CA2076037A1 - Method of site-specific alteration of rna and production of encoded polypeptides - Google Patents
Method of site-specific alteration of rna and production of encoded polypeptidesInfo
- Publication number
- CA2076037A1 CA2076037A1 CA002076037A CA2076037A CA2076037A1 CA 2076037 A1 CA2076037 A1 CA 2076037A1 CA 002076037 A CA002076037 A CA 002076037A CA 2076037 A CA2076037 A CA 2076037A CA 2076037 A1 CA2076037 A1 CA 2076037A1
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- rna
- rna molecule
- segment
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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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- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
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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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/823—Reproductive tissue-specific promoters
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/312—Phosphonates
- C12N2310/3125—Methylphosphonates
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
Abstract
A method of site-directed alteration (removal or removal followed by replacement) of selected nucleotides in an RNA molecule, as well as to mixed phosphate backbone oligonucleotides useful in the method. It further relates to a method of producing polypeptides or proteins encoded by the RNA molecule altered by the present method. Through use of the present method, site-directed cleavage of an RNA molecule is effected, followed by excision of the selected or target segment of the RNA molecule.
Description
U'091/12323 PCT/US9ltO0968 HETHOD O~ SITE SPECIFIC ALTERATION O~ RNA AND
PRODUCTION OF ENCODED POLYPEPTIDES
Description Back~round ____ _____ 05 Several aspects of contemporary molecular genetics and biotechnology make it desirable to be able to produce genetically-altered proteins. For example, mu~ated protein domains are sometimes hyper-immunogenic, facilitating the production of neutralizing antibody-based vaccines. Moreover, site-directed mutations, ideally one amino ~cid at a time, csn be a powerful approach to deciphering protein structure and/or enzyme-substrate reaction mechanisms.
Typically, deletions or substitutions of amino acids are made at the gene or DNA level, by recombinant DNA
techniques ~hich rely on the use of restriction endonucleases. However, restriction endonucleases available have a limited array of target sites in DNA , (usually palindromic hexanucleotide or octanucleotide z~ sequences). Deletion of a particular in-frame trinucleo-tide or trinucleotides ~ay not be possible because there ~ay be no suitably located restriction sites. As a result, presently-available methods of altering an amino . . . .: .
. . ~ .
.
WO9~/1232~ PCT/US91/00968 acid sequence by altering the DNA sequence which encodes it, are limited in their applicsbility.
Summary of the Inventlon ______ _ _ _______._ __ The present invention relates to a method of site-05 directed alteration (removal or re~o~al followed by replacement) of selected nucleotides in an RNA ~o~ecule, as well ns to ~i~ed phosphate backbone oligonucleotides useful in the method. It further relates to ~ method of producing polypept~des or proteins encoded by the RNA
molecule altered by the present-method. Through use of the present method, site-directed cleavage of an RNA
molecule is effected, followed by e~cision of the selected or target segment of the RNA molecule.
Follo~ing cleavage and excision, in onç embodiment, the two segmen~s of the resulting interrupted RNA.molecule ~re ~oined, through the sction of an appropriate ligase.
This results in production of a continuous RNA molecule, referred to as an altered RNA molecule, which is the same as the original RNA molecule except that it lacks the nucleotides originally pres~ent in the target segment of the RNA molecule. In a second embodiment, selected nucleotides can be introduced into the space or gap created by removal of the target RNA segment; a continuous RNA molecule is created by ligating the selected nucleotides introduced in this manner to the nYcleotide on each side of the gap.
ln the present ~etkod, an RNA molecule ~hose nucleo-tide sequence is to be altered in a site-directed manner -: . .
' .
, 3 PCT/~S91/00968 2076~37 is brou~ht into contact with an oligonucleotide, referred to as a mixed phosphate backbone oligonucleotide, in the presence of RN~se H. The mixed phosphste backbone oligonucleotide is complementary to all or a portion of 05 an RNA ~olecule ~hich includes a target segment to be altered. ln addition, the mixed phosphate backbone oligonucleotide includea ~n internal portion or ~egment of deoxynucleotides which is capsble of sctivatin~ RNase H ~nd is flanked on each side by a cequence of nucleo-tides ~hich is unable to sctivate RN~se H. The internalsequence includes two or ~ore consecutive phosphodiester linka~es, ~hich may be unmodified or modified. The flanking sequences are modified deo~yribonucleotide or ribonucleotide sequences. It has been shown that when lS such a mixed phosphate backbone oligonucleotide is contacted with a target segment of an RNA molecule, according to the method of th,e present invention~ the result is RNase H metiated excision of RNA target nucleotides complementary only to the internal sequence of oligonucleotides. This mekes it possible to excise precisely any tesired nucleotide or oligonucleotide from an RNA molecule. Followed by ~NA ligation, this results in a desired altered messenger RNA or other type of RNA. ;~
Thus, for the first time, it is possible to carry out -2S precise excision of a selected segment of sn RNA
molecule.
As a result, it is possible to selectively telete any desired number of nucleotides and, if desired, to ~ntroduce replacement nucleotides. The encoded amino acid sequence or polypeptide can be produced by express-ing the ~ltered RNA in vitro or in vivo. As a result, a ... .. .
.
WO91/12323 PCT/USgl/00968 selected a~ino acid sequence or selected polypeptide csn be produced by the present method by: l) producing an altered RNA ~olecule encoding a elected ~mino acld sequence or selected polypeptide snd 2) expressing the O5 ~ltered RNA ~olecule under appropr1ate conditlons.
Cell-free translation of the altercd RNA molecule can be cnrried out to produce deslred mutant proteins use~ul, for e~ample, for ~tudies of protein structure or func-tion. Aleernati~ely, an appropriate ~ixed phosphate backbone oli~onucleotide can be -taken up by or introduced into cultured cells or into c"eils of an animal or a plsnt, in ~hich endogenoùs RNase H and RNA ligase activities can produce altered RNAs; upon translation, corresponding genetically altered protein~ are produced.
lS This is useful, for example, as a means of producing defective viral or infectious/pathogenlc agent seplication or gene expression, which can be useful therapeutically or prophylactically.
Brief Descri~tion of the Drawings Figure l is a schematic representation of the present method for excision of a tar~eted segment of an RNA molecule in ~hich sequence I is RNA from which a targeted segment is to be excised and sequence II is a mixed phosphate backbone oligonucleotide.
Subscript "m designates an RNase H-resistant internucleoside phosphate; subscript "s" indicates an RNase H-suscept~ble internucleoside phosphate; "X"
designates any of the four ribonucleotides A, C, G or U;
and ~Y~ des~gnates a deoxyribonucleotide complementary to 30 the ribonucleotide directly abo~e it. ' Figure 2 is a schematic representatlon of repair of a genetic defect by the method of the present invention, in ~hlch IVa and IVb represent a double-stranded hybrid consisting of a smaller unmodified ribonucleotide (IVa) 05 hybr~dized to B lsrger nodifled, RNase H-resistant deo~yribonucleot~de (IVb) and V is the repa~red RNA
hybridized to the RNase N-r6sistant deo~yr~bonucleot~de.
Detailed Descri~tion of the Invention The present invent~on relstes to a method of site-specific or site-directed alteration (removal or removal follo~ed by replacement) of selected nucleotides (i.e., a target segment) in an RNA molecule, to produce an altered RNA sequence, as well as to mixed phosphate back~one oligonucleotides useful in the method. It further xelates to a method of produc~ng altered amino acid sequences or polypept~des by translating the altered RNA
seq~ence, which results in production of the encoded ~olecule.
