gene for staphylococcal nuclease is cloned adjacent to the TAT or REV gene in plasmids pSV2TAT 72 (or pgTAT) and pCREV, respectively.
  • the resultant plasmids encoding hybrid proteins are transfected into Hela cells carrying either pHIV-CAT or pHIV-env depending on the type of hybrid nuclease.
  • the biological effects of the in vivo expression of TAT24C-nuclease is evaluated using the CAT assay as described above.
  • the effect of the hybrid nuclease containing the gene product of REV will be assayed in Hela cells by quantitating the repression of the production of viral envelope protein as assayed using antibodies against envelope proteins.
  • attaching staphylococcal nuclease to an RNA binding polypeptide e.g., based on TAT and REV
  • a short tether of several amino acids may generate a sequence-specific ribonuclease.
  • Constitutive expression of such an RNA-specific nuclease in an un-infected cell, contained in a population of cells infected with an RNA virus that contains the target RNA binding sequence may confer resistance of the un-infected cells against viral infection.
  • peripheral blood mononucleocyte cells are isolated from the blood of an HIV-positive patient.
  • T-cells are isolated and transformed to carry a TAT-nuclease hybrid protein encoding gene.
  • the cells are amplified and replaced in the patients blood stream.
  • Such an approach may lead to a gene therapy for the treatment of AIDS: providing HIV-resistant T- cells.
  • RNA cleaving reagents combined with the above- described oligonucleotide cleaving agents may provide a two-pronged attack against viral diseases by providing cleavage of viral RNA and pro-viral DNA.
  • Synthetic oligonucleotide linkers and primers were prepared using commercially available auto- mated oligonucleotide synthesizers. Alternative ⁇ ly, custom designed synthetic oligonucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA) .
  • Oligonucleotide sequences encoding peptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of cloning steps involving a tandem array of multiple oligonucleotide fragments correspond ⁇ ing to the coding sequence (Crea; Yoshio et al.; Eaton et al.). Oligonucleotide coding sequences can be expressed by standard recombinant proce ⁇ dures (Sambrook et al.; Ausubel et al.)
  • peptides can be synthesized directly by standard in vitro techniques (Applied Biosystems, Foster City CA) .
  • T7 RNA polymerase was purchased from Promega (Madison, WI) and used as per the manufacturer's instructions.
  • Polynucleotide kinase and restriction enzymes were obtained from Boehringer Mannheim (Indianapolis IN) or New England Biolabs (Beverly MA) and were used as per the manufacturer's directions.
  • Ribonuclease Tl, Ribonuclease CL3, and SI nuclease were obtained from Boehringer Mannheim and were used as per the manufacturer's direc ⁇ tions.
  • Uranyl nitrate was obtained from Mallencrodt (Paris KY) .
  • Radionuclides were obtained from New England Nuclear (Boston, Mass.), ICN (Costa Mesa CA) or Amersham (Arlington Heights IL) .
  • TAT and REV proteins are isolated as previously described (Weeks et al., 1990, herein incorporated by reference; Brown et al., 1990, herein incorporated by reference) .
  • the entire protein coding sequences of the TAT and REV proteins are presented in Figures 2A (SEQ ID N0:1) and 4 (SEQ ID NO:6), respectively, polypeptides derived from these proteins were synthesized at SRI's polypeptide synthesis facility using a Beck an model 990 polypeptide synthesizer, as per the manufacturer's instructions.
  • Polypeptides having the same C-terminal sequence but truncated at different N-terminal sites are recovered from a single solid-phase synthesis by removing some of the reaction bed at different stages of the syn- thesis.
  • cysteine residue can be added at any position internal to the polypeptide sequence or at either the amino- or carboxy- terminal ends of the protein.
  • the insertion of cysteine groups must be consistent with the maintenance of RNA-binding activity; such binding properties can be tested as described below.
  • TAT24C The TAT polypeptide designated TAT24C was chemically synthesized as just described.
  • TAT24C consists of amino acid residues 49-72 of the TAT protein (Figure 2A, underlined sequence) and an additional cysteine residue at the C-terminus ( Figure 2B, SEQ ID NO:2).
  • the TAT24C polypeptide was purified by standard HPLC.
  • the resulting polypeptides were separated from un-reacted iodo compound by passing the reaction mixtures through Sephadex G-50 spin columns (Pharmacia, Piscataway NJ) .
  • HIV-1 TAR is the 57-nt RNA stem-loop structure found in HIV-1 mRNA (nt 1-57); HIV-2 TAR includes the region of HIV-2 RNA essential for transactivation by HIV-2 TAT (nt 13-91, Arya et al., 1988); and ⁇ TAR is a truncated RNA containing the minimum TAT binding site (nt 17-43, Weeks et al., 1990 ).
  • the three RNA substrates are shown in Figures 1A, IB, and lC.
  • RNA substrates are presented in the sequence listing as follows: HIV-1 TAR, SEQ ID NO:3; HIV-2 TAR, SEQ ID NO:4; and ⁇ TAR, SEQ ID NO:5.
  • the RNA substrates were synthesized as follows. Synthetic DNA templates were formed by standard phosphoramidate synthesis using a Model 381B synthesizer (Applied Biosystems, Foster City, CA) . The synthetic DNA templates, containing T7 promoter sequences (Stahl et al. , 1981; Davanloo et al.
  • RNA molecules were purified by size fractionation on denaturing 10% polyacrylamide gels (Maniatis et al. ; Sambrook et al.) followed by electroelution. The isolated RNA molecules were then heated to 70'C and slowly cooled to room temperature to facilitate formation of the native secondary structure.
  • WT-RRE wild-type RRE
  • ⁇ RRE truncated version of RRE
  • ⁇ RRE truncated version of RRE
  • RNAs are synthesized by in vitro transcription using T7 RNA polymerase as described above for TAR RNAs.
  • WT-RRE can also be transcribed using a "BLUESCRIPT" plasmid (Stratagene, La Jolla, CA) carrying a 280 bp insert containing base pairs 7333-7612 of the RRE region (Daly et al. , 1989) . Transcribed RNAs are purified and end-labeled as described above.
  • RNA substrate Approximately 10 3 cpm of each uniformly labelled RNA substrate (Example 2) was incubated with either TAT24C or TAT24C-phen in a buffer containing 70 mM NaCl, 0.2 mM EDTA, 10 mM Tris-HCl (pH 7.5), 5% glycerol and 0.1% "NONIDET P40" (Sigma, St. Louis MO) for 20 minutes at 25°C. The samples were then run on a 10% native polyacrylamide gels in TBE (Tris-Borate-EDTA) buffer (Maniatis et al.; Sambrook, et al.) at room temperature. To obtain autoradiograms the gels were exposed to X-ray film. Figure 3 shows a photograph of the resulting autoradiogram.
  • TBE Tris-Borate-EDTA
  • panel A using HIV-1 TAR-RNA substrate: lane 1, in the presence of TAT24C (200 ng) ; lane 2, no polypeptide was added; lane 3, in the presence of TAT24C-phen (200 ng) .
  • panel B using HIV-2 TAR-RNA substrate: lane 1, in the presence of TAT24C (200 ng) ; lane 2, no polypeptide was added; lane 3, in the presence of TAT24C-phen (200 ng) .
  • panel C using ⁇ TAR-RNA as substrate: lane 1, no polypeptide was added; lane 2, in the presence of TAT24C-phen (200 ng) .
  • panel D using yeast tRNA (Bethesda Research Laboratories, Gaithersburg MD) as the substrate: lane 1, in the presence of TAT24C-phen (200 ng) ; lane 2, no protein was added; lanes 3 and 4 were with 200 ng and 400 ng of TAT24, respectively.
  • yeast tRNA Bethesda Research Laboratories, Gaithersburg MD
  • Figure 3C lane 2
  • These results show binding of polypeptides to all three RNAs containing the TAT responsive TAR element.
  • the mobility shift of samples containing modified and unmodified polypeptides are virtually identical, indicating that the attachment of the phenanthroline moiety at the C-terminus of the polypeptide does not affect binding to the TAR site.
  • Example 4 Cleavage of TAR-Site Containing Substrates
  • Cleavage of HIV-1 TAR Cleavage reactions using the polypeptide- cleaving reagents of the present invention were typically performed as follows. RNA substrates were 5' end-labeled (Maniatis et al. ; Sambrook, et al.) employing T4 polynucleotide kinase (Boehringer Mannheim) using 32 P ⁇ 7-ATP (Amersham) and purified by gel electrophoresis as described above.
  • RNA was incubated in 10 ⁇ l of buffer A in the presence of TAT24C-phen, and cleavage was initiated by adding CuS0 4 (to a final concentration of 10 ⁇ M) and mercaptopropionic acid (to a final concentration of 2.5 mM) after 10 minutes at 22 * C. After incubating for 17 hours, the reaction was stopped by adding the following to the indicated final concentrations: 2,9-dimethyl-l,10-phenanthroline to 3 mM, tRNA to 0.2 mg/ml, and sodium acetate (NaOAc) to 0.3 M.
  • NaOAc sodium acetate
  • TAT24C-phen (20 pmol); lane 3, TAT24C-phen (30 pmol) ; lane 4, SI nuclease (1 U) , incubated for 5 min at room temperature; lane 5, G-specific reaction, ribonuclease Tl (1 U) , incubated for 10 min at 37"C in 10 ⁇ l of buffer A (70 mM NaCl, 10 mM Tris-HCl (pH 7.5)); lane 6, C-specific reac ⁇ tion, ribonuclease CL3 (0.2 U) , incubated for 20 min at 37°C in 10 ⁇ l of buffer A; lane 7, cleavage at every nucleotide by irradiating RNA with 350-nm light (1.2 J in a "STRATALINKER", Stratagene, La Jolla, CA) at 25°C in the presence of 20 mM uranyl nitrate; lane a, 40 ⁇ M Cu(II)-1,10
  • Figure 8B presents fine mapping analysis of the cleavage sites at the 3' half of HIV-1 TAR. All reactions were conducted as described above.
  • lane 1 incubation with TAT24C-phen (90 ng) ;
  • lane 2 incubation with TAT24C-phen (60 ng) ;
  • Figure 8B. represents a gel subjected to longer electrophoresis time in order to separate larger fragments.
  • RNAs from all reactions were ethanol precipitated and analyzed on a denaturing (8.3 M urea) 15% polyacrylamide gel. The gel was dried and autoradiographed.
  • RNAs from all reactions were ethanol precipitated and analyzed on a denaturing (8.3 M urea) 15% polyacrylamide gel. The gel was dried and autoradiographed.
  • RNAs from all reactions were ethanol precipitated and analyzed on a denaturing (8.3 M urea) 15% polyacrylamide gel. The gel was dried and autoradiographed. The cleavage sites on the HIV-2 TAR RNA are indicated by arrows.
  • RNAs from all reactions were ethanol precipitated and analyzed on a denaturing (8.3 M urea) 15% polyacrylamide gel. The gel was dried and autoradiographed.
  • E. Analysis of Cleavage Results The chemical nuclease activity of Cu(II)-com ⁇ plexed 1,10-phenanthroline derives from an oxidative attack on the sugar ring by a copper-oxo species generated in the presence of a reducing agent (Sigman et al., 1990).
  • the nucleolytic activity of TAT24C-phen on HIV-1 TAR is shown in Figure 8A. As seen in lanes 2 and 3, cleavage occurs primarily in the loop of the target RNA (structure shown between Figures 8A and 8B) , especially at the uridine (U 30 ) in the 5' side of the loop.
  • a secondary cleavage site can also be seen on the stem, at nt 12-14 and 18 (indicated by the short arrows in the RNA structure shown in Figure 8A) and at nt 43-45 on the complementary region ( Figure 8B) .
  • the cleavage pattern on opposite sides of the stem is shifted to the 5' side, an indication that the cleaving moiety is occupying the major groove of the duplex RNA stem (Dervan,
  • the cleavage sites lie on either side of the bulge where the TAT protein is known to bind to the TAR target site ( Roy et al., 1990; Cordingley et al., 1990; Dingwall et al., 1989; Weeks et al., 1990). Because Cu(II)-phenanthroline is known to preferentially cleave unpaired bases of RNA (Murakawa et al., 1989), HIV-1 TAR was incubated with free Cu(II)-1,10-phenanthroline, i.e., not bound to RNA-binding protein, as a control.
  • TAT24C-phen produces no cleavage (lane B, Figure 8A) .