In the present method, a selected or target segment of an RNA molecule, such as pre-mRNA, mRNA or viral RNA, is altered as follows: an RNA molecule which includes the target segment (l.e., a nucleotide or a nucleotide sequence to be altered) is combined ~ith an appropri-ately-selected mixed phosphate backbone oligonucleotide in the presence of RNase H. The mixed phosphate backbone oligonucleotide is ;complementary to all or a portion of the RNA ~olecule which includes the target BNA segment;
it is of sufficient length to hybridize to the target RNA
segment and sequences on either side and remain - . ~' ' U'091/12323 PCT/US91/~0968 2076~37 hybridized under the conditions used. The mixed phosphate backbone oli~onucleotide has ~o key components: an internsl portion or segment o f deoxynucleotides which is capable of acei~ating RN~se H
05 and t~o nucleotide cequences, ~hich flank the i~ternal seg~ent, ~h~ch are unable to act~vate RNase H. The internal deoxynucleotide segment includes two or ~ore phosphodieçter linkages, ~hlch ~ay be un~odLfied or modifled. The fianking nucleot~de sequence3 m~y be deo~yribonucleotide or ribonucleotide sequences and is modified. That is, some or all of the internucleoside bridging phosphate residues are modified phosphates, such as methyl phosphonates, phosphoromorpholidates, phosphoropiperzaidates and phosphoramidates. An essential feature of the mi~ed phosphate backbone olig'onucleotide is that the internal complementary segment is RNase H acti~ating and the flanking complementary sequences are unable to activate RNase H.
As demonstrated herein, site-directed alteration occurs ~hen the RNA molecule which includes the target segment and an appropriately selected mixed phosphate backbone oligonucleotide are combined in the presence of RNase H and maintained under appropriate conditions ~e.~., temperaturè, time, salt concentration) for 2~ complementary nucleotide sequences to hybridize and RNase H to be activated (i.e., to be able to clea~e aDd excise). That is, as a result, the nucleotides in the RNA molecule to ~hich tbe internal segment of the mixed phosphate backbone oligonucleotide i5 complementary are excised precisely from the RNA molecule.
.,. , ", . : :. .
, WO9l/1'~1~ Pcr/ US91/00968 2~760~7 , In one embodiment of the present ~ethod, the gap resulting from e~cision of the target RNA ~e~ent can be closed by the sct~it; of an appropriate ligase(s), resulting in a continuous RNA ~olecule refarred to as an ;05 altered RNA oolecule. The resultlng alter~d RNA ~olec~le differs from the RNA molecule only as to the target regment, Yhich ~s not pr~sent in the altered RNA ~ole-cule.
In ~ ~econd embodiment the gap created by the action 10 of RNase H as described above can be f~lled in by inero- -ducing a segment of replacement nucleotides, which can be of any length appropriate to fit into the gap created in the RNA molecule. The se~ent of replacement nucleotides is subsequently linked to the stjacent nucleotides of the RNA molecule by an sppropriPtely selected ligsse(s). The nucleotide present at each end of the segment of replace-ment nucleotides is ligated to the nucleotide present on the respecti~e end~ of the gap created in the RNA
molecule (underlined in Fi~ure 2(111~). The resulting altered RNA molecule tiffers from the RNA ~olecule in that the target segment (present in the RNA ~olecule) has been removed snd a replacement nucleotide cequence introduced in its place.
Altered RNA produced as des~ribed herein can be expressed, either in_vitro or i__~i~o, to produce the encoded polypeptide or protein; ~s used herein, the term polypeptide includes proteins. For example, the altered RNA can be introduced into an appropr~ate ~ector, which is in turn introduced into a host cell capable of trans-lating the alt~red RNA ~olecule and producing the encodedpolypeptide. ~olypeptides produced in this ~anner can be .... ~: .
' ~
WO9t/12323 PCr/US9l/00968 used for assessment of thelr structur~l/functional characteristics and a comparative assess~ent of polypep-tides ~hich d~ffer ln ~ defined ~anner ~e.~., by selected a~ino acids). Such polypeptides c~n also be used 05 therapeutically or prophylactically.
The following is M description, with reference to the fi~ures, of two enbodimenes of the present ~ethod of site-directed alteration of an ~NA molecule: a firfit embodiment in which a selected nucleotide qequence ~target segment) is re~oved and the resulting fragments ligated to produce sn altered RNA molecule lacking the target segment and 8 second embodiment in which a selected nucleotide sequence ~s removed and replaced by a selected nucleot~de or nucleotides, ~hich are ligsted to the nucleotide at each side of the gap created by removal of the target RNA segment.
~ethod for Excisin~ a SPecific Small Se~ment of Rlbo-_________ _ _______ ____ _______________ _____________ nucleotides from an RNA Molecule Figure l ls a schematic representation of sn embodi-ment of the present method.~ The sequence designated I is a segment of an RNA molecule, ~hich includes a t~rget segcent (~GACGUCA) to be altered. The RNA may be a pre-RNA, a viral ~NA, or one of a Yariety of RNAs present in animal, plant or bacterlal cells. The sequence designated II is a se~ment of DNA which is a mixed phosphate backbone oli~odeoxynucleotide complementary to the RNA molecule I. It can be synthesized us~ng known techniques, such as chemical or enzymatic methods. The mixed phosphate backbone oligodeoxynucleotide includes an inte~nal sequence which is capable of activating RNase H
.
.
. . - . .
. , ~ . .
WO 91/12323 PCI/US91/OOg68 g and is flanked on each side by a DNA ~egment which is incapable of activating RNase h, ~he total length of the ~i~ed phosphate backbone oligodeoxynucleotides varies, dopending on the length of the target RNA segment to be 05 gltered, but must be sufficient to hybrid$ze to the RNA
nolecule containing the target RNA segment Hnd remain hybrldiz~d under the cond~tions u~ed. The ~nternal ~egDent ~ust be of sufficient length--et least two nucleotides--to be capable of actl~ating RNase H, ~s de~onstrstet herein and by others (Ualder, ~.Y. snd J.A.
~alder, Proc. Natl. acad. Sci USA, 85:5011-5015 ~1988);
Furdon, P.J. et al., Nucleic Acids_Res., 17:9193 9204 (1989)).
In the internal segment, the internucleoside bridg-lS ing phosphate residues may be unmodified phosphates or any phosphate modific~tion capable of activa~ing RNase ~, such a~ phosphorothioates. The flanking n~cleotide sequences can be deo~yribonucleotides, as descr~bed in this embodiment or can be ribonucleotides and their modifications. The flanking sequences sre connected by methyl phosphonates (PC), phosphoromorpholldates (PM), phosphoropiperaz~dates, phosphoramidates, or other modifications of in~ernucleoside phosphates which are not able to activate RNase H. The RNA molecule I and the ~odified backbone oligodeoxynuclestide II are combinet in the presence of RNase H, such as endogenous RNase H in a cell, and excision of the target segment occurs. A
hairpin loop may form by pairing of complementary nucleo-tides, with the result that the two newly-formed segments are brought into proximity to each oeher. lt ~ay not be necessary that a hairpin loop form, however, in order for '~ ' - ' '~ '.:
, WO 91/1232~ PCI~US91/00968 2076037 -~
the se~ments produced by the activity of RNase H to be brought together becauce the two RNA segments are tether-ed by their base-pairing with the ~xed phosphate backbone oligodeo~ynucleotide. In the presence of an 05 appropriate ligase(s), ~uch as an endogenous ligase(s), the two newly-created cegments are ~oined, by ligation of the nuclcotide (~nterlined ~n Fi~ure 1~ on ~ach ~ide of the gap.
The definition of ~activating RNsse H" ls based on the induced-fit theory of Koshland, in which "enzyme sites Yere envisaged as somewhat flexible and undefined before binding occurred, locking actlve site residues into defined positions around the substrate" (Koshlant, D.E., Jr., Proc. Nael _Acad. Sci. USA, 44:98 (1958);
Zeffren, E. and P.L. Hall, The Study of_Enzyme Mechsn isms, p. 201, John Wiley and Sons, New York, (1973)).
Method_for excisi R_a_d_re~lacin~_a_s~ecific_small se~ment of ribon~cleotides from_an RNA molecule A second e~bodiment of the present invention is represented schenatically ~n Figure 2. This embodiment is useful, as described below, in repairing a genetic defect by re~o~ing the defective nucleotides and replac-in~ them with others, such as those present in the normal RNA or those wh~ch result in RNA cncoding a desired polypept~de. As shown, sn RNA molecule to be altered, such as pre-mRNA or ~RNA in which a tefect is present, is combined with an appropriate mixed phosphate backbone oligodeoxyr~bonucleotide ln which the internal segment is capable of activ~ting RNase H and the flankin~ nucleotide sequences are unable to acti~ate RNase H, in the presence of RNase H. As described above, the target se~ment is e~cised in a site-d~rected msnner, producing a spl$t RNA
molecule, as represented in Figure 2 as sequence lIIa.
In this embodinene, a replacement oligomer, ~hich ls the S scries of nucleotides to be inserted into the gAp created by excision cf the target ~egment, is ~ntroduced into cells. $hrough hybridizstion e~ch-nge (as described ln detail belo~), the r-pl-cement oligoner ~s introduced into the g~p created as a result of the RNase H activity.
E~pression of the resulting altered RNA (which includes the desired/nondefective cequence in place of the defecti~e sequence) results in production of the desired/nondefective polypeptide.
Certa$n genetic tisorders (inborn errors of ~etabol-ism~ can be corrected by the method of the presentin~ention. For example, cystic fibrosis is usually due to a 8ene mutation in which a specific phenylalanine codon is deleted (Riordon, J.R. et al., Science, 245:
1066-1073 (1989)). Through use of the present me~hod, ~he mutant cystic fibrosLs mRNA present ln an indiv~du-al's cells can be cleaved at the missing codon by site-directed RNase H alteration, as described below. Intro-duction into the individual's cells of the appropriate oligoribonucleotide (i.e., one encodin~ a phenylalanine), followed by endo~enous RNA ligase acti~ity results in ligation and, thus, production of a wild-type mRNA
encoding a normsl protein product.
Other genetic tefects can be remedied in a similar manner. Examples of genetic defects for which this would be appropriate are: the substitution of serine for glyc~ne 844 in a severe ~ariant of osteogenesis , . ' ~ : -.
' ' .
Wo 91/12323 PCI/l,'S~1/00968 inperfecta (Pack, ~. et al., J. Biol. Chem., 264:
19694-19699 (1989)); a glycine to serine substitution in pro ~ (II) collagen in a for~ of dwarfism (Vissing, H. et al., J. Blol. Chem., 264:18265-18267 (1989)); ~ glyc~ne _ _ _ 05 833 conversion to aspartate in a mild varlant of Ehlers-Danlos syndro~e IV (Tromp, G. et al., J. Biol.
Che~., 264:19313-19317 (1989)); an ~ to G translt~on in an lnit~tion codon ~utction in the Apo C~ ene Df ~
patient with a deficièncy of apolipoprotein C-II (Fo~o.
S.S. et Al., J Biol Chem., 264:10839-10842 (1989)); and an aberrant GT splice-donor signal flanking exon 19 in retinoblastoma RB-88 (Ysndell, D.W. et al., N.E. J. Med., 321:1689-1695 (1989)). Such precise genet~c tefects can be repaired at the RNA level by the method described.
Correction of the s~ecific defect sssoc~ste_ with cystic fibrosis Riordan et al. ha~e found that in approximately 70%
of the cases of cystic fibrosis, a trinucleotide (TTT) is missing fro~ a seg~ent of the gene of chromosome 7, as compared with its normal, wild-type counterpart, Thus, the ~NA transcription prod~ct of TTT, na~ely VUU, is also missing.
An approach to correcting the defect associated with cystic ibrosis is ~s follows: a mixed phosphate backbone oligodeoxyribonucleotide is synthesized, which consists of an ~nternal segment composed of either ~nmodified bridging phosphodiester bonds, or of modified ones, such as phosphorothioates, (or certain other modific~tlons) Yhich ~re c~pable of activating RNase H.
, .. .
- . . .
.
; ~
2076~37 As an example, Figure 2(I) illustrates 8 section of pre-mRNA from cystic fibrosis in which the UUU trinucleo-tide is ~iSsiDg. The ni~ed phosphate backbone oligamer A-TAG-T~A-CCA-CA~-~ ls cynthesized. It incl~des an 05 internAl se~me~t, bearln~ the subscript ~s~, ~hich ~ncludes the ~nternucleotido phosphates (unmodified, phosphorothioate or other) ~hlch re capable of ~cti~at-lng RNase H. The flanking se~ents have the subscrlpt designation ~m~, ~hich s~gnifies that the internucleoside bridging phosphate oodific~tions render this ~e8ment of DNA incapable of activating RNase H. These ~m~ ~odifica-tions may be ~ethyl phosphonates, phosphoromorpholidates, phosphoropiperazidates, phosphoro-n-butylamidates, or other modifications of the lnternucleoside phosphate which result in inability to ~ctlvate RNase H. In Figure 2(1I) is shown a synthetic oligodeoxynucleotide consist-ing of six internal deoxynucleotides (subscript s) capable of activatin~ RNase H in the portion of RNA to which they are hybridized. These residues are flanked on both sides by oligod~oxynucleotides whose lnternucleoside bridging phosphate modificitions are designated by subscript ~m~, to indicate that these segments of the synthetic oligomer are incapable of activating RNase H.
RNase H acti~ation requires a DNA-RNA hybrid in which at least 2 consecutive nucleotides of the DNA strand have RNase H sensiti~e internucleoside linkages, as demonstrated herein (See Exemplification) and by others.
Walder R.Y. and J.A. Walder, Proc__ atl-~_ca_. Sci. USA~
85:5011-5015 (1988); Furdon, P.~. et al., Nucleic Acids Res., 17:9193 9204 (1989)~. Alternati~ely the sequence designated II can be a central RNase H sensitive section, ~ 091/12323 PCT/~S91/00968 ss described abo~e, flanked by two segments of ribonucleotides (subscript r), one on either side of the internal seg~ent. In this case, the oligomer of Figure 2(II) would be represented ~s follows:
05 ~-VAG-TAA-CCA-CCA-A. Hybridlzed ribonucleotide oligomers do not activate RNsse H; therefore, the sbove oligoocr ~ould also consist of ~Na~e H ~ensitive and reslstant ~egments.
As is described ~above ~lth reference to Figure l, the target segment i5 e~cised ln a site-spec~fic ~anner, resulting in an interrupted RNA ~olecule; in this case, AUC-GGU have been removed,;producin~ a 6-nucleotide gap.
For exa~ple, in an indivitual, the synthetic oligomer (II), dissolved ~n physiological saline solution (e.g., at a concentration of l x lO - l x lO M) is injected (e.g, intravenously) into an individual with cyst~c fibrosis. The oligomer ~ould become distributed lnto various organs and tissues of the individusl. HybrLdiza- ;
tion of the synthetic oligomer with pre-mRNA or mRNA at 20 the targetet site ~ould occur, activation of RNase H
would follow, and cleavage and excision of the six nucleotide segment of RNA w~uld result.
Subsequently, another construct, such as that designated IVa in Figure 2, is administered to the individual in such a manner that it enters cells (e.g., by Intravenous injection). This construct is a hybrid, partially double-stranded synthetic oligomer which consists of a r~bonucleo~ide unmodified nonamer ~ith a 5'-phosphate, hybridized to a 27-~er deoxyribonucleotide, in which all of the residues are modified so as to be nuclease resistant.
......