  • HIV-2 TAR In contrast to cleavage of HIV-1 TAR, for which the primary site is at the loop adjacent to the TAT binding site, cleavage of HIV-2 TAR takes place mainly at the stem, roughly midway between the two loops ( Figure 9A, lanes 1 and 2) .
  • HIV-2 TAR has two 2-nt bulges, both of which have the consensus TAT binding motif (Weeks et al. , 1990; Green et al., 1988; Frankel et al. , 1988; Milligan et al.,1987; Arya et al. , 1988; Weeks et al. , 1991; Murakawa et al., 1989).
  • HIV-1 TAT can transactivate HIV-2 LTR-directed gene expression when either stem-loop I or stem-loop II is present (although the TAT product of HIV-2 requires both stem-loops for efficient transactivation (Arya et al. , 1988; Emerman et al. , 1987). Accordingly, the HIV-1 encoded TAT protein appears to bind to either stem-loop. Because the TAT24C polypeptide is based on HIV-1 TAT, the cleavage of both loops probably results from binding of the polypeptide to both elements. The major cleavage site for HIV-2 thus corresponds to the minor cleavage site for HIV-1 TAR (i.e., approximately 3-8 base pairs from the bulge in the direction away from the loop(s)).
  • TAT24C-phen might bind only to stem-loop I and the molecule could fold to bring the two loops close together.
  • tRNA was used as the substrate for cleavage, the cleavage pattern induced by TAT24C-phen was identical with that caused by free Cu(II)-phenanthroline ( Figure 9B) , indicating that TAT24C-phen does not induce site-specific cleavage on RNA lacking a TAR site.
  • ADDRESSEE SRI International
  • MOLECULE TYPE RNA (genomic)
  • MOLECULE TYPE RNA (genomic)
  • (C) INDIVIDUAL ISOLATE a peptide derived from the REV protein of HIV-1
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE RNA (genomic)
  • MOLECULE TYPE RNA (genomic)
  • CAGUGGGAAU AGGAGCUUUG UUCCUUGGGU UCUUGGGAGC AGCAGGAAGC ACUAUGGGCG 60
  • CAGCGUCAAU GACGCUGACG GUACAGGCCA GACAAUUAUU GUCUGGUAUA GUGCAGCAGC 120
  • MOLECULE TYPE RNA (genomic)

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Abstract

The present invention describes the generation of site-directed RNA cleaving agents. These agents consist of RNA-binding proteins, or polypeptides derived thereof, which are modified to contain a moiety capable of cleaving RNA backbones. Alternatively, the agents are oligonucleotides having nuclease resistant backbones to which a moiety capable of cleaving RNA backbones has been attached. The present invention also describes a method of cleaving target RNA substrates using the cleaving agents described herein. Further, the invention describes a method for inhibiting RNA virus expression in infected cells.

Description

ANTIVIRAL REAGENTS BASED ON R A-BINDING PROTEINS
Field of the invention
The present invention relates to RNA-binding proteins modified to contain nucleic acid cleaving moieties, in particular, viral trans-acting pro¬ teins can be used as specific antiviral reagents. The invention further includes methods for genera¬ ting said antiviral reagents, methods of cleaving viral nucleic acid, and methods of inactivating viral nucleic acid in cells.
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Background of the invention
Chemical systems for the oxidative cleavage of DNA have been developed and used in recent years (Dervan, 1986; Youngquist et al., 1987;
Sluka et al . , 1987; Strobel et al. , 1990; Chen et al., 1986; Francois et al. , 1989; Ebright et al. , 1990; Bruice et al., 1991; Sigman et al., 1990; Chen et al., 1987). For example, phenanthroline attached to an oligonucleotide or polypeptide will bind cupric ion and this complex can be used to cleave DNA. In the presence of a reducing agent the bound cupric ion is reduced to cuprous ion, which reduces molecular oxygen to produce hydrogen peroxide. The H-jO-, reacts with the cuprous complex to form a copper-oxo species that is directly responsible for cleavage. Chen et al. (1987) used this approach to convert the E. coli trp repressor to a site-specific deoxyribonuclease.
Copper-phenanthrolene has also been tethered to oligonucleotides to induce sequence-specific cleavage of single-stranded and double-stranded DNA (Francois et al., 1989). An alternative but chemically analogous system (Dreyer et al., 1985; Dervan, 1986; Moser et al., 1987; Maher et al., 1989) utilizes EDTA-chelated iron tethered to an oligonucleotide to cleave DNA. By attaching an
Fe-EDTA group to the DNA-binding domain of the Hin recombinase, Sluka et al. (1987) achieved site-specific DNA cleavage at Hin recombination sites. In addition to the above chemical systems for the oxidative cleavage of DNA, Corey and Schultz (1987) have converted the nonspecific nuclease staphylococcal nuclease to a site-specific nuclease by attaching it to an oligonucleotide. In this hybrid molecule, the relatively short oli¬ gonucleotide is able to confer binding specificity on the target DNA via hybridization (Corey et al., 1987) or triplex formation (Pei et al., 1990). Although the conversion of DNA-binding molecules to site-specific chemical deoxyribonucleases has been an area of active investigation, efforts to achieve site-specific cleavage of RNA have been mainly limited to the use of ribozymes. Experiments performed in support of the present invention have demonstrated site-specific cleavage of RNA using sequence specific RNA binding proteins. Summary of the Invention
The present invention describes a polypeptide having site-specific RNA binding, where the polypeptide is modified to contain a moiety capable of cleaving an RNA backbone, in particular, viral polypeptides having site- specific viral RNA-binding. Exemplary of such polypeptides are the polypeptides presented as SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:22.
A number of cleaving moieties are useful in the practice of the present invention including the following: phenanthroline Cu(II) , Zn(II) , Fe(II)-EDTA, Cu(II)-bipyridine, and Cu(II)-terpyridine.
In one embodiment of the present invention the RNA-binding polypeptide can be either the HIV TAT or REV proteins, or polypeptides derived therefrom. A preferred embodiment of the present invention is the polypeptide having the sequence presented as SEQ ID N0:1, which further contains an end-terminal cysteine residue, and where the cleaving moiety is phenanthroline Cu(II) . Another preferred embodiment is the polypeptide having the sequence presented as SEQ ID NO:2 where the cleaving moiety is phenanthroline Cu(II) .
A further embodiment of the polypeptide cleaving reagents of the present invention is the generation of fusion polypeptides containing the RNA-binding polypeptide coding sequence fused in frame to a non-specific nuclease. Alternatively, the non-specific nuclease may be covalently bound to the RNA-binding polypeptide. One preferred embodiment of this aspect of the present invention is the polypeptide having the sequence presented as SEQ ID NO:2 coupled to Staphylococcal non¬ specific nuclease. The present invention also includes a method of cleaving a target RNA. The method involves contacting an RNA molecule containing a cognate RNA-binding site with the RNA-binding polypeptide cleaving reagent. Both chemical and nuclease cleaving reagents are useful in this aspect of the present invention.
In one embodiment, the RNA molecule is an HIV RNA and the RNA-binding polypeptide is an HIV- encoded RNA-binding protein, such as TAT, REV, or polypeptides derived therefrom. The RNA-binding polypeptide reagent is supplied at a concentration effective to produce cleavage of the target RNA molecule. Cleavage reactions may further include the addition of a reducing agent, such as mercaptopropionic acid, N-acetyl cysteine, or ascorbate. In one embodiment, for the cleavage of HIV RNA, the cleavage reactions include the addition of the polypeptide reagent, CuS04 and mecaptopropionic acid. The invention further includes a method of inhibiting expression of viral antigens in infected cells. The method in¬ volves exposing the infected cells to the above described polypeptide cleaving reagent where the polypeptide reagent binds, site-specifically, to the RNA target and is able to be taken up by the infected cells. The cells are exposed to a concentration of the polypeptide reagent which is effective to produce reduction in (i) viral antigen expression or (ii) viral transcription in the infected cells. The cells may be exposed to the polypeptide reagent in the presence of a reducing agent, such as N-acetyl cysteine and ascorbate. In one embodiment of the present invention the viral RNA is HIV RNA and the polypeptide reagent is based on the HIV-encoded TAT or REV proteins, or polypeptides derived therefrom. When the polypeptide reagent includes a non-specific nuclease, expression of the polypeptide reagent in infected and/or un-infected cells provides a useful gene therapy to fight viral disease.
In another aspect of the present invention the cleaving agents of the present invention are oligonucleotides having nuclease resistant backbones to which a moiety capable of cleaving RNA backbones has been attached.
Brief Description of the Figures
Figure 1 illustrates the sequence and structure of a -variety of TAR elements. Figure 1A shows the TAR element of HIV-1 (also presented as SEQ ID NO:3) . Figure IB shows a TAR element of HIV-2 (also presented as SEQ ID NO:4) . Figure 1C shows a truncated HIV-1 TAR element designated ΔTAR (also presented as SEQ ID NO:5). Figure ID illustrates the binding of a TAT-peptide, having an attached cleaving moiety, to a TAR element- containing target RNA.
Figure 2A presents the primary coding sequence of the HIV-1 TAT protein. This sequence is also presented as SEQ ID N0:1. The region in bold represents the nuclear targeting domain. The underlined polypeptide, a proteolytic product of wild type TAT protein, binds specifically to TAR- element-containing RNA (Weeks et al. , 1990) . Figure 2B presents the sequence of the TAT-derived polypeptide, TAT24C (SEQ ID NO:2), which is derived from the underlined sequence of Figure 2A. Figure 2C illustrates a phenanthroline moiety attached to a cysteine residue of a polypeptide. Figures 3A-D show the results of gel shift mobility assays using modified and unmodified polypeptides and different RNA substrates. Figure 4 presents the sequence of the HIV-1 encoded REV protein. This sequence is also presented as SEQ ID NO:6.
Figure 5 illustrates the chemistry of the at- tachment of a phenanthroline moiety to a cysteine- containing polypeptide.
Figure 6 illustrates the use of 2-iminothiolane for attachment of cleaving moieties to amino groups of polypeptides. Figure 7 shows one scheme for attachment of iminodiacetic acid to polypeptides for Zn(II) binding.
Figures 8A and 8B show the results of cleavage assays using a variety of cleaving agents and an HIV-1 TAR-element containing substrate. The drawing of the RNA substrate indicates cleavage sites (arrows) of TAT-based cleaving agents. Figure 8C shows the results of cleavage assays using a variety of cleaving agents and a truncated HIV-1 TAR-element-containing substrate. The drawing of the RNA substrate indicates cleavage sites (arrows) of TAT-based cleaving agents.
Figure 9A shows the results of cleavage assays using a variety of cleaving agents and an HIV-2 TAR-element containing substrate. The drawing of the RNA substrate indicates cleavage sites (arrows) of TAT-based cleaving agents. Figure 9B shows the cleavage products resulting from using tRNA as a substrate RNA.
Figure 10 provides an overview of a method for targeting and inactivation of HIV mRNA using oligonucleotides which contain cleaving moieties.
Figure 11 shows, in bold, potential triplex target sites within the HIV-1-LTR region: A (SEQ ID NO:8), B (SEQ ID NO:9), and C (SEQ ID NO:10).
Figure 12 shows oligonucleotides [A 1 (SEQ ID N0:11) and 2 (SEQ ID NO:12), B 1 (SEQ ID NO:13) and 2 (SEQ ID NO:14), and C 1 (SEQ ID NO:15) and 2 (SEQ ID NO:16)] designed to target the sequences presented in Figure 11 A, B, and C, respectively. Next to each of these oligonucleotides is the general pattern of base triplets expected when triplexes are formed. Oligonucleotides K (SEQ ID N0:17), L (SEQ ID N0:18), and M (SEQ ID N0:19) are the control oligonucleotides. Figure 13 shows a schematic representation of triplex formation at an mRNA target site (bold) using a linear complementary oligonucleotide where the end loop contains basic oligonucleotides (-X- ) . The RNA sequence is presented as SEQ ID NO:21 and the oligonucleotide is presented as SEQ ID NO:20, where N is an basic residue.
Figure 14 shows a schematic representation of the HIV-1 REV response element (RRE) ; this sequence is also presented as SEQ ID NO:23. SEQ ID NO:24 presents the sequence of a truncated RRE binding site which corresponds to Stem II in the figure.
Detailed Description of the Invention I. Selection of RNA-Binding Proteins
Experiments performed in support of the present invention demonstrate that RNA-specific binding proteins can be adapted to perform site-specific cleavage of a target RNA when the target RNA contains the cognate binding site to which the RNA-binding protein binds. One important application of these protein-based cleaving agents is the inactivation of mammalian viruses; in particular, RNA binding proteins modified to accomplish site specific cleavage can be used for inactivating RNA viruses, including the human immunodeficiency viruses (HIV) . Among the criteria for choosing an RNA- binding protein or polypeptide for use in the present invention are the following: RNA target site specific recognition; known or identifiable recognition sequence; the ability to get the protein or polypeptide into cells containing the target RNA; a relatively small protein binding domain is preferable; and fragments containing the protein binding domain should compete well for binding at the RNA target site with the native protein from which they are derived.
Two examples of RNA-binding proteins useful in the practice of the present invention are the TAT and REV proteins encoded by HIV-l. TAT consists of 86 amino acids and is a potent transactivator of long terminal repeat (LTR)-directed viral gene expression and is essential for viral replication (Dayton et al., 1986; Fisher et al., 1986). The amino acid sequence of the TAT protein is presented as SEQ ID NO:l. TAT-induced transactivation requires the presence of the TAR (transactivation response) element, located at the untranslated 5' end of the viral mRNA element. The RNA sequence of the TAR element is presented as SEQ ID NO:3. The TAR element is capable of forming a stable stem-loop structure (Muesing et al., 1987) in the native viral RNA (Figure 1) . A 3 nucleotide (nt) bulge on the stem of TAR has been demonstrated to be essential for specific and high-affinity binding of the TAT protein to the TAR element (Roy et al., 1990; Cordingley et al . , 1990; Dingwall et al., 1989; Weeks et al., 1990). Further, the ability of (i) purified TAT protein and (ii) polypeptide fragments derived from TAT, which contain the nuclear targeting domain, to bind in vitro to RNA containing the TAR element (Roy et al. , 1990; Cordingley et al r 1990; Dingwall et al. , 1989; Weeks et al. , 1990) make TAT a useful model for the RNA-cleaving reagents of the present invention.
In the TAR element, the integrity of the stem and the initial U22 of the bulge (see Figure 1) are important or TAT protein binding (Roy et al. , 1990b) . in vivo other sequences that do not affect the binding of the TAT protein to the TAR site are needed for trans-activation of tran¬ scription. One such region is the sequence at the loop, which is required for the binding of cellular factors that may interact with the TAT protein to mediate transactivation (Gatignol et al. , 1989; Gaynor et al. , 1989; Marciniak et al. , 1990a; Gatignol et al., 1991).
For the purposes of the present invention, the important components of the protein/RNA interaction are (i) the portion of the protein involved in binding to the specific target sequence, and (ii) the RNA target sequence required for binding. In the case of the TAT protein, both full- length TAT (86 amino acids) and a truncated TAT protein (the N-terminal 72 amino acids) have been expressed as fusion proteins in Escherichia coli (Weeks et al., 1990). The TAT proteins were linked to the E . coli catabolite activator protein via a protease recognition site. When the fusion proteins were purified and cleaved by the appropriate protease, polypeptide fragments were recovered from each fused protein which resulted from an unexpected cleavage between Gly48 and Arg49 (Figure 2) , at the start of the lysine/arginine-rich RNA-binding region. The fragments resulting from this cleavage, TRF38 and TRF24, corn-prise the regions extending from Arg49 to residue 86 and Arg49 to residue 72, respectively. Both of these polypeptide fragments specifically bound RNA containing the TAR site (TAR-RNA) . Further, a synthetic 14-residue polypeptide spanning the basic region (amino acid residues 48-61 of the TAT protein) bound TAR-RNA as well.
Up to three copies of TRF38 could bind to the wild- type TAR (wt-TAR) site, the first with an apparent dissociation constant of 5 nM and a second copy with an apparent dissociation constant of 20 nM. Only one copy of the TRF38 polypeptide could bind to a truncated version of TAR consisting of the minimal TAT binding site: the minimal site consists of 26 bases containing the upper part of the wt-TAR (Figure 1C; Muesing et al., 1987). Similar binding characteristics were found for TRF24.
A polypeptide similar to TRF24, designated TAT24C, was chemically synthesized (Example 1A) . TAT24C consists of amino acid residues 49-72 of the TAT protein (Figure 2A) and an additional cysteine residue at the C-terminus (Figure 2B, SEQ ID NO:2). The TAT24C polypeptide was purified by HPLC and reacted with 5-iodoacetamido-l,10- phenanthroline to attach a 1,10-phenanthroline moiety to the cysteine residue (peptide designated TAT24C-phen, see below) .
The above described synthetic polypeptides were then evaluated for TAR-RNA binding. In general, the ability of synthetic polypeptides (both with and without the cleaving moiety) to bind specifically to the target binding site is evaluated using a gel mobility shift assay typically involving two nucleic acid substrates. One substrate contains the wild-type target binding site (e.g., the TAR element of HIV-1 viral RNA) ; the other substrate is an RNA that lacks part or all of the recognition sequence and is therefore unable to bind the cognate protein (e.g., a TAR element mutated at the bulge to which TAT is unable to bind, or a tRNA molecule) . The second substrate provides a negative control. RNA substrates are prepared as described in Example 2. Several TAR-containing RNAs were synthesized to use as substrates in binding assays to test the binding activity of the modified polypeptide
TAT24C-phen (Example 2) . The predicted secondary structures of the target RNAs are shown in Figure 1C. The RNA substrate designated HIV-1 TAR is the 57-nt RNA stem-loop structure found in native HIV-1 mRNA (Sharp et al. , 1989). The RNA substrate designated ΔTAR is a truncated RNA containing the minimum TAT binding site (nt 17-43) (Weeks et al., 1990). In addition to HIV-1, the etiologically associated virus of AIDS, another retrovirus, termed type 2 (HIV-2) has been reported (Clavel et al., 1986). HIV-2 also possess a functional TAT gene (Arya et al. , 1985; Arya and Grallo, 1986) . Although the transactivation of genes under the HIV-1 LTR by HIV-2 encoded TAT is quite inefficient, the TAT gene product of HIV-I can effectively transactivate genes under the HIV-2 LTR (Arya et al., 1988; Emer an et al. , 1987). Two subelements responsible for the TAT-mediated transactivation in the HIV-2 TAT responsive element (TAR) have been identified and contain two stem-loop struc¬ tures confined to +1 - +103 nucleotides (Arya et al., 1985). The sequence of HIV-2 TAR RNA used in the present study is from +13 - 90 nucleotides and contains both stem-loop structures.
The above TAT24C polypeptides and RNA substrates were then employed in gel mobility-shift assays (Example 3) to evaluate the binding of the polypeptide to the binding site. Figure 3 shows a photograph of a representative gel mobility shift assay. In Figure 3 discrete bands having retarded mobility for samples containing either phenanthroline- odified or un¬ modified polypeptides demonstrate the binding of both polypeptides to each of the three RNAs con¬ taining TAR elements (Figure 3A-C) . The attach- ment of the extra cysteine and the phenanthroline moiety at the C-terminus of the TAT24 polypeptide does not substantially affect binding to the TAR site. Essentially no retardation of tRNA was observed when it was incubated with high levels of the TAT24C polypeptide (Figure 3D) , indicating relatively specific binding to TAR-site-containing RNA substrates.
The entire TAT protein, as well as fragments of TAT containing the nuclear targeting region, are rapidly taken up by cells. The TAT protein taken up by cells in this fashion specifically activates HIV-1 LTR-linked gene expression (Green et al., 1988; Frankel et al., 1988). The entry of polypeptides containing (i) the nuclear targeting region of TAT and (ii) nucleic acid cleaving agents can be evaluated as described below.
A second HIV protein that is useful in the present invention is the REV protein. REV protein is a regulatory factor essential for viral replication; it is required for the production of viral structural proteins. It appears to exert its effect at the level of splicing and perhaps transport of viral mRNA into the cytoplasm (Malim et al., 1989a, 1989b; Felber et al., 1989); further REV appears to increases the stability of unspliced HIV mRNA (Felber et al., 1989). The REV protein consists of 116 amino acids (Figure 4, SEQ ID NO:6), encoded by two exons. An arginine-rich domain (residues 38-51) acts as the nuclear tar¬ geting domain (Malim et al., 1989b). Mutational analysis has demonstrated that some C-terminal deletion mutants of the REV protein are non¬ functional, in terms of normal REV functions, but are trans-dominant as illustrated by competitive inhibition of wild-type REV functions.
The action of REV requires the presence of a target sequence termed the REV response element
(RRE; Figure 14, SEQ ID NO:23), located in the HIV envelope gene (Malim et al. , 1989a, 1989b) . RRE has been mapped to a 234-nucleotide region capable of forming four stem-loop structures and one branched stem-loop structure (Malim et al.,
1989a) . Footprinting data (Holland et al. , 1990; Kjems et al., 1991) suggest that REV binds to six base pairs in one stem structure and to three nucleotides in an adjacent stem-loop structure of the RRE. A 40 nucleotide region in stem-loop II (SEQ ID NO:24) has been implicated as the minimum REV binding region (Cook et al. , 1991). Thus, REV offers another potential means of targeting a cleaving agent specifically to HIV RNA and has a potential advantage over the TAT protein in that REV has more potential binding sites.
Other RNA-binding proteins and their cognate binding sites can be characterized as has been de¬ scribed above for the TAT protein and TAR site. Truncated versions of any such protein and/or binding site can be evaluated for protein/-peptide binding using, for example, the gel mobility shift assay or standard filter binding assays (Sauer et al. ; Radding et al.). Further, cellular uptake can be evaluated. If the protein is not adequately taken up by cells at concentrations useful to provide intracellular catalytic function, i.e., cleavage, then alternative methods of cellular uptake, such as targeted liposomal delivery where the liposomes carry target cell surface recognition moieties, can be employed to get polypeptide fragments into target cells.
II. Generation of Trans-Acting Proteins Having
Cleaving Moieties A. Chemical Nucleases
A number of chemical moieties are capable of cleaving nucleic acid substrates including phenan¬ throline (Chen et al., 1986, 1987; Francois et al., 1989; Ebright et al., 1990), Fe(II)-EDTA (Dreyer et al., 1985; Dervan, 1986; Moser et al., 1987; Maher et al., 1989; Sluka et al. , 1987), Cu(II)-bipyridine, Cu(II)-terpyridine, and Zn(II) (Modak et al., 1991; Eichhorn et al., 1971; Ikenaga et al., 1974; Breslow et al., 1989). These chemical cleaving moieties can be employed in the present invention as exemplified below with reference to the TAT24 C-phen protein.
Nucleic acid cleaving moieties are attached to the TAT derived polypeptides as described in Example IB. The chemically synthesized
HPLC-purified polypeptides are reacted with 5-iodoacetamido-l,10-phenanthroline (phenanthroline moiety) to obtain polypeptides containing the phenanthroline moiety uniquely attached to the side chain of the cysteine residue. Figure 5 illustrates the attachment of a phenanthroline moiety to cysteine-containing polypeptides.
The TAT24-C polypeptide (SEQ ID NO:2), consisting of that 24-residue domain plus a single cysteine residue at the C-terminus, was chemically synthesized (Example 1A) . A phenanthroline moiety was then attached to the sulfhydryl group of the cysteine to obtain TAT24C-phen (Example IB) . The cleaving agent can also be attached at residues other than cysteines. For example, phenanthroline can be attached to the side chain- amino groups of lysines and arginines, as well as to the amino group at the N-terminus, by reacting the protein first with 2-iminothiolane hydro¬ chloride, followed by coupling with 5-iodoace- tamido-l,10-phenanthroline (Chen et al. , 1987). This coupling is illustrated in Figure 6 and described in Example IB. Because of the higher loading of cleaving moieties per protein/peptide molecules, such protein/peptide molecules are expected to be efficient reagents for cleavage and are expected to be resistant to in vivo degradation.