'' ' , 1 :' -' :' ' ' ' ': '' ' ", 2~76o37 As diagrammed in Figure 2, a hybridization exch~nge occurs, in ~hich the longer, double-stranded oligomer construction IV would replace the shortsr oligomer IIIb (A-IAG-T~A-CCA-CAA-A) in hybridizing ~ith the natursl RNA
05 (IIIa) at.the cr~tical, excised defect~Ye slte shown in Fi~ure 2(IIIa). The latter bears a 5iX nucleotide gap;
the tYo ~egments are held ~n place by the ol~gomer A-~AG-TAA-CCA-CAA-A. Due to the greHter hybridization length of the hybrld shown ln F~gure 2(IVb), a hybritiza-tion exchange occurs and the latter oligomer constructreplaces the A-TAG-TAA-CCA-CAA~A. The nonamer (IVa) fills in the gap left by the nucleotide excision. The complementary pattern of the oligomer construct with the cystic fibrosis R~A ~ill separate its two seg~ent.s sufficiently to permit a nine nucleotide segment to be inserted ~here a six segment nucleotide was removed. The 5' phosphate on this unmod~fied RNA nonamer will permit ATP and RNA ligase, both present endogenously, perhaps assisted by other intracellular factors, to make the two co~alent phosphodiester bonds necessary to repair the excision defect. The f~nal result is to insert a trinucleotide UUU, at the tesired location into the RNA
transcript of the cystic fibrosis patient's genome.
~ybridization exchange occurs because of the greater hybridization association constant and higher Tm of the 18 complementary nucleotides in the oligo~e-r construct as comparet ~ith that of the eight nucleotides in the A-TA~-TAA.-CCA-CAA-A. It is slso possible that a natural RNA repair process may fill in the gap, if the unmodified rebonucleotide nonamer is omitted, and just the oligomer : . .. : .~ . .
W091/l23~3 PCT/US91/00968 2076037 _ TTA-TAG-TAG-AAA-CCA-CAA-ACG-A~A ls used for the transhybridization and repair steps.
node of administration of ~odified backbone olI~onucleo-tide 05 The ~anner in ~hich ~odified backbone ollgonucleo-tides ~re provided will depend on the context ~n ~hlch they are used ~i.e., in vitro, in ~o).
The modified backb~one oligonucleotide is generally dissolved in water or'a suitable buffèred medium, such as Dulbecco's medium, Eagle's medium, or a similar physio-logical saline ~edium, typically at a concentr~tion of 10 5 to 10 8 mol~r. In the case of a tissue cultuxe system, the dissolved oligomer is ster~lized by filtra-tion through a bacterial filter, nnd is added to the other components of the tissue culture incubation medium.
In the case of a seed, the oligomer is d~ssolved in ~ater and added to the seeds spread on sterile filter paper iDside a sterile covered glass or plastlc dish. In the case of a plant, dissolved oligomèr, in aqueous media, is added to the soil or other nutrient material in which the plant is gro~ing. In the case of an animal or man, the oligomer, dissolved ln physiological saline, may be iniected subcutaneously, intraper~toneally, intra-muscularly, intravenously, or possible by capsule orally.
It has been shown that oligomers such as the above-described enter living cells, and are found in signifi-cant concentrations both in the cytoplasm and in the nucleus ~ithin mi~utes after administration (Zamecnik,' P.C. et al., Proc._Natl. Acad._Sci._USA, 83:4143-4146 (1986); Coodchild, J- et_al., C rre_t_Co m__ica ions in , :
.;
- ::
WO 91/123'3 PCI/l!S91/00968 .
Molecular Biolo~v Aneisense RNA and DNA, Cold Spring Harbor, pp. 135 139 (1988~; Wickstrom, E.L. et al., Proc.
Natl._Acad. Sci. USA, 85:1028-1032 (1988)).
The follo~ing exempllfication demonstrates that S site-spec~f$c excision of nucleotides ~t the RNA le~el ha~e been carried out, us~n~ the present ~ethod ~nd ~odified bac~bone oligonuclsotides ~s described herein.
E~EMPLIFICASION
Materials and Methods _____________________ Oli~odeoxynucleotide Synthesis ___ _____ ____________ _______ Oligodeoxynucleotides were synthesized on an auto-matet instrument (model 8700, Milligen, MA~. Nor~al phosphodiester (PO) oligodeoxynucleotides and the analogous phosphorothioate (PS) or phosphoramidate oligodeoxynucleotides were synthesized using H-phos-phonate chemistry (Agrawal, S. et_al., Proc Nael. Acad.
Sci _~SQ, 86:7790-7794 (1989); and Agrawal, S. et al., Proc Natl. Acad. Sci. USA, 85:7079-7083 (1988)).
01igodeoxynucleos~de methylphosphonate (PC) ~nalogues uere a~sembled by using nucleoside methylphosphonamidites (Agrawal. S. snd J. Goodchild, Tetra_edron Lett., 28:
3539-3542 (1987~). Oligodeoxynucleotides containing both PO and PC internucleoside linkages were assembled by using nucleoside-~-cyanoethylphosphoramidites and nucleo-side methylphosphonamidites, and oligodeoxynucleot-ides containing both PC and PS linkages were synthesized from nucleoside methylphosphonamidites (Agrawal, S. and J.
Goodchild, Tetrahe_ro__Lett., 28:3539-3542 (19B7)~ and , WO 91/12323 PC~/l,'S9t/00968 nucleoside H-phosphonates. Oligodeoxynucleotides con-taining both P0 and phosphor~midate linka~es were syn-thes~zed by us~ng nucleoside-B-cyanoethylphosphora~idites and nucleoside H-phosphonates.
05 RNase H Assays __ _ _ The 13-~er oli~odeoxynucl~otides complement ry to nucleotides 2-14 of hunan Ul s~all nuclear RNA ~count~ng the G cap as nucleotide (0)) ~ere stded at 100 ~g/~l to a HeLa cell nuciear extract (Dignam, J.D. et_al., Nucleic Acids_Res., 11:1475 1479 (1983)~ coneaining 0.5 mM ATP, 20 mM crestine phosphate, 3.2 ~M MgC12, ant 1000 units of RNase per ml and ~ere incubated under the cond~tions specified. RNA was isolated from the nucle~r extract by phenol~chloroform extraction ~nd ethanol precipitation, followed by electrophoresis In a 10~ polyacrylamid~ ~et con~aining 8.3 M urea. The RNAs were visualized by ethidiu~ bromide staining.
A second RNase H assay was also carried out in nuclear extracts by using an exogenous P-labeled RNA.
A 5}4-nucleotide test RNA (here~fter ter~ed ~514 RNA~ for convenience) was generated by SP6 RNA polymerase trans-cription of a HindIII-linearized pGEM-2 clone, pT7H~6.
514 RNA is antisense to the first two exons and intron of human ~-globin pre-mRNA ~nd was chosen for the reasons described below. 514 RNA labeled with a ¦~- 2P~GTP, l~-32PlCTP, snd l~-32P]UTP was added to the nuclear extract containing the specified oligodeoxynucleotide at an oligo~er-to-514 RNA ~olar ratio of 3000:1, unless other~ise noted. After incubation as specified, RNA was extracted and the P-labeled 514 RNA or its clea~age :' , ~ : '~
U'091~1232~ PCT/~;S91/~0968 2076~37 -l9-products were visualized by electrophore~is and auto-radiography.
~esules Act~on of RNase H on Oli~odeoxynucleotide Ul Small 05 Nuclear RNA Hybrids HeLa cell ~uclear e~tracts cont~in RNase H sctivity that can ~ct on DNA-RNA hybrids that form after addit-on of oligodeoxynucleotides complementary to rertain endo-genous nuclear RNAs (Krainer, A.R. and T. ~aniatis, Cell, 42:725-736 (1985)). The ability of this RNase H activity __ to cleave the'5'-terminal nucleotides of endo~enous Ul small nuclear RNA, after incubation of the various ~odi-fied oligomers ~n the nuclear extract, uas investigated.