Other available nucleic acid cleaving agents can also be used in the methods of the present invention, including Fe(II)-EDTA,
Cu(II)-bipyridine, and Cu(II)-terpyridine. If the intracellular reduction potential is not sufficient to recycle metal atoms to its reduced state, the intracellular reduction potential can be affected using N-acetyl cysteine, which increases the intracellular glutathione level (Roederer et al. , 1990; Kalebic et al. , 1991), in order to assist in keeping the metal atom of the cleaving agent in the reduced state. Alternatively, different chemical cleaving mechanisms are available. RNA degradation is known to be induced by divalent metal ions, especially Zn(II) , in the absence of a reducing agent. The reduced hydrolytic cleavage compared to reaction in the presence of a reducing agent can be at least partly of set by having more than one chelating molecule attached to the polypeptide. One technique for tethering Zn(II) to a protein is via coordination by iminodiacetic acid (Aldrich, Milwaukee WI) : one possible scheme for such attachment is shown in Figure 7. Briefly, iminodiacetic acid is converted to its diethyl ester to protect carboxylic function¬ alities. The resulting product is condensed with iodoacetic acid in the presence of dicyclohexyl carbodiimide (DCC) to obtain compound 3 (Figure 7) . Compound 3 is then reacted with, for example, a polypeptide containing a cysteine residue.
The ability of proteins/peptides, with and without the nucleic acid cleaving moiety, to bind their cognate nucleic acid substrate is evaluated as described above.
Once, the polypeptides are screened for specific binding to their cognate nucleic acid binding site, the polypeptides carrying nucleic acid cleaving moieties are next evaluated for their ability to cleave the target nucleic acid.
B. Hybrid-Protein Nucleases
In addition to the above chemical cleaving moieties, a non-specific enzymatic nuclease can be attached to a sequence specific RNA binding protein to form a sequence-specific ribonuclease. Staphylococcal nuclease, a non-specific nuclease that attacks both RNA and DNA, has been converted to site- specific DNA endonuclease by attaching the protein to an oligonucleotide (Corey et al., 1987; Pei et al., 1990).
For the present invention, hybrid fusion pro¬ teins are generated between the RNA sequence- specific protein and the coding sequence of Staphylococcal nuclease. The hybrid proteins can be expressed using any number of standard expres¬ sion systems (e.g., "CLONTECH" commercially available vectors) . Typically, the hybrid protein is expressed in E. coli using the OmpA-derived expression system (plasmid pONFl) already adopted for the secretion of staphylococcal nuclease (Takahara et al. , 1985) . In this construct, the signal peptide required for the secretion of o pA protein is fused to the nuclease gene to obtain large amounts of secreted nuclease: the nuclease is then released from the signal polypeptide by appropriate processing. DNA sequences encoding the TAT, REV, or derivative RNA-binding poly¬ peptides are cloned adjacent to and in-frame with the staphylococcal nuclease gene in plasmid pONFl. The RNA-binding protein coding sequence can be generated by any number of methods including polymerase chain reaction amplification (Mullis et al. ; Mullis) and standard cloning technology (Ausubel et al. ; Maniatis et al.; Sambrook, et al.). The insertion of the RNA-binding protein/- peptide coding sequence is performed by standard cloning methods (Ausubel et al. ; Maniatis et al.; Sambrook, et al.) . The resulting hybrid proteins are then expressed in E. coli and purified (Takahara et al., 1985).
The ability of hybrid proteins/peptide nucleases to bind their cognate nucleic acid substrate is evaluated as described above using, for example, the gel mobility shift or filter binding assays.
Once the hybrid protein/peptide nucleases are screened for specific binding to their cognate nucleic acid binding site, they are next evaluated for their ability to cleave the target nucleic acid.
III. Targeted Cleavage of Nucleic Acids
The chemical nuclease activity of Cu(II)-com- plexed l,10-phenanthroline derives from an oxida- tive attack on the sugar ring by a copper-oxo species generated in the presence of a reducing agent (Sigman et al., 1990).
The above described polypeptides were tested for their ability to cleave RNA substrates which contained cognate binding sites. Typically, RNA target molecules containing the binding sites were 5'-end- labeled, purified on denaturing polyacryl- amide gels, and annealed for use as substrates for cleavage (Example 2) . A typical cleavage reaction contained 103 cpm of end-labeled RNA and 40-100 ng of polypeptide. Example 4A describes RNA cleavage reactions utilizing the TAT24C-phen polypeptide. The target RNA was incubated with TAT24C-phen at 25"C for 10 min before the cleavage was initiated by adding CuS04 and mercaptopropionic acid. After incubating for a given period of time, the reac¬ tion was stopped by the addition of 2,9-dimethyl- 1,10-phenanthroline. RNAs were then isolated and analyzed on denaturing 15% sequencing gels. The results of this cleavage reaction, when HIV-1 TAR was used as the substrate, are shown in Figure 8A and Figure 8B. As seen in lanes 2 and 3 (Figure 8A) , there is a specific cleavage occurring pri a- rily at the beginning of the loop of the target stem and loop structure (Figure 1) . This is an expected site of cleavage based on binding of the polypeptide at the three- nucleotide bulge. A possible secondary cleavage site may be due to flexibility of the phenothroline-containing protein of the polypeptide, allowing it to reach to stem on the other side of the binding site.
Since Cu(II)-phenanthroline is known to be biased in favor of cleaving unpaired bases of RNA (Murakawa et al., 1989), HIV-1 TAR was incubated with l,10-phenanthroline-Cu(II) as a control and the cleavage pattern induced by free phenanthro- line is shown in lane a (Figure 8A) . As expected, free phenanthroline cleaves nonspecifically, how¬ ever, the unpaired bases at the loop and the bulge are somewhat hyperreactive. The reactivity of loop nucleotides towards free phenanthroline-
Cu(II) is different from their reactivity toward the polypeptide.
The cleavage induced by TAT24C-phen on the ΔTAR sequence is illustrated in Figure 8C (Example 4B) . The cleavage pattern of this truncated substrate is consistent with the cleavage pattern observed for the full-length TAR. With ΔTAR, the cleavage is restricted to the loop. The reacti¬ vity of nucleotides occupying the loop towards the polypeptide is different from that of free phenan¬ throline (compare lane 1 with lanes 5 and 6, Figure 8C) .
The effect of the TAT24C-phen was examined relative to the HIV-2 TAR site (Example 4C) . The primary cleavage site found on the HIV-2 TAR is somewhat unexpected (Figure 9, lanes 1 and 2). Based on the results observed with the HIV-1 TAR substrates, the cleavage site of the HIV-2 target was anticipated to be predominantly at the loop close to the TAT binding site (loop 1, Figure 9A) . However, the TAT24C-phen polypeptide appears to cleave the HIV-2 TAR-RNA at a site located towards the 5' end of its binding site (Figure 9A) . Unlike the HIV-1 TAR RNA and Δ TAR RNA substrates, the primary cleavage site in the HIV-2 TAR site is not in the loop and does not appear to have unpaired bases (as implied by the SI nuclease cleavage reactions the results of which are shown in lane 4, Figure 9A) . The primary site of cleavage in the HIV-2 substrate is likely the consequence of the tertiary structure of the HIV-2 TAR RNA. Unlike HIV-1 TAR RNA sequence which has a single stem- loop, the HIV-2 target consists of two stem- loop structures: the HIV-2 TAR RNA sequence may have a complex tertiary structure reduces the otherwise favorable interaction of the polypeptide-bound cleaving moiety with the loop. The TAT24C-phen polypeptide does, however, cleave both HIV-2 target loops. The cleavage of the loop 1 is ex¬ pected and the cleavage of the remote second loop may be the result of the two loops being in a spatially close orientation within the overall secondary structure of the HIV-2 substrate.
Overall, the cleavage sites of the RNA tar¬ gets lie on either side of the bulge where the TAT protein is known to bind (Roy et al., 1990;
Cordingley, et al., 1990; Dingwall et al., 1989; Weeks et al., 1990). As shown in Figures 8A and 8C, lane a and lane 1, respectively, free phenan¬ throline moieties cleave relatively randomly, but the unpaired bases at the loop (especially G32) and the bulge are most reactive. This is consistent with previous work which showed that Cu(II)-phen¬ anthroline preferentially cleaves unpaired bases of RNA (Murakawa et al., 1989). In view of this result, the above experiments performed in support of the present invention indicated that tethering phenanthroline to the TAT24C polypeptide suppres¬ ses non-specific cleavage on TAR RNA. In the absence of cupric ions, TAT24C-phen produces no cleavage (Figure 8A, lane b) .
The cleavage pattern on ΔTAR, which lacks the base of the stem, is consistent with that of full- length TAR (Figure 8C) , suggesting that the mini¬ mum TAT binding site is sufficient to be recog- nized by the TAT24C-phen polypeptide. On ΔTAR, the cleavage is restricted to the loop, but the pattern is very different from that for cleavage by free phenanthroline (Figure 8C, compare lane 1 with lanes 5 and 6) .
In contrast to cleavage of HIV-1 TAR, for which the primary site is at the loop adjacent to the TAT binding site, cleavage of HIV-2 TAR takes place mainly at the stem, roughly midway between the two loops (Figure 9A, lanes 1 and 2) . HIV-2 TAR has two 2-nt bulges, both of which have the consensus TAT binding motif (Weeks et al. , 1990; Green- et al., 1988; Frankel et al. , 1988; Milligan et al., 1987; Arya et al. , 1988; Weeks et al., 1991; Murakawa et al. , 1989).
When tRNA was used as the substrate for clea¬ vage, the cleavage pattern induced by TAT24C-phen was identical with that caused by free Cu(II)- phenanthroline (Figure 9B) , indicating that TAT24C-phen does not induce site-specific cleavage on RNA lacking a TAR site.
In the absence of the reducing agent mercap- topropionic acid, Cu(II)-complexed TAT24C-phen still cleaves TAR-containing RNA. When mercapto- propionic acid was replaced by ascorbic acid, a reducing agent more suitable for in vivo studies, the rate of cleavage increased. Experiments performed in support of the pre¬ sent invention which demonstrate the specific cleavage induced by TAT24C-phen on TAR-containing RNAs, taken in combination with previous reports that some mutations of the TAT protein are trans- dominant (Green et al., 1989; Pearson et al.,
1990) support the potential efficacy of using the TAT-24C-phen polypeptide for the inactivation of HIV RNA even in the presence of native TAT, as would be found inside an HIV-infected cell. The above described method can be applied to test the cleavage of a number of RNA target mole¬ cules using modified binding polypeptides, i.e., proteins known to have binding sites in a selected RNA target molecule. For example, REV-derived polypeptides encompassing the nuclear targeting domain (underlined sequence, Figure 4) are synthesized and attached to cleaving agents as described for reagents based on the TAT protein. A synthetic peptide spanning the basic domain of the REV protein (SEQ ID NO:22) has been shown to bind specifically to the RRE target (Kje s et al., 1991) ; in vitro the same peptide inhibits the splicing of mRNA containing RRE.
Alternatively, as described above, target RNA molecules can be cleaved using polypeptides modi¬ fied with iminodiacetic acid in the presence of ZnCl2.
The efficiency of cleavage reactions using any RNA binding proteins or polypeptides, which are derived from these proteins, depends on seve¬ ral factors: (a) the binding affinity and speci- ficity of the reagent; (b) the spatial positioning of the cleaving moiety; (c) the nature of the cleaving reagent; and (d) reaction conditions.
First, if a binding protein is to be used as a truncated reagent, the specificity and the binding affinity of a binding polypeptide to its cognate binding site in an RNA target molecule may potentially be increased by increasing the length of the polypeptide, for example: (i) by extending the polypeptide length from the N-terminus, (ii) the C-terminus, or (iii) both ends of the basic nuclear targeting domain of a transactivator.
In the case of the TAT and REV proteins the maximum number of residues needed to maximize specific binding is not expected to be more than 50 residues, because both trans-activators are relatively small, on the order of 100 residues, and both have functional domains besides the RNA binding domain. Two other approaches to increa¬ sing the binding specificity of TAT24C-phen to the target TAR site are as follows: (i) evaluating in vitro generated mutations (Ausubel et al.) for mutations that increase the binding specificity of the protein/peptide to the cognate binding site (Sauer et al.); (ii) chemical alterations of the polypeptides which can effect a general improve¬ ment of RNA binding, such as replacing Asp and Glu with Asn and Gin. Alternatively, chemical modifi¬ cation can be used to convert Asp and Glu to esters or amides to increase the net positive charge of the polypeptide. Chemical modification reactions that occur in solution can be performed on the polypeptide bound to the target RNA where possible, so that sites critical for binding are protected against alteration (Galas et al. ; Siebenlist et al.).