Incubation of the P0 oligomer $n the nuclear extract led to cleavage of a large proportion of the Ul RNA to a product (Ul~) hav~ng the mobility expected for removal of the first 15 nucleotides (cap, nucleotide l, and nucleotides 2-14). There was a lack of effect on thP
mobility of any other RNAs present, demonstrating the high ~equence specificity of the Ul oligomer-directed RNase H cleavage. Ul cleavage ~as also observed with the PS oligomer, although to a consistently lesser extent than with the P0 oligomer. In contrast, no cleavage was obser~ed ~ith the PG, phosphoro~-morpholidate (PM), or phosphoro-N-butylam~dAte (PB) oligomers. Reducing the temperature of ~ncubation to 20-C and extending the time to 60 minutes did ~ot increase the extent of cleavage observed with the PS oligomer, nor did it reveal cleavages with the PC, PM or PB oligomers. The pattern .
:-- . :
WOgl/12323 PCT/US91/00968 of UI cleavages seen with the vario~s oligomers w~s also not aleered by adding E._coli RNase H (Yharmacia, final concentrstion 8 units/ml~ to the nuclear ~xtract at the outset of incubation.
S RNase H Action at an Internal RNA Site Hybridized with __ ___ ____ _________ __________ _____ _______ or~al and Modified Oligodeo~ynucleotides She foregoing Ul RNA cloavsges lnvolve the 5' estFe~ity of a s~all RNA. Indeed, the 5' end of Ul RNA
is sn exceptionally fsvored target for ollgodeoxy-Ducleotide-directed RN~se H cleavage aince al~ost all the other regions of this RNA molecule are tightly complexed vith proteins in the Ul small nuclear ribonucleoprotein particle (Patton, J.R. et al., Mol. Cell. Biol., 7:
4030-6037 (1987); Patton, J.R. et al., Proc. Natl. Acad.
lS Sci. USA, 85:747-751 (1988); Patton, J.R. et al., Mol.
Cell. Biol., 9:3360-3368 (1989)). Therefore, the action of RNase H on hybrids formed by normal or modified oligomers at internal sites in a longer RNA was also in-~estigated.
For this purpose a test RNA which is 514 nucleotides long (termed 514 RNA) ~as uged. 514 RNA is antisense to the first t~o exons and intron of human ~-glo~in pre-mRNA. The underlying reasoning was that, because it is antisense to a pre-mRNA, 514 RNA ~ould not undergo splicing when ~dded to nuciear extract (the cleava~e-ligation steps of whlch ~ould otherwise complicate analysis of oligodeoxynucleotide-directed RNase H clea~-ages).
A series of normal and modified lS-~er oligodeoxy-nucleotides complementary to nucleotides 349-363 of 514 ': . . , ' `'i:
. .
WO91~12323 PCT/US91/00968 RNA vas synthesized chemically (Table 1, oligomers A-E).
This particular 514 RNA site ~as selected because oligomer-directed RNase H cleavage would generate two frdgments of readlly distinguishable lengths (lS0 and 348 05 nucleotides) ~nd also because this 15-nucleotide ~equence does ~ot occur else~here in 514 RNA (Lawn, R.M. et al., Cell, 21:647-651 (1980)). ~elting curves ln 0.16 M Na f~r four of these oli~odeo~yDucleotides ~fter duplex formstion with the co~plementnry (P0) oligomer reve~led that the P0, PC, PS and P~ oligomer-containing duplexes had tms of 53-C, 46-C, 43-C and 38-C, respectively, indicating a lower duplex ~tability for the modified oligomers, in part confir~lng previous reports (Stein, C.A. et al., Nucleic_Acids Res., 16:3209 3221 (1988);
lS Froehler, B. et_al., Nucleic_Ac~ds Res., 16:4831 4839 (1988); Quartin, R.S. ~nd J.G. Wetmur, Biochemistry, 2B:1040-1047 (1989); Agrawal, S. et_al., Nucleosides Nucleot~_es, 8:819-823 (1989)), . , . :
:: . .
. :- - . . :
, .
, ~'091/12323 PCT/US9l/0096~
2~7 ~ ~ I 22-___ ___________ _____ ___ ___ _ ___ __ _____ _ __ _ .
Ineernucleos~de Oli~omer Sequence Linka~e ___ __ _ _ _ __ ____ _ _ _ _ __ __ _ _ _ AGTA TCA ~GG TTA CAA PO
05 ~ GIA TCA AGG TTA CAA PS
CGTA TCA AGG TTA CAA PM
DGTA TCA ~GG TTA CAA PB
EGTA TCA AGG TTA CAA PC
F*GTA TCA TAT GAG ACA PO
G* GTA GCA AGG CTA CAA PO
H*GTA TGA GAC ATA TAC PO
__ _ _____ ___________________ ________________ Underlined nucle~tides indicate base pairing mismatches; PB is pbospho-N-butylamidate.
The beries of normal snd modified ol$godeoxy-nucleotides shown in Table 1 were incubated with P-labeled 514 RNA in nuclear extracts, under the conditions specified tn the ~aterisls and ~ethods section, for either 30 minutes or 3.5 hours.
The results of incubating these oligomers with 514 RNA in HeLa nuclear extract ~re as follows: After 30 minutes of incubation without any oligomer, intact input 514 RNA was the only labeled ~pecies v~sualized. After 3.5 hours of incubation without any oligomer, the 514 RNA
~as completely degraded by the action of ribonuclease kno~n to be present ~n the extract. Incubation of 514 RNA in the extract for 30 minutes together ~ith either the PO or PS oligomers resulted in precise cleavage of the substrate RNA into two fragments of the sizes . .
, ... . ~ ,.
., , . ~ , . .
:
W~91/123'~ PCT/~IS91/0096~
2~76037 23- , expected fro~ ehe location of the oligomer-complementary sequence. Surprisingly, in the case of the PS oligomer, these two fragments vere still present, albeit ln sli~htly degrnded form, after 3.5 hours of incubatlon.
05 Incubation of 514 RNA with PM ol~omer for 30 minutes resulted in partial cleavage (see the figure legend for additional details). No cleavage was observed with the PB or PC oligomers under these conditions.
PO oligomers that vere only pareially complementary eo 514 RNA (oligomers F, G and H ~n Table 1) were also tested. None of these oligomers, containing 4, 5 or 6 uninterrupted complementary nucleotides out of the 15 (Table 1), elicited RNase H cleavage of 514 RNA.
The finding that the PS oligomer was less effective than the P0 oligomer in eliciting RNase H cleavage in the Ul RNA assay raised the possibility that the more complete RNase H cleavages observed with both P0 and PS
oligomers in the S14 RNA assay might reflect the particular reaction conditions employed. Therefore, a ran~e of oligomer-to-514 RNA molar ratios tests (0.1:1-1000:1), all below that used in the above-described (3000:1), was investigated. Results showed that virtually complete 514 RNA cleavage occurred with the PO oligomer at an oligomer-to-~NA ratio of lO0:1, whereas a comparabl~ extent of 514 cleavage with the PS oli~omer occurred at a oligomer-to-RNA ratio of 1000:1. A very similar, ~ncomplete extent of cleavage was obser~ed with the P0 and PS oligomers at ratios of 10:1 and 100:1, respectively. The possibility that these results might reflect a preferential instability of the PS oligomer turing incubation in ~he nuclear extracts was ', .
WO91~1232~ PCT/US91/00968 2076~'~7 examined by experiments in which either the P0 or the PS
oligomer ~as preincubated in nuclear extract or 30 minutes, followed by addition of P-labeled 514 RNA and incubation for an atditional 30 min. This revealed the 05 same e~tent of difference in RNase H cleavage as described above.
~Restriction Endonuclease.Like~ Cleava~e with Oli~omers Containin~ RNase H Sensitive and -Resistant Internucleo-s~de_Linka~es-The extreme differences be~ween the R~ase H sensi-tivity of DNA-RNA hybrids containing PO or PS oligodeoxy-nucleotides, contrasted with ones with PC, PM or PB
oligomers, led to an investlgat~on of how RNase H acts on a DNA-RNA hybrid in which only a small proportion of lS internucleoside linkages in the DNA strand were RNase H-sensitive. Table 2 shows the series of oligomers that were synthesized to address this issue,.
WO91t12323 PCT/US91/00968 2076~37 _________ ____ _ _____ ___ ______ Oli~omer Sequence __ ___ _ _ _ ____________ AGG T
J A AGG TT
L GTA ICA ~GG TTA CAA
~ GTA TCA AGG TTA CAA
N GTA TCA AGG TTA CAA
O GTA TCA AGG TTA CAA
p GTA TCA AGG TTA CAA
___ _ __ Q GTA TCA AGG TTA CAA
R GTA TCA AGG TTA CAA
__ _____________ _ _ _ _ ____ _ _ Vnderlined nucleotides are PC; dashed nucleotides are PS; Boxed nucleotides are phosphoromorpholidates;
lS Double-underlined nucleotites are PM (oligomer Q) or PB;
the remaining nucleotides are PO.
The oligomers listed yere tested for their capacity to elicit RNase H action after hybridization to 514 RNA, as in the preced1ng experiments.
20 Results demonstrated that neither a tetramer nor a hexa~er (all PO) oligodeoxynucleoeide co~plementary to ~14 R~A was able to induce RNase H cleavage in this nuclear extract system. ~hen PO/PC-containing penta-decamers containing either two or four coDsecutive PO
linkages were used, a ~ow but readily detectable level of RNase H cleavage occurred. In contrast, a pentadecamer containing six consecutive PO linkages elicited complete RNase H cleavage of the substrate RNA. Note thst the six WO91/123~3 PCT/~'S91/00968 207~37 PO nucleotides in this oligomer (oligomer M in Table 2) are identical in sequence to the RNase H-inactive hexamer (oligomer J), from which it is inferred that the potency of the pentadec~mer reflects its increased hybrid ~5 stability with 514 RNA owing to the additional nine complenentary nucleotides.
Similar tests ~ere performed ~ith mixed PO~PC
pent-dec-mers co~pl~mentsry to a different site ~n 514 RNA (i,e,, nucleotides 463-477). These tests revealed an effect of the number of PO linksges on RNase H cleavage similar to that described above.
Additional variants of mixed PO/PC oligomers were also tested. Pentadecamer PO/PC oligomers with five or six consecutive PO linkages at either the extreme 5' or 3' end were highly effective in eliciting RNase H cleav-age, A PS/PC pentadecamer with six consecutive PS
linkages at the extreme 5' end ~oligomer P in Table 2) was only partially active. Comparison of these results with lanes 2 and 3 in Figure 4B confirms the above-described sesults showing that all-PS oligomers are less effective than all-P0 oligomers in eliciting RNase H
cleavage. RNase H cleavage~of 514 RNA was also observed with a 15-mer containing nine consecutive PM or PB
linkages followed by six PO-linked nucleotides (oli~omers Q and R in Table 2).
. . .
' ' ' : . '"' ~ ~ . ,. `
.: '' ' ' . , , ~ ' :
... . .. . .
PRODUCTION OF ENCODED POLYPEPTIDES
Description Back~round ____ _____ 05 Several aspects of contemporary molecular genetics and biotechnology make it desirable to be able to produce genetically-altered proteins. For example, mu~ated protein domains are sometimes hyper-immunogenic, facilitating the production of neutralizing antibody-based vaccines. Moreover, site-directed mutations, ideally one amino ~cid at a time, csn be a powerful approach to deciphering protein structure and/or enzyme-substrate reaction mechanisms.
Typically, deletions or substitutions of amino acids are made at the gene or DNA level, by recombinant DNA
techniques ~hich rely on the use of restriction endonucleases. However, restriction endonucleases available have a limited array of target sites in DNA , (usually palindromic hexanucleotide or octanucleotide z~ sequences). Deletion of a particular in-frame trinucleo-tide or trinucleotides ~ay not be possible because there ~ay be no suitably located restriction sites. As a result, presently-available methods of altering an amino . . . .: .
. . ~ .
.
WO9~/1232~ PCT/US91/00968 acid sequence by altering the DNA sequence which encodes it, are limited in their applicsbility.
Summary of the Inventlon ______ _ _ _______._ __ The present invention relates to a method of site-05 directed alteration (removal or re~o~al followed by replacement) of selected nucleotides in an RNA ~o~ecule, as well ns to ~i~ed phosphate backbone oligonucleotides useful in the method. It further relates to ~ method of producing polypept~des or proteins encoded by the RNA
molecule altered by the present-method. Through use of the present method, site-directed cleavage of an RNA
molecule is effected, followed by e~cision of the selected or target segment of the RNA molecule.
Follo~ing cleavage and excision, in onç embodiment, the two segmen~s of the resulting interrupted RNA.molecule ~re ~oined, through the sction of an appropriate ligase.
This results in production of a continuous RNA molecule, referred to as an altered RNA molecule, which is the same as the original RNA molecule except that it lacks the nucleotides originally pres~ent in the target segment of the RNA molecule. In a second embodiment, selected nucleotides can be introduced into the space or gap created by removal of the target RNA segment; a continuous RNA molecule is created by ligating the selected nucleotides introduced in this manner to the nYcleotide on each side of the gap.
ln the present ~etkod, an RNA molecule ~hose nucleo-tide sequence is to be altered in a site-directed manner -: . .
' .
, 3 PCT/~S91/00968 2076~37 is brou~ht into contact with an oligonucleotide, referred to as a mixed phosphate backbone oligonucleotide, in the presence of RN~se H. The mixed phosphste backbone oligonucleotide is complementary to all or a portion of 05 an RNA ~olecule ~hich includes a target segment to be altered. ln addition, the mixed phosphate backbone oligonucleotide includea ~n internal portion or ~egment of deoxynucleotides which is capsble of sctivatin~ RNase H ~nd is flanked on each side by a cequence of nucleo-tides ~hich is unable to sctivate RN~se H. The internalsequence includes two or ~ore consecutive phosphodiester linka~es, ~hich may be unmodified or modified. The flanking sequences are modified deo~yribonucleotide or ribonucleotide sequences. It has been shown that when lS such a mixed phosphate backbone oligonucleotide is contacted with a target segment of an RNA molecule, according to the method of th,e present invention~ the result is RNase H metiated excision of RNA target nucleotides complementary only to the internal sequence of oligonucleotides. This mekes it possible to excise precisely any tesired nucleotide or oligonucleotide from an RNA molecule. Followed by ~NA ligation, this results in a desired altered messenger RNA or other type of RNA. ;~
Thus, for the first time, it is possible to carry out -2S precise excision of a selected segment of sn RNA
molecule.
As a result, it is possible to selectively telete any desired number of nucleotides and, if desired, to ~ntroduce replacement nucleotides. The encoded amino acid sequence or polypeptide can be produced by express-ing the ~ltered RNA in vitro or in vivo. As a result, a ... .. .
.
WO91/12323 PCT/USgl/00968 selected a~ino acid sequence or selected polypeptide csn be produced by the present method by: l) producing an altered RNA ~olecule encoding a elected ~mino acld sequence or selected polypeptide snd 2) expressing the O5 ~ltered RNA ~olecule under appropr1ate conditlons.