Second, in order to improve the binding of a protein or polypeptide to its cognate RNA binding site the spatial positioning of the cleaving moi¬ ety may be adjusted. Because of the three-dimen¬ sional folding of polypeptides, the position of the cleaving agent within the polypeptide molecule can be crucial for the cleavage. In the cases of TAT and REV proteins the three dimensional struc¬ ture of the proteins is not known. Accordingly, favorable locations for the placement of the cleaving moiety can be empirically determined; the single cysteine residue can be placed at several positions, including the N-terminus, C-terminus, and internal positions of the polypeptide which do not affect RNA-binding ability.
Third, in order to improve the efficiency of cleavage reactions using RNA binding proteins or polypeptides derived from these proteins the nature of the cleaving reagent can be modified as described above, using chemical or enzymatic cleaving moieties.
Lastly, reaction conditions can be modified to improve cleavage of RNA substrates by, for example, increasing reduction potential in the reaction mixture or intracellularly by, for example, adding N-acetyl cysteine (or perhaps ascorbate) to the system. In vitro reaction conditions can also be modified by altering temperature, ionic conditions, the amount and type of reducing agent, and pH.
The cleavage assay will be used to assess the effects of the above factors on the efficiency of the cleavage reaction. Single variables will be modified to evaluate efficacy. For example, to assess the cleavage induced by different peptides, an identical concentration of the different pep¬ tides are used in reaction mixtures containing the other reagents at fixed concentrations. After the cleavage reaction has been carried out for a specified period, digested end-labeled RNAs (e.g., HIV-1 TAR) will be resolved on sequencing gels, the gels will be autoradiographed, and bands corresponding to starting material (intact RNA) and cleavage products will be excised. The radioactive counts present in the excised bands will be determined by scintillation counting. The relative concentrations of cleavage product to starting material is then determined ( (cpmprod/cpm Pro +cPinintact)10°) • Densitometry scanning can also be used to evaluate efficiency of cleavage reac¬ tions by using films which have not been overex¬ posed.
Oligonucleotides as Anti-Viral Target
Agents
An alternative method to target cleaving agents to RNA targets are oligonucleotides. The chemistry for attaching cleaving groups to DNA fragments in order to sequence-specifically cleave single- and double-stranded DNA targets has been described (Chen et al. , 1986; Francois et al. , 1989; Dreyer et al., 1985; Dervan, 1986; Moser et al. , 1987; Maher et al., 1989). RNA molecules contain both single- and double-stranded regions that can offer targets for oligonucleotide binding (Zamecnik et al., 1986; Rittner et al., 1991). To be effective, RNA oligonucleotide agents must con¬ tinuously bind to the target molecules in such a way as to inactivate them. However, when a clea¬ vage agent is attached to RNA oligonucleotides, the oligonucleotides only need to bind the target RNA long enough to cleave it in order to achieve permanent inactivation.
In order to generate RNA-binding oligonucleo¬ tides which contain cleaving agents, a chosen cleaving group (see above for chemical and enzyma- tic cleaving groups) is attached to oligonucleo¬ tides which are resistant to cleavage by endo¬ genous nucleases. Such backbones include deoxy- ribose or ribose sugar moieties connected by methyl phosphonate or phosphorothioate linkages (Miller et al., 1985).
Cleaving agents are attached to either end of the oligonucleotide, or, if necessary, to both ends of the molecule to maximize cleavage and minimize exonucleolytic degradation. RNA-binding oligonucleotides have sequences of the following two types: (i) sequences designed to form a du¬ plex with putatively single-stranded regions of a target RNA, and (ii) sequences designed to form triplexes with homopurine regions of the DNA which encodes the RNA-target, for example, a DNA pro- virus. In the case of the HIV virus, one mRNA target site is the TAR region, because base pairing at this site by a complementary oligonucleotide is expected to block formation of the stem-loop structure required for binding and transactivation by TAT. An inter-molecular duplex is potentially more stable than the intra-molecular stem-loop duplex due to the absence of unpaired bases. Further, such an inter-molecular duplex may be able to displace the stem-loop structure by pairing initially with the loop or with single-stranded regions adjacent to the stem, particularly in view of the observation that alteration of non-essential sequences adjacent to TAR create competing secondary structures which inhibit TAR function (Berkhout et al., 1989).
A major advantage of targeting the DNA pro- virus associated with an RNA virus is that typically only one, or a few copies, of integrated, transcriptionally active DNA are present per cell in contrast to many copies of mRNA which may be present in an infected cell (Soma et al., 1988). Homopurine-homopyrimidine regions of duplex DNA can bind single-stranded oligonucleotides having the same sequence as either the homopurine or the homopyrimidine strand of the target DNA but with the reverse polarity (Dervan, 1986) , forming purine-purine-pyrimidine or pyrimidine-purine-pyrimidine triplexes, respectively. The purine-purine-pyrimidine triplexes typically require a divalent cation such as Mg++ or Zn++for their stability but are relatively independent of pH (Lyamichev et al., 1991) . The pyrimidine-purine-pyrimidine triplexes require divalent cation but are favored by slightly acid pH.
The triple helix approach for targeting DNA to inhibit expression has had limited use due to the requirement for homopurine target sequences. Triplex formation at an oligopurine*oligopyrimi¬ dine tract can be induced by a single strand consisting of either only pyrimidines or only purines. Sequence-specific recognition by the oligopyrimidine strand relies on the formation of PyPuPy (C+«GC and TΑT) base triplets (Moser et al., 1987). In this case, the oligopyrimidine strand is parallel to the purine tract of the duplex. An oligopurine strand, on the other hand, lies anti-parallel to the purine tract of the duplex, and sequence-specific recognition in this case is brought about by Pu«PuPy (G«GC and A*AT) base triplets (Kohwi et al., 1988; Beal et al.,
1991) . A sequence of 15-18 purines is required to achieve sufficient specificity, and this requirement limits the triplex approach in controlling the expression of a particular gene. Although long homopurine stretches do occur in viral genomes, finding such a sequence within a gene vital to the virus can be difficult.
Experiments performed in support of the present invention support that triplex formation can occur at tandem oligopurineOligopyrimidine sequences using normal DNA, without any unnatural linkages or synthetic base analogues. Briefly, such sequences utilize both types of base triplets, Pu'PuPy and PyPuPy, in forming a triplex. In other words, this approach allows the formation of triplexes at base sequences made up of both purines and pyrimidines. In addition, the incorporation of Pu«PuPy base triplets has the advantage that triplex formation does not demand low pH, which is usually the case when the C+«GC base triplet is involved.
For use in the present invention, triplex- forming oligonucleotides are designed to interact with ho opurine-homopyrimidine sequences in the pro-virus. For example, within the HIV-1-LTR region are three potential targeting sites (sequences in bold, Figure 11A, 11B, 11C) for targeting with single-stranded oligonucleotides. These cleaving-oligonucleotide reagents bind and cleave the DNA provirus as well as the mRNA of HIV, increasing the likelihood of preventing viral replication.
All three target sites are located in the control region of the HIV-LTR (i.e., upstream of the transcription initiation site) and therefore do not interact with mRNA sequences to function as anti-sense mediators. The potential target sites A, B, and C have different triplex forming motifs:
(i) Site A, consisting exclusively of purines, is targeted for triplex formation using oligonucleotides A-l and A-2 (Figure 12) , which are capable of forming triplexes with Pu«PuPy and PyPuPy base triplets, respectively.
(ii) Site B consists of a tract of pyrimidine residues flanked by two purine tracts and is targeted with oligonucleotides B-1 and B-2 (Figure 12) , having the correct polarity to bind with two strands of the target (see above) .
(iii) Site C has some pyrimidines scattered within a highly purine-rich sequence, and oligonu¬ cleotides C-l and C-2 (Figure 12) are directed toward site C.
In Figure 12 oligonucleotides K, L, and M, correspond, respectively, to sites A, B, and C in the reverse polarity and are therefore blocked from triplex formation; these oligonucleotides are used as controls. Test oligomers with and without phenanthroline are used to assess the effect of cleavage. Attachment of 1,10-phenanthroline to oligo¬ nucleotides is achieved as follows. During chemi¬ cal synthesis each oligonucleotide is synthesized with a thiol group at the 5' end by use of the "C6-thiol modifier™11 reagent from Clontech (Palo Alto, CA) according to the manufacturers instruc¬ tions. The oligonucleotides are de-protected with NH4OH and treated with silver nitrate to expose the thiol group. The oligonucleotide is immediately reacted with 5-iodoacetamido 1,10-phenanthroline as described above to covalently link 1,10-phenan¬ throline to polypeptides.
In vitro triplex formation by the oligonucle¬ otides at their designated target sites is assayed by determining site-specific cleavage induced at the target sequences by the test oligonucleotides . equipped with the phenanthroline moiety. pHIV-lLTR-CAT is linearized with Hindlll, end-labeled with 32P~7~ATP using polynucleotide kinase, and subjected to a second restriction digest to obtain a uniquely labeled DNA fragment containing the duplex target sequence. After gel purification, this DNA fragment is mixed with an appropriate modified oligonucleotide in a buffer containing 10 mM Tris-HCl, 100 mM NaCl, 100 μM spermine, and 10 mM MgCl2. The pH of the buffer is adjusted depending on the sequence of the target (a lower pH is used for the formation of C+«GC base triplets) . After incubation at 20°C for 30 min, cleavage is initiated by adding CuSo4 (to 10 μM) and mercaptopropionic acid (to 2.5 mM) . Cleavage products are resolved on sequencing gels along with the products of sequencing reactions. This method allows the mapping of the site of triplex formation and the cleavage efficiency (detected by counting the radioactivity of excised gel bands) ; cleavage efficiency is used to quantitate the efficiency of triplex formation.
To evaluate in vivo triplex formation the CAT gene is transiently expressed under the direction of HIV-1 LTR in HeLa cells. HeLa cells are trans- fected with pHIV-lLTR-CAT, using the DEAE-dextran technique (Queen et al., 1983). Twelve hours after transfection, the cells are incubated with an oligonucleotide, as described by Postel et al. (1991) , and itomycin C (SIGMA) is added to the medium to induce CAT expression. Cells are har¬ vested at 12 and 24 hr and CAT activities deter¬ mined as described by Gorman et al. (1982) and compared to controls, i.e., cells that have been exposed to control oligonucleotides (K, L, and M) and cells without oligonucleotide treatment.
To assess the effect of target sequence clea¬ vage, oligonucleotides carrying phenanthroline are complexed with CuS04 before being introduced to the cell medium. Ascorbic acid (or mercaptopropionic acid) is supplied to the medium 12 hr after the oligonucleotide treatment and cells are harvested and assayed for CAT activity after another 24 hr. The effect of oligonucleotides in the pre- sence of TAT protein is assayed using p-HIV-lLTR- CAT under stable expression conditions (described above) . CAT-active clones are transfected with a TAT expression vector and these cells, which are transiently expressing TAT are used for oligonu- cleotide treatment followed by the measurement of CAT activity.
An alternative to the above described triplex helix methods is to use an oligonucleotide-based approach where a single-stranded oligonucleotide is capable of forming a triplex with HIV mRNA by contributing two "strands" connected by a hairpin loop (Figure 10; Figure 13) . This triplex- directed anti-sense approach is expected to be more effective in arresting biological processes such as translation and reverse transcription than is the convention anti-sense approach where a DNA- RNA duplex is formed. Triplex formation in this fashion is highly selective and of high affinity and may not be a substrate for enzymes such as helicases. The action of such helicases has been a potential problem in the conventional anti-sense approach (Bass et al. , 1987).
For cleavage of target RNA substrates, oligonucleotides have a chemical cleaving group attached to one end (Figure 10) and an inter- calator linked to the other end. Because de- protection procedures are different and indepen¬ dent from each other, derivatization at the two ends can be performed at two stages of oligonu¬ cleotide synthesis.
IV. Utility
The cleaving reagents of the present inven¬ tion provide means for a method of cleaving RNA targets at specific sites. Such cleavage is useful for the analysis of RNA structure and function as well as diagnostic analyses. One example of a diagnostic application is to isolate RNA from a cell infected with a particular RNA virus. Total or poly-A+ RNA (Ausubel et al.) is end labeled. The RNA is then isolated away from free label and the amount of incorporated label estimated, for example, by scintillation counting. The labeled RNA is then treated with an RNA cleaving agent, such as an RNA-binding protein combined with a chemical cleaving moiety, and the amount of liberated label is used as an indicator of the concentration of RNA contain the RNA- binding protein cognate binding site. The cleaving reagents of the present invention are particularly desirable for use with RNA virus tar¬ gets or their pro-viral DNA forms: for example, cleaving HIV genomic RNA or pro-viral DNA. The cleaving reagents of the present inven¬ tion are also useful in a method of inhibiting expression of RNA viral (e.g., HIV) antigens in cells infected with the virus. For this applica¬ tion, the infected cells are exposed to an RNA binding protein or polypeptide modified to contain a cleaving moiety (i.e., the reagent), at a re¬ agent concentration effective to produce reduction in viral antigen expression in the infected cells. Examples of such reagents for anti-HIV agents have been described above.