Cell-free translation of the altercd RNA molecule can be cnrried out to produce deslred mutant proteins use~ul, for e~ample, for ~tudies of protein structure or func-tion. Aleernati~ely, an appropriate ~ixed phosphate backbone oli~onucleotide can be -taken up by or introduced into cultured cells or into c"eils of an animal or a plsnt, in ~hich endogenoùs RNase H and RNA ligase activities can produce altered RNAs; upon translation, corresponding genetically altered protein~ are produced.
lS This is useful, for example, as a means of producing defective viral or infectious/pathogenlc agent seplication or gene expression, which can be useful therapeutically or prophylactically.
Brief Descri~tion of the Drawings Figure l is a schematic representation of the present method for excision of a tar~eted segment of an RNA molecule in ~hich sequence I is RNA from which a targeted segment is to be excised and sequence II is a mixed phosphate backbone oligonucleotide.
Subscript "m designates an RNase H-resistant internucleoside phosphate; subscript "s" indicates an RNase H-suscept~ble internucleoside phosphate; "X"
designates any of the four ribonucleotides A, C, G or U;
and ~Y~ des~gnates a deoxyribonucleotide complementary to 30 the ribonucleotide directly abo~e it. ' Figure 2 is a schematic representatlon of repair of a genetic defect by the method of the present invention, in ~hlch IVa and IVb represent a double-stranded hybrid consisting of a smaller unmodified ribonucleotide (IVa) 05 hybr~dized to B lsrger nodifled, RNase H-resistant deo~yribonucleot~de (IVb) and V is the repa~red RNA
hybridized to the RNase N-r6sistant deo~yr~bonucleot~de.
Detailed Descri~tion of the Invention The present invent~on relstes to a method of site-specific or site-directed alteration (removal or removal follo~ed by replacement) of selected nucleotides (i.e., a target segment) in an RNA molecule, to produce an altered RNA sequence, as well as to mixed phosphate back~one oligonucleotides useful in the method. It further xelates to a method of produc~ng altered amino acid sequences or polypept~des by translating the altered RNA
seq~ence, which results in production of the encoded ~olecule.
In the present method, a selected or target segment of an RNA molecule, such as pre-mRNA, mRNA or viral RNA, is altered as follows: an RNA molecule which includes the target segment (l.e., a nucleotide or a nucleotide sequence to be altered) is combined ~ith an appropri-ately-selected mixed phosphate backbone oligonucleotide in the presence of RNase H. The mixed phosphate backbone oligonucleotide is ;complementary to all or a portion of the RNA ~olecule which includes the target BNA segment;
it is of sufficient length to hybridize to the target RNA
segment and sequences on either side and remain - . ~' ' U'091/12323 PCT/US91/~0968 2076~37 hybridized under the conditions used. The mixed phosphate backbone oli~onucleotide has ~o key components: an internsl portion or segment o f deoxynucleotides which is capable of acei~ating RN~se H
05 and t~o nucleotide cequences, ~hich flank the i~ternal seg~ent, ~h~ch are unable to act~vate RNase H. The internal deoxynucleotide segment includes two or ~ore phosphodieçter linkages, ~hlch ~ay be un~odLfied or modifled. The fianking nucleot~de sequence3 m~y be deo~yribonucleotide or ribonucleotide sequences and is modified. That is, some or all of the internucleoside bridging phosphate residues are modified phosphates, such as methyl phosphonates, phosphoromorpholidates, phosphoropiperzaidates and phosphoramidates. An essential feature of the mi~ed phosphate backbone olig'onucleotide is that the internal complementary segment is RNase H acti~ating and the flanking complementary sequences are unable to activate RNase H.
As demonstrated herein, site-directed alteration occurs ~hen the RNA molecule which includes the target segment and an appropriately selected mixed phosphate backbone oligonucleotide are combined in the presence of RNase H and maintained under appropriate conditions ~e.~., temperaturè, time, salt concentration) for 2~ complementary nucleotide sequences to hybridize and RNase H to be activated (i.e., to be able to clea~e aDd excise). That is, as a result, the nucleotides in the RNA molecule to ~hich tbe internal segment of the mixed phosphate backbone oligonucleotide i5 complementary are excised precisely from the RNA molecule.
.,. , ", . : :. .
, WO9l/1'~1~ Pcr/ US91/00968 2~760~7 , In one embodiment of the present ~ethod, the gap resulting from e~cision of the target RNA ~e~ent can be closed by the sct~it; of an appropriate ligase(s), resulting in a continuous RNA ~olecule refarred to as an ;05 altered RNA oolecule. The resultlng alter~d RNA ~olec~le differs from the RNA molecule only as to the target regment, Yhich ~s not pr~sent in the altered RNA ~ole-cule.
In ~ ~econd embodiment the gap created by the action 10 of RNase H as described above can be f~lled in by inero- -ducing a segment of replacement nucleotides, which can be of any length appropriate to fit into the gap created in the RNA molecule. The se~ent of replacement nucleotides is subsequently linked to the stjacent nucleotides of the RNA molecule by an sppropriPtely selected ligsse(s). The nucleotide present at each end of the segment of replace-ment nucleotides is ligated to the nucleotide present on the respecti~e end~ of the gap created in the RNA
molecule (underlined in Fi~ure 2(111~). The resulting altered RNA molecule tiffers from the RNA ~olecule in that the target segment (present in the RNA ~olecule) has been removed snd a replacement nucleotide cequence introduced in its place.
Altered RNA produced as des~ribed herein can be expressed, either in_vitro or i__~i~o, to produce the encoded polypeptide or protein; ~s used herein, the term polypeptide includes proteins. For example, the altered RNA can be introduced into an appropr~ate ~ector, which is in turn introduced into a host cell capable of trans-lating the alt~red RNA ~olecule and producing the encodedpolypeptide. ~olypeptides produced in this ~anner can be .... ~: .
' ~
WO9t/12323 PCr/US9l/00968 used for assessment of thelr structur~l/functional characteristics and a comparative assess~ent of polypep-tides ~hich d~ffer ln ~ defined ~anner ~e.~., by selected a~ino acids). Such polypeptides c~n also be used 05 therapeutically or prophylactically.
The following is M description, with reference to the fi~ures, of two enbodimenes of the present ~ethod of site-directed alteration of an ~NA molecule: a firfit embodiment in which a selected nucleotide qequence ~target segment) is re~oved and the resulting fragments ligated to produce sn altered RNA molecule lacking the target segment and 8 second embodiment in which a selected nucleotide sequence ~s removed and replaced by a selected nucleot~de or nucleotides, ~hich are ligsted to the nucleotide at each side of the gap created by removal of the target RNA segment.
~ethod for Excisin~ a SPecific Small Se~ment of Rlbo-_________ _ _______ ____ _______________ _____________ nucleotides from an RNA Molecule Figure l ls a schematic representation of sn embodi-ment of the present method.~ The sequence designated I is a segment of an RNA molecule, ~hich includes a t~rget segcent (~GACGUCA) to be altered. The RNA may be a pre-RNA, a viral ~NA, or one of a Yariety of RNAs present in animal, plant or bacterlal cells. The sequence designated II is a se~ment of DNA which is a mixed phosphate backbone oli~odeoxynucleotide complementary to the RNA molecule I. It can be synthesized us~ng known techniques, such as chemical or enzymatic methods. The mixed phosphate backbone oligodeoxynucleotide includes an inte~nal sequence which is capable of activating RNase H
.
.
. . - . .
. , ~ . .
WO 91/12323 PCI/US91/OOg68 g and is flanked on each side by a DNA ~egment which is incapable of activating RNase h, ~he total length of the ~i~ed phosphate backbone oligodeoxynucleotides varies, dopending on the length of the target RNA segment to be 05 gltered, but must be sufficient to hybrid$ze to the RNA
nolecule containing the target RNA segment Hnd remain hybrldiz~d under the cond~tions u~ed. The ~nternal ~egDent ~ust be of sufficient length--et least two nucleotides--to be capable of actl~ating RNase H, ~s de~onstrstet herein and by others (Ualder, ~.Y. snd J.A.