It has been demonstrated that chemically synthesized full-length TAT protein as well as truncated polypeptides consisting of nuclear targeting domain are rapidly taken up by cells and have biological effects (Green et al., 1988;
Frankel et al., 1988). Since efficient cellular uptake is relevant for an anti-viral reagent, both modified and unmodified polypeptides are assayed for their ability to enter the cell. One method to evaluate cellular uptake is to label the poly¬ peptides with a fluorescent dye, such as fluores- cein isothiocyanate (FITC) (Pierce, Rockford, IL) at the single cysteine residue. The fluores¬ cent-labeled polypeptides are added to the cell culture medium and the cellular distribution analyzed by fluorescence microscopy. For modified polypeptides, fluorescent labeling is carried out at a single cysteine residue before reacting amino groups with 2-iminothilane for attachment of the cleaving moiety. Alternatively, uptake of the re¬ agent polypeptide can be evaluated using radio¬ active label since any polypeptide can be easily made radioactive during synthesis (Chen et al. , 1986) . Another alternative is to perform an immuno-fluorescence assay on fixed cells after incubation with the reagent using rabbit anti- peptide- antibodies and rhodamine-conjugated goat anti-rabbit antibodies (Malim et al., 1989).
The above methods to evaluate cellular uptake of an RNA-binding protein can be applied to any protein or polypeptide under investigation, e.g., TAT or REV. As has been demonstrated above, TAT covalently attached to the chemical cleaving group, 1,10-phenanthroline results in cleavage of target TAR sequences consistent with polypeptide binding to the 3-nt bulge. These results, along with existing evidence of the rapid cellular up¬ take of TAT polypeptides (Green et al., 1988; Frankel et al., 1988), suggest that chemical nucleases based on TAT may be useful for inacti¬ vating HIV mRNA in vivo. In vivo cell systems allowing the expression of a selected RNA can be used to test the anti¬ viral effects of RNA-cleaving protein/peptide reagents. For example, the in vivo usefulness of the TAT24C-phen polypeptide is tested using a number of cell systems including the following:
(i) Chloramphenicol acetyltransferase (CAT) assays — HIV-1 LTR-directed CAT activity is mea¬ sured under transient expression as well as stable expression conditions. For the transient assay, HeLa cells will be transfected with an expression vector containing the entire U3 region and 78 base pairs of the R region of the HIV LTR (e.g., pHIV- 1LTR-CAT (S. Miller, SRI International, Menlo Park CA) ; or Gendelman et al., 1985). The LTR region contains the enhancer, promoter and TAR elements. Transfection is performed using the DEAE-dextran technique (Queen et al. , 1983). Twelve hours after transfection, the cells are incubated with the polypeptide reagent, over a range of polypeptide concentrations. Mitomycin C is added to the medium to induce CAT expression. Since the HIV-1 LTR is under the influence of NF- kB, the expression of CAT activity can be induced by treating with either UV or mitomycin C (Nabel et al., 1987). After 12 and 24 hours the cells are harvested and CAT activities are determined as described by Gorman et al. (1982) . CAT activities are compared between (i) cell samples which were not treated with the polypeptide reagent, and (ii) cells samples which were treated with the poly¬ peptide reagent. Cleavage of the target substrate by the polypeptide reagent is expected to result in a decrease of CAT activity. Polypeptide re¬ agents containing phenanthrolene are complexed with CuS04 before addition to the cell samples. If the cellular reduction potential is not sufficient for the cleavage to occur, ascorbic acid (or mercaptopropionic acid) is added to the medium.
To assess the activity of polypeptide reagents in the presence of wild-type TAT protein, a stable CAT expression system is used. HeLa cells are cotransfected with a 1:5 ratio of pSV2neo (a mammalian integration plasmid which confers neomycin-resistance; Southern et al., 1982) and pHIV-lLTR-CAT plasmids using DEAE- dextran procedure. Cells are selected for G418 resistance, and individual colonies are picked, expanded, and tested for CAT expression. CAT- active clones are transfected with a wild-type TAT expression vector (e.g., pcDEBtat, S. Miller, SRI International; or pAR, available from the AIDS Re¬ search and Reference Program) . Cells now expressing TAT transiently are used for polypeptide treatment followed by the measurement of CAT activity.
The TAT24C-phen polypeptide reagent is added to the culture media over a range of concentra- tions. The ability of TAT24C-phen to block the transactivation by endogenous TAT protein is determined by measuring chloramphenicol acetyl transferase (CAT) activity over time after the addition of the TAT24C-phen polypeptide. (ii) Chronically HIV-infected cell lines — The ability of TAT24C-phen to block induction of expression is also evaluated using chronically-HIV infected cell lines that produce HIV constitu- tively, e.g., ACH-2 (T-cells; Clouse et al., 1988), Ul (pro-monocytes; Folks et al. , 1987), and H-9 cells. As above, the cells are treated using a range of polypeptide concentrations. Expression of HIV is monitored by one or more of the follow¬ ing standard methods: (a) HIV antigen levels, including p24, asso¬ ciated with HIV-infected cells (e.g., by ELISA (Wang et al. , 1988, 1989; Crowe et al., 1990);
(b) the reverse-transcriptase activity associated with HIV-infected cells; or (d) the level of replication of the HIV-I virus as identified by RNA transcription levels of the viral genome (e.g., slot-blot hybridization (Crowe et al. , 1989)).
(iii) Blocking acute infection — The abil- ity of TAT24C-phen to prevent acute infection by HIV of the following cells will be assessed: PHA-stimulated human peripheral blood lymphocytes, MT4 and Jurkat cells (both CD4+ lymphocyte cell lines) , macrophages, and monocytes infected by monocytropic HIV isolates.
In addition, cell toxicity of the TAT24C-phen polypeptide is evaluated using, for example, killing of Jurkat cells. Also, mutagenicity is evaluated with a standard Ames test.
For cleavage reactions carried out in cells in culture, the intracellular reduction potential can be modulated using N-acetyl cysteine, which increases the intracellular glutathione level (Roederer et al., 1990; Kalebic et al., 1991). Such manipulation of the intracellular reduction potential assist in keeping, for example, a copper atom of a cleaving agent in the reduced state. RNA cleaving reagents composed of an RNA- binding protein and a non-specific nuclease also have important in vivo applications. A specific RNA cleaving-hybrid nuclease can be evaluated as described above when the hybrid nuclease is taken up into cells. Alternatively, CAT expression in Hela cells harboring target RNA-CAT fused genes are assayed in the presence and absence of hybrid- nuclease expressed from an independent promoter. For example, for expression in human cells, the gene for staphylococcal nuclease is cloned adjacent to the TAT or REV gene in plasmids pSV2TAT 72 (or pgTAT) and pCREV, respectively. The resultant plasmids encoding hybrid proteins are transfected into Hela cells carrying either pHIV-CAT or pHIV-env depending on the type of hybrid nuclease. The biological effects of the in vivo expression of TAT24C-nuclease is evaluated using the CAT assay as described above. The effect of the hybrid nuclease containing the gene product of REV will be assayed in Hela cells by quantitating the repression of the production of viral envelope protein as assayed using antibodies against envelope proteins. Thus attaching staphylococcal nuclease to an RNA binding polypeptide (e.g., based on TAT and REV) using a short tether of several amino acids may generate a sequence-specific ribonuclease. Constitutive expression of such an RNA-specific nuclease in an un-infected cell, contained in a population of cells infected with an RNA virus that contains the target RNA binding sequence, may confer resistance of the un-infected cells against viral infection. For example, peripheral blood mononucleocyte cells are isolated from the blood of an HIV-positive patient. T-cells are isolated and transformed to carry a TAT-nuclease hybrid protein encoding gene. The cells are amplified and replaced in the patients blood stream. Such an approach may lead to a gene therapy for the treatment of AIDS: providing HIV-resistant T- cells.
Finally, a combined use of polypeptide-based RNA cleaving reagents combined with the above- described oligonucleotide cleaving agents may provide a two-pronged attack against viral diseases by providing cleavage of viral RNA and pro-viral DNA.
The following examples illustrate, but in no way are intended to limit the present invention.
Materials and Methods
General molecular genetic manipulations were carried out as described by Ausubel et al. and Sambrook et al. General manipulations involving protein/nucleic acid interactions are described in Protein/DNA Interactions, edited by Robert T. Sauer (Methods in Enzymology, Academic Press, 1991) .
Synthetic oligonucleotide linkers and primers were prepared using commercially available auto- mated oligonucleotide synthesizers. Alternative¬ ly, custom designed synthetic oligonucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA) .
Oligonucleotide sequences encoding peptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of cloning steps involving a tandem array of multiple oligonucleotide fragments correspond¬ ing to the coding sequence (Crea; Yoshio et al.; Eaton et al.). Oligonucleotide coding sequences can be expressed by standard recombinant proce¬ dures (Sambrook et al.; Ausubel et al.)
Alternatively, peptides can be synthesized directly by standard in vitro techniques (Applied Biosystems, Foster City CA) . T7 RNA polymerase was purchased from Promega (Madison, WI) and used as per the manufacturer's instructions.
Polynucleotide kinase and restriction enzymes were obtained from Boehringer Mannheim (Indianapolis IN) or New England Biolabs (Beverly MA) and were used as per the manufacturer's directions.
Ribonuclease Tl, Ribonuclease CL3, and SI nuclease were obtained from Boehringer Mannheim and were used as per the manufacturer's direc¬ tions. Uranyl nitrate was obtained from Mallencrodt (Paris KY) .
Radionuclides were obtained from New England Nuclear (Boston, Mass.), ICN (Costa Mesa CA) or Amersham (Arlington Heights IL) .
Mammalian, yeast, and bacterial expression vectors are commercially available from a number of sources including Bethesda Research Laboratories (Gaithersburg MD) and Clontech (Palo Alto CA) . Example 1 Preparation of TAT Protein and Derivative
TAR-Binding polypeptides A. Preparation of the polypeptides. The TAT and REV proteins are isolated as previously described (Weeks et al., 1990, herein incorporated by reference; Brown et al., 1990, herein incorporated by reference) . The entire protein coding sequences of the TAT and REV proteins are presented in Figures 2A (SEQ ID N0:1) and 4 (SEQ ID NO:6), respectively, polypeptides derived from these proteins were synthesized at SRI's polypeptide synthesis facility using a Beck an model 990 polypeptide synthesizer, as per the manufacturer's instructions. Polypeptides having the same C-terminal sequence but truncated at different N-terminal sites are recovered from a single solid-phase synthesis by removing some of the reaction bed at different stages of the syn- thesis.
For the attachment of cleavage groups a cysteine residue can be added at any position internal to the polypeptide sequence or at either the amino- or carboxy- terminal ends of the protein. The insertion of cysteine groups must be consistent with the maintenance of RNA-binding activity; such binding properties can be tested as described below.
The TAT polypeptide designated TAT24C was chemically synthesized as just described. TAT24C consists of amino acid residues 49-72 of the TAT protein (Figure 2A, underlined sequence) and an additional cysteine residue at the C-terminus (Figure 2B, SEQ ID NO:2). The TAT24C polypeptide was purified by standard HPLC.
B. Attachment of Nucleic Acid Cleaving Chemical Moieties. Commercially available 5-nitro-l,10-phenanthroline (Sigma) is converted to 5-iodoacetamido-l,10-phenanthroline (Chen et al., 1986, herein incorporated by reference) (Figure 5) .
HPLC-purified cysteine-containing polypeptides are reacted with 5-iodoacetami¬ do-l,10-phenanthroline to obtain polypeptides containing the phenantroline moiety (Figure 2C) uniquely attached to the side chain of the cysteine residue contained in the polypeptide sequence (Chen et al., 1987, herein incorporated by reference) .
After the addition of the phenanthroline moiety, the resulting polypeptides were separated from un-reacted iodo compound by passing the reaction mixtures through Sephadex G-50 spin columns (Pharmacia, Piscataway NJ) .
Example 2
Preparation of RNA Substrates The sequence of RNA target molecules were chosen based on previous studies characterizing the binding properties of the HIV-encoded TAT protein and the TAR target region from both HIV-1 and HIV-2: HIV-1 TAR is the 57-nt RNA stem-loop structure found in HIV-1 mRNA (nt 1-57); HIV-2 TAR includes the region of HIV-2 RNA essential for transactivation by HIV-2 TAT (nt 13-91, Arya et al., 1988); and ΔTAR is a truncated RNA containing the minimum TAT binding site (nt 17-43, Weeks et al., 1990 ). The three RNA substrates are shown in Figures 1A, IB, and lC. Further, the sequences of the RNA substrates are presented in the sequence listing as follows: HIV-1 TAR, SEQ ID NO:3; HIV-2 TAR, SEQ ID NO:4; and ΔTAR, SEQ ID NO:5. The RNA substrates were synthesized as follows. Synthetic DNA templates were formed by standard phosphoramidate synthesis using a Model 381B synthesizer (Applied Biosystems, Foster City, CA) . The synthetic DNA templates, containing T7 promoter sequences (Stahl et al. , 1981; Davanloo et al. , 1984) in addition to the RNA substrate sequences, were then used to generate uniformly labeled RNAs in vitro transcription of the synthetic DNA templates using T7 RNA polymerase (Milligan et al., 1987). In order to facilitate isolation of the newly generated RNA molecules, transcription was carried out in the presence of 32P-α-CTP (Amersham) . The RNA molecules were purified by size fractionation on denaturing 10% polyacrylamide gels (Maniatis et al. ; Sambrook et al.) followed by electroelution. The isolated RNA molecules were then heated to 70'C and slowly cooled to room temperature to facilitate formation of the native secondary structure.
Further, both the wild-type RRE (WT-RRE) element (SEQ ID NO:23) and a truncated version of RRE (ΔRRE, SEQ ID NO:24) which contains the minimum domain (stem-loop II; Cook et al. , 1991) of RRE, will be used. These RNAs are synthesized by in vitro transcription using T7 RNA polymerase as described above for TAR RNAs. WT-RRE can also be transcribed using a "BLUESCRIPT" plasmid (Stratagene, La Jolla, CA) carrying a 280 bp insert containing base pairs 7333-7612 of the RRE region (Daly et al. , 1989) . Transcribed RNAs are purified and end-labeled as described above.
Example 3 Target Binding Assay
Approximately 103 cpm of each uniformly labelled RNA substrate (Example 2) was incubated with either TAT24C or TAT24C-phen in a buffer containing 70 mM NaCl, 0.2 mM EDTA, 10 mM Tris-HCl (pH 7.5), 5% glycerol and 0.1% "NONIDET P40" (Sigma, St. Louis MO) for 20 minutes at 25°C. The samples were then run on a 10% native polyacrylamide gels in TBE (Tris-Borate-EDTA) buffer (Maniatis et al.; Sambrook, et al.) at room temperature. To obtain autoradiograms the gels were exposed to X-ray film. Figure 3 shows a photograph of the resulting autoradiogram.
In Figure 3, panel A, using HIV-1 TAR-RNA substrate: lane 1, in the presence of TAT24C (200 ng) ; lane 2, no polypeptide was added; lane 3, in the presence of TAT24C-phen (200 ng) . In Figure 3, panel B, using HIV-2 TAR-RNA substrate: lane 1, in the presence of TAT24C (200 ng) ; lane 2, no polypeptide was added; lane 3, in the presence of TAT24C-phen (200 ng) . In Figure 3, panel C, using ΔTAR-RNA as substrate: lane 1, no polypeptide was added; lane 2, in the presence of TAT24C-phen (200 ng) . In Figure 3, panel D, using yeast tRNA (Bethesda Research Laboratories, Gaithersburg MD) as the substrate: lane 1, in the presence of TAT24C-phen (200 ng) ; lane 2, no protein was added; lanes 3 and 4 were with 200 ng and 400 ng of TAT24, respectively.
Discrete bands demonstrating retarded gel mobility were observed in samples containing both modified and unmodified polypeptides (Figure 3A, lanes 1 and 3; Figure 3B, lanes 1 and 3; and
Figure 3C, lane 2) . These results show binding of polypeptides to all three RNAs containing the TAT responsive TAR element. The mobility shift of samples containing modified and unmodified polypeptides are virtually identical, indicating that the attachment of the phenanthroline moiety at the C-terminus of the polypeptide does not affect binding to the TAR site. There is no, or very low level, mobility shift with either polypeptide when tRNA was used as the substrate (Figure 3D) indicating that the binding for TAR-containing RNA is specific.
Example 4 Cleavage of TAR-Site Containing Substrates A. Cleavage of HIV-1 TAR. Cleavage reactions using the polypeptide- cleaving reagents of the present invention were typically performed as follows. RNA substrates were 5' end-labeled (Maniatis et al. ; Sambrook, et al.) employing T4 polynucleotide kinase (Boehringer Mannheim) using 32P~7-ATP (Amersham) and purified by gel electrophoresis as described above. Approximately 30 pmol of RNA was incubated in 10 μl of buffer A in the presence of TAT24C-phen, and cleavage was initiated by adding CuS04 (to a final concentration of 10 μM) and mercaptopropionic acid (to a final concentration of 2.5 mM) after 10 minutes at 22*C. After incubating for 17 hours, the reaction was stopped by adding the following to the indicated final concentrations: 2,9-dimethyl-l,10-phenanthroline to 3 mM, tRNA to 0.2 mg/ml, and sodium acetate (NaOAc) to 0.3 M. Typically 5' end-labeled, gel purified, HIV-1 TAR RNA (103 cpm) was used as the substrate in the following reactions. In Figure 8A, lane 1, RNA only; lane 2,
TAT24C-phen (20 pmol); lane 3, TAT24C-phen (30 pmol) ; lane 4, SI nuclease (1 U) , incubated for 5 min at room temperature; lane 5, G-specific reaction, ribonuclease Tl (1 U) , incubated for 10 min at 37"C in 10 μl of buffer A (70 mM NaCl, 10 mM Tris-HCl (pH 7.5)); lane 6, C-specific reac¬ tion, ribonuclease CL3 (0.2 U) , incubated for 20 min at 37°C in 10 μl of buffer A; lane 7, cleavage at every nucleotide by irradiating RNA with 350-nm light (1.2 J in a "STRATALINKER", Stratagene, La Jolla, CA) at 25°C in the presence of 20 mM uranyl nitrate; lane a, 40 μM Cu(II)-1,10-phenanthroline; lane b, TAT24C-phen (30 pmol) in the absence of Cu(II) , same as lane 3 except that no CuS04 was added.
Figure 8B presents fine mapping analysis of the cleavage sites at the 3' half of HIV-1 TAR. All reactions were conducted as described above. In Figure 8B, lane 1, incubation with TAT24C-phen (90 ng) ; lane 2, incubation with TAT24C-phen (60 ng) ; lane 3, SI cleavage; lane 4, uranyl nitrate cleavage; and lane 5, Ribonuclease CL3 digestion. Figure 8B. represents a gel subjected to longer electrophoresis time in order to separate larger fragments.
RNAs from all reactions were ethanol precipitated and analyzed on a denaturing (8.3 M urea) 15% polyacrylamide gel. The gel was dried and autoradiographed.
A summary of the cleavage sites on HIV-l TAR RNA are indicated by arrows in the RNA structure shown between Figures 8A and 8B.
B. Cleavage of HIV-1 ΔTAR RNA.
Cleavage reactions were carried out as above except that 5' end-labeled, gel purified, ΔTAR RNA (103 cpm) was used as the substrate for the following reactions. Figure 8B, lane 1, cleavage with Cu(II)-1,10-phenanthroline; lane 2, Uranyl nitrate ladder; lane 3, SI cleavage; lane 4, RNA only; lanes 5 and 6, TAT24C-phen cleavage (20 pmol, lane 5 and 30 pmol, lane 6).
As above, RNAs from all reactions were ethanol precipitated and analyzed on a denaturing (8.3 M urea) 15% polyacrylamide gel. The gel was dried and autoradiographed.
A summary of the cleavage sites on ΔTAR RNA are indicated by arrows in Figure 8C.
C. Cleavage of HIV-2 TAR RNA. Cleavage reactions were performed as described above except that 5' end-labeled, gel purified HIV-2 TAR RNA (103 cpm) was used as substrate for the following reactions. In Figure 9A, lane 1, TAT24C-phen (30 pmol); lane 2, TAT24C-phen (20 pmol); lane 3, RNA only; lane 4, SI nuclease; lane 5, uranyl nitrate ladder; lane 6, C-specific reaction; lane 7, G-specific reaction.
RNAs from all reactions were ethanol precipitated and analyzed on a denaturing (8.3 M urea) 15% polyacrylamide gel. The gel was dried and autoradiographed. The cleavage sites on the HIV-2 TAR RNA are indicated by arrows.
D. Cleavage of tRNA. Cleavage reactions were performed as described above except that 5' end-labeled, gel purified tRNA (yeast tRNA; Bethesda Research
Laboratories, Gaithersburg MD) (103 cpm) was used as substrate for the following reactions. In Figure 9B, lane 1, Cu(II)-1,10-phenanthroline (40 mM) ; lane 2, TAT24C-phen (60 pmol); lane 3, TAT24C-phen (30 pmol) ; lane 4, RNA only; lane 5, SI nuclease.
RNAs from all reactions were ethanol precipitated and analyzed on a denaturing (8.3 M urea) 15% polyacrylamide gel. The gel was dried and autoradiographed. E. Analysis of Cleavage Results. The chemical nuclease activity of Cu(II)-com¬ plexed 1,10-phenanthroline derives from an oxidative attack on the sugar ring by a copper-oxo species generated in the presence of a reducing agent (Sigman et al., 1990). The nucleolytic activity of TAT24C-phen on HIV-1 TAR is shown in Figure 8A. As seen in lanes 2 and 3, cleavage occurs primarily in the loop of the target RNA (structure shown between Figures 8A and 8B) , especially at the uridine (U30) in the 5' side of the loop.
A secondary cleavage site can also be seen on the stem, at nt 12-14 and 18 (indicated by the short arrows in the RNA structure shown in Figure 8A) and at nt 43-45 on the complementary region (Figure 8B) . The cleavage pattern on opposite sides of the stem is shifted to the 5' side, an indication that the cleaving moiety is occupying the major groove of the duplex RNA stem (Dervan,
1986; Sluka et al., 1987). A possible alternative binding mode, where the basic region of TAT24C-phen polypeptide binds to the minor groove and the phenanthroline moiety reaches over to the major groove to effect cleavage, is unlikely based on evidence suggesting that TAT-derived poly¬ peptides bind in the major groove (Weeks et al., 1991) . Further, binding of the TAT24C-phen poly¬ peptide to the minor grove is unlikely since, at least in the case of B-DNA, Cu(II)-1,10-phenan¬ throline prefers to bind to the minor groove (Sigman et al., 1990). Overall, the cleavage sites lie on either side of the bulge where the TAT protein is known to bind to the TAR target site ( Roy et al., 1990; Cordingley et al., 1990; Dingwall et al., 1989; Weeks et al., 1990). Because Cu(II)-phenanthroline is known to preferentially cleave unpaired bases of RNA (Murakawa et al., 1989), HIV-1 TAR was incubated with free Cu(II)-1,10-phenanthroline, i.e., not bound to RNA-binding protein, as a control. As shown in lane A (Figure 8A) , free phenanthroline cleaves everywhere, but the unpaired bases at the loop (especially G32) and the bulge are most reactive. Thus, tethering phenanthroline to the polypeptide suppresses nonspecific cleavage on TAR RNA.
In the absence of cupric ions, TAT24C-phen produces no cleavage (lane B, Figure 8A) .
The cleavage pattern on ΔTAR, which lacks the base of the stem, is consistent with that of full-length TAR (Figure 8C) , suggesting that the minimum TAT binding site is sufficient to be recognized by TAT24C-phen. Using the ΔTAR substrate, the cleavage is restricted to the loop, but the pattern is very different from that for cleavage by free phenanthroline (compare lane 1 with lanes 5 and 6 of Figure 8C) .
In contrast to cleavage of HIV-1 TAR, for which the primary site is at the loop adjacent to the TAT binding site, cleavage of HIV-2 TAR takes place mainly at the stem, roughly midway between the two loops (Figure 9A, lanes 1 and 2) . HIV-2 TAR has two 2-nt bulges, both of which have the consensus TAT binding motif (Weeks et al. , 1990; Green et al., 1988; Frankel et al. , 1988; Milligan et al.,1987; Arya et al. , 1988; Weeks et al. , 1991; Murakawa et al., 1989). HIV-1 TAT can transactivate HIV-2 LTR-directed gene expression when either stem-loop I or stem-loop II is present (although the TAT product of HIV-2 requires both stem-loops for efficient transactivation (Arya et al. , 1988; Emerman et al. , 1987). Accordingly, the HIV-1 encoded TAT protein appears to bind to either stem-loop. Because the TAT24C polypeptide is based on HIV-1 TAT, the cleavage of both loops probably results from binding of the polypeptide to both elements. The major cleavage site for HIV-2 thus corresponds to the minor cleavage site for HIV-1 TAR (i.e., approximately 3-8 base pairs from the bulge in the direction away from the loop(s)). The higher level of cleavage at this site could be partly due to a superimposition of the cleavage resulting from TAT24C-phen binding on both stem-loops I and II. Alternatively, TAT24C-phen might bind only to stem-loop I and the molecule could fold to bring the two loops close together. When tRNA was used as the substrate for cleavage, the cleavage pattern induced by TAT24C-phen was identical with that caused by free Cu(II)-phenanthroline (Figure 9B) , indicating that TAT24C-phen does not induce site-specific cleavage on RNA lacking a TAR site.
In the absence of the reducing agent mercaptopropionic acid, Cu(II)-complexed TAT24C-phen still cleaves TAR-containing RNA, presumably via a Cu+2-induced hydrolytic pathway (Modak et al., 1991) but with lower efficiency. When mercaptopropionic acid was replaced by ascorbic acid, a reducing agent more suitable for in vivo studies, the rate of cleavage increased. While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jayasena, Sumedha D. Johnston, Brian H.
(ii) TITLE OF INVENTION: Antiviral Reagents Based on
RNA-Binding Proteins
(iii) NUMBER OF SEQUENCES: 24 (iv) CORRESPONDENCE ADDRESS:
CA) ADDRESSEE: SRI International
(B) STREET: 333 Ravenswood Avenue
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(F) ZIP: 94025
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25
( i) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/808,452
(B) FILING DATE: 13-DEC-1991
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Fabian, Gary R.
(B) REGISTRATION NUMBER: 33,875
(C) REFERENCE/DOCKET NUMBER: P-2962 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 859-4550
(B) TELEFAX: (415) 859-3880
(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: the sequence of the TAT protein of
HIV-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser 1 5 10 15
Gin Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe 20 25 30
His Cys Gin Val Cys Phe lie Thr Lys Ala Leu Gly lie Ser Tyr Gly 35 40 45
Arg Lys Lys Arg Arg Gin Arg Arg Arg Pro Pro Gin Gly Ser Gin Thr 50 55 60
His Gin Val Ser Leu Ser Lys Gin Pro Thr Ser Gin Ser Arg Gly Asp 65 70 75 80
Pro Thr Gly Pro Lys Glu 85
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: the sequence of the TAT24C peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Arg Lys Lys Arg Arg Gin Arg Arg Arg Pro Pro Gin Gly Ser Gin Thr 1 5 10 15
His Gin Val Ser Leu Ser Lys Gin Cys 20 25
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: the sequence of the TAR site of HIV-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GGUCUCUCUG GUUAGACCAG AUCUGAGCCU GGGAGCUCUC UGGCUAACUA GAGAACCC 58
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 77 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: the sequence of the TAR site of HIV-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GGGAGGCUGG CAGAUUGAGC CCUGGGAGGU UCUCUCCAGC CUAGCAGGUA GAGCCUGGGU 60
GUUCCCUGCU AGCUCCC 77
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: the sequence of a truncated TAR site of HIV-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GCAGAUCUGA GCCUGGGAGC UCUCUGC 27
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 116 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: the sequence of the REV protein of HIV-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ala Gly Arg Ser Gly Asp Ser Asp Glu Asp Leu Leu Lys Ala Val 1 5 10 15
Arg Leu lie Lys Phe-Leu Tyr Gin Ser Asn Pro Pro Pro Asn Pro Glμ 20 25 30
Gly Thr Arg Gin Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg 35 40 45
Gin Arg Gin lie His Ser lie Ser Glu Arg lie Leu Ser Thr Tyr Leu 50 55 60
Gly Arg Ser Ala Glu Pro Val Pro Leu Gin Leu Pro Pro Leu Glu Arg 65 70 75 80
Leu Thr Leu Asp Cys Asn Glu Asp Cys Gly Thr Ser Gly Thr Gin Gly 85 90 95
Val Gly Ser Pro Gin lie Leu Val Glu Ser Pro Thr lie Leu Glu Ser 100 105 110
Gly Ala Lys Glu
115
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE : peptide
( iii ) HYPOTHETICAL : NO
( iv ) ANTI -SENSE : NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: a peptide derived from the REV protein of HIV-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Leu lie Lys Phe Leu Tyr Gin Ser Asn Pro Pro Pro Asn Pro Glu Gly 1 5 10 15
Thr Arg Gin Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg Gin 20 25 30
Arg Gin lie His Ser lie Ser Glu Arg lie Leu Ser Thr Tyr Leu Gly 35 40 45
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: HIV-LTR TARGET REGION, 11A
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
TTTAAAAGAA AAGGGGGGAC TGG 23 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: HIV-LTR TARGET REGION, 11B
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GCTGGGACTT TCCAGGGAGG CGT 23
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: HIV-LTR TARGET REGION, 11C
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CCTGGGCGGG ACTGGGGAGT GGCGAGCCC 29
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SINGLE STRAND OLIGONUCLEOTIDE, 12/Al
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
AGGGGGGAAA AGAAAA 16
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SINGLE STRAND OLIGONUCLEOTIDE, 12/A-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
TTTTCTTTTC CCCCCT 16
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SINGLE STRAND OLIGONUCLEOTIDE, 12/B-l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GGAGGGACCT TTCAGGGG 18
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SINGLE STRAND OLIGONUCLEOTIDE, 12/B-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CCCCTGAAAG GTCCCTCC 18
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: SINGLE STRAND OLIGONUCLEOTIDE, 12/C-l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GAGGGGAGAG GGGAGAGGGG GGG 23
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SINGLE STRAND OLIGONUCLEOTIDE,
Figure imgf000065_0001
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
CCCGCCCTGA CCCCTCACCG CTC 23
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SINGLE STRAND OLIGONUCLEOTIDE, 12/K (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
AAAAGAAAAG GGGGGA 16
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SINGLE STRAND OLIGONUCLEOTIDE, 12/L
(xi) SEQUENCE.DESCRIPTION: SEQ ID NO:18:
GGGGATCCCT TAGGGAGG 18
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SINGLE STRAND OLIGONUCLEOTIDE, 12/M
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GGGTGGGATC GGGGAGCGGT GGAGAG 26 (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SINGLE STRAND OLIGONUCLEOTIDE,
FIGURE 13
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION: 25..29
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "WHERE N=BASIC RESIDUE"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
TCTTCTCCTC CTTTTTTCCT TTTTNNNNNT TTTTCCTTTT TTCCTCCTCT TCT 53
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: TARGET RNA, FIGURE 13
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GGUAGAAGAG GAGGAAAAAA GGAAAAACUG 30 (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: polypeptide containing the basic domain of HIV-REV protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Thr Arg Gin Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg Gin 1 5 10 15
Arg
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 244 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: HIV-1 REV RESPONSE ELEMENT, FIGURE 14
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CAGUGGGAAU AGGAGCUUUG UUCCUUGGGU UCUUGGGAGC AGCAGGAAGC ACUAUGGGCG 60 CAGCGUCAAU GACGCUGACG GUACAGGCCA GACAAUUAUU GUCUGGUAUA GUGCAGCAGC 120
AGAACAAUUU GCUGAGGGCU AUUGAGGCGC AACAGCAUCU GUUGCAACUC ACAGUCUGGG 180
GCAUCAAGCA GCUCCAGGCA AGAAUCCUGG CUGUGGAAAG AUACCUAAAG GAUCAACAGC 240
UCCU 244
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: STEM II OF THE HIV-1 REV RESPONSE ELEMENT, FIGURE 14
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
AGCACUAUGG GCGCAGCGUC AAUGACGCUG ACGGUACAGG CCAGACAAUU AUUGUCUGGU 60
AUAGUGCAG 69

Claims

IT IS CLAIMED:
1. A viral polypeptide having site-specific viral-RNA binding, where said polypeptide is modified to contain a moiety capable of cleaving an RNA backbone.
2. The polypeptide of claim 1, wherein said polypeptide is selected from the group of polypep¬ tides having the amino acid sequences presented as SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:6, SEQ ID N0:7 and SEQ ID NO:22.
3. The polypeptide of claim 1, wherein said moiety is selected from the group consisting of phenanthroline Cu(II) , Zn(II) , Fe(II)-EDTA, Cu(II)-bipyridine, and Cu(II)-terpyridine.
4. The polypeptide of claim 2, wherein the polypeptide has the sequence presented as SEQ ID N0:1, which further contains an end-terminal cysteine residue, and the moiety is phenanthroline.
5. The polypeptide of claim 2, wherein the polypeptide has the sequence presented as SEQ ID NO:2 and the moiety is phenanthroline.
6. The polypeptide of claim 2, wherein said moiety is a non-specific nuclease.
7. The polypeptide of claim 6, wherein the polypeptide has the sequence presented as SEQ ID NO:2 and the non-specific nuclease is Staphylococcal nuclease.
8. A method of cleaving HIV RNA, comprising contacting an HIV RNA molecule with a modified HIV polypeptide which is capable of site- specific binding to HIV RNA, where said modified HIV polypeptide contains a moiety capable of cleaving the backbone linkages of the HIV RNA; further, where said modified HIV polypeptide is at a polypeptide concentration effective to produce cleavage of said HIV genomic RNA.
9. The method of claim 8, wherein said contacting further includes a reducing agent selected from the group consisting of mercapto¬ propionic acid, N-acetyl cysteine, and ascorbate.
10. The method of claim 8, wherein said polypeptide is selected from the group of polypeptides having the amino acid sequences presented as SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:22.
11. The method of claim 8, wherein said moiety is selected from the group consisting of phenanthroline Cu(II) , Zn(II) , Fe(II)-EDTA, Cu(II)-bipyridine, and Cu(II)-terpyridine.
12. The method of claim 11, wherein the polypeptide has the sequence presented as SEQ ID NO:l, which further contains an end-terminal cysteine residue, and the moiety is phenanthroline.
13. The method of claim 11, wherein the polypeptide has the sequence presented as SEQ ID NO:2 and the moiety is phenanthroline.
14. The method of claim 12, wherein said contacting further includes the addition of CuS04 and mecaptopropionic acid.
15. The method of claim 8, wherein said moiety is a non-specific nuclease.
16. The method of claim 15, wherein the polypeptide has the sequence presented as SEQ ID NO:2 and the non-specific nuclease is Staphylococcal nuclease.
17. A method of inhibiting expression of HIV antigens in cells infected with HIV, comprising exposing the infected cells to a modified HIV polypeptide capable of
(i) site-specific binding to HIV RNA, where said modified HIV polypeptide contains a moiety capable of cleaving the backbone linkages of the HIV RNA, and
(ii) uptake into infected cells; further, where said exposing is at a concentration of modified polypeptide effective to produce reduction in viral antigen expression in HIV infected cells.
18. The method of claim 17, wherein said exposing further includes a reducing agent selected from the group consisting of N-acetyl cysteine and ascorbate.
19. The method of claim 17, wherein said polypeptide is selected from the group of polypeptides having the amino acid sequences presented as SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:6, SEQ ID N0:7, and SEQ ID N0:22.
20. The method of claim 17, wherein said moiety is selected from the group consisting of phenanthroline Cu(II) , Zn(II) , Fe(II)-EDTA,
Cu(II)-bipyridine, and Cu(II)-terpyridine.
21. The method of claim 17, wherein said moiety is a non-specific nuclease.
PCT/US1992/010770 1991-12-13 1992-12-11 Antiviral reagents based on rna-binding proteins WO1993012234A1 (en)

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