~alder, Proc. Natl. acad. Sci USA, 85:5011-5015 ~1988);
Furdon, P.J. et al., Nucleic Acids_Res., 17:9193 9204 (1989)).
In the internal segment, the internucleoside bridg-lS ing phosphate residues may be unmodified phosphates or any phosphate modific~tion capable of activa~ing RNase ~, such a~ phosphorothioates. The flanking n~cleotide sequences can be deo~yribonucleotides, as descr~bed in this embodiment or can be ribonucleotides and their modifications. The flanking sequences sre connected by methyl phosphonates (PC), phosphoromorpholldates (PM), phosphoropiperaz~dates, phosphoramidates, or other modifications of in~ernucleoside phosphates which are not able to activate RNase H. The RNA molecule I and the ~odified backbone oligodeoxynuclestide II are combinet in the presence of RNase H, such as endogenous RNase H in a cell, and excision of the target segment occurs. A
hairpin loop may form by pairing of complementary nucleo-tides, with the result that the two newly-formed segments are brought into proximity to each oeher. lt ~ay not be necessary that a hairpin loop form, however, in order for '~ ' - ' '~ '.:
, WO 91/1232~ PCI~US91/00968 2076037 -~
the se~ments produced by the activity of RNase H to be brought together becauce the two RNA segments are tether-ed by their base-pairing with the ~xed phosphate backbone oligodeo~ynucleotide. In the presence of an 05 appropriate ligase(s), ~uch as an endogenous ligase(s), the two newly-created cegments are ~oined, by ligation of the nuclcotide (~nterlined ~n Fi~ure 1~ on ~ach ~ide of the gap.
The definition of ~activating RNsse H" ls based on the induced-fit theory of Koshland, in which "enzyme sites Yere envisaged as somewhat flexible and undefined before binding occurred, locking actlve site residues into defined positions around the substrate" (Koshlant, D.E., Jr., Proc. Nael _Acad. Sci. USA, 44:98 (1958);
Zeffren, E. and P.L. Hall, The Study of_Enzyme Mechsn isms, p. 201, John Wiley and Sons, New York, (1973)).
Method_for excisi R_a_d_re~lacin~_a_s~ecific_small se~ment of ribon~cleotides from_an RNA molecule A second e~bodiment of the present invention is represented schenatically ~n Figure 2. This embodiment is useful, as described below, in repairing a genetic defect by re~o~ing the defective nucleotides and replac-in~ them with others, such as those present in the normal RNA or those wh~ch result in RNA cncoding a desired polypept~de. As shown, sn RNA molecule to be altered, such as pre-mRNA or ~RNA in which a tefect is present, is combined with an appropriate mixed phosphate backbone oligodeoxyr~bonucleotide ln which the internal segment is capable of activ~ting RNase H and the flankin~ nucleotide sequences are unable to acti~ate RNase H, in the presence of RNase H. As described above, the target se~ment is e~cised in a site-d~rected msnner, producing a spl$t RNA
molecule, as represented in Figure 2 as sequence lIIa.
In this embodinene, a replacement oligomer, ~hich ls the S scries of nucleotides to be inserted into the gAp created by excision cf the target ~egment, is ~ntroduced into cells. $hrough hybridizstion e~ch-nge (as described ln detail belo~), the r-pl-cement oligoner ~s introduced into the g~p created as a result of the RNase H activity.
E~pression of the resulting altered RNA (which includes the desired/nondefective cequence in place of the defecti~e sequence) results in production of the desired/nondefective polypeptide.
Certa$n genetic tisorders (inborn errors of ~etabol-ism~ can be corrected by the method of the presentin~ention. For example, cystic fibrosis is usually due to a 8ene mutation in which a specific phenylalanine codon is deleted (Riordon, J.R. et al., Science, 245:
1066-1073 (1989)). Through use of the present me~hod, ~he mutant cystic fibrosLs mRNA present ln an indiv~du-al's cells can be cleaved at the missing codon by site-directed RNase H alteration, as described below. Intro-duction into the individual's cells of the appropriate oligoribonucleotide (i.e., one encodin~ a phenylalanine), followed by endo~enous RNA ligase acti~ity results in ligation and, thus, production of a wild-type mRNA
encoding a normsl protein product.
Other genetic tefects can be remedied in a similar manner. Examples of genetic defects for which this would be appropriate are: the substitution of serine for glyc~ne 844 in a severe ~ariant of osteogenesis , . ' ~ : -.
' ' .
Wo 91/12323 PCI/l,'S~1/00968 inperfecta (Pack, ~. et al., J. Biol. Chem., 264:
19694-19699 (1989)); a glycine to serine substitution in pro ~ (II) collagen in a for~ of dwarfism (Vissing, H. et al., J. Blol. Chem., 264:18265-18267 (1989)); ~ glyc~ne _ _ _ 05 833 conversion to aspartate in a mild varlant of Ehlers-Danlos syndro~e IV (Tromp, G. et al., J. Biol.
Che~., 264:19313-19317 (1989)); an ~ to G translt~on in an lnit~tion codon ~utction in the Apo C~ ene Df ~
patient with a deficièncy of apolipoprotein C-II (Fo~o.
S.S. et Al., J Biol Chem., 264:10839-10842 (1989)); and an aberrant GT splice-donor signal flanking exon 19 in retinoblastoma RB-88 (Ysndell, D.W. et al., N.E. J. Med., 321:1689-1695 (1989)). Such precise genet~c tefects can be repaired at the RNA level by the method described.
Correction of the s~ecific defect sssoc~ste_ with cystic fibrosis Riordan et al. ha~e found that in approximately 70%
of the cases of cystic fibrosis, a trinucleotide (TTT) is missing fro~ a seg~ent of the gene of chromosome 7, as compared with its normal, wild-type counterpart, Thus, the ~NA transcription prod~ct of TTT, na~ely VUU, is also missing.
An approach to correcting the defect associated with cystic ibrosis is ~s follows: a mixed phosphate backbone oligodeoxyribonucleotide is synthesized, which consists of an ~nternal segment composed of either ~nmodified bridging phosphodiester bonds, or of modified ones, such as phosphorothioates, (or certain other modific~tlons) Yhich ~re c~pable of activating RNase H.
, .. .
- . . .
.
; ~
2076~37 As an example, Figure 2(I) illustrates 8 section of pre-mRNA from cystic fibrosis in which the UUU trinucleo-tide is ~iSsiDg. The ni~ed phosphate backbone oligamer A-TAG-T~A-CCA-CA~-~ ls cynthesized. It incl~des an 05 internAl se~me~t, bearln~ the subscript ~s~, ~hich ~ncludes the ~nternucleotido phosphates (unmodified, phosphorothioate or other) ~hlch re capable of ~cti~at-lng RNase H. The flanking se~ents have the subscrlpt designation ~m~, ~hich s~gnifies that the internucleoside bridging phosphate oodific~tions render this ~e8ment of DNA incapable of activating RNase H. These ~m~ ~odifica-tions may be ~ethyl phosphonates, phosphoromorpholidates, phosphoropiperazidates, phosphoro-n-butylamidates, or other modifications of the lnternucleoside phosphate which result in inability to ~ctlvate RNase H. In Figure 2(1I) is shown a synthetic oligodeoxynucleotide consist-ing of six internal deoxynucleotides (subscript s) capable of activatin~ RNase H in the portion of RNA to which they are hybridized. These residues are flanked on both sides by oligod~oxynucleotides whose lnternucleoside bridging phosphate modificitions are designated by subscript ~m~, to indicate that these segments of the synthetic oligomer are incapable of activating RNase H.
RNase H acti~ation requires a DNA-RNA hybrid in which at least 2 consecutive nucleotides of the DNA strand have RNase H sensiti~e internucleoside linkages, as demonstrated herein (See Exemplification) and by others.
Walder R.Y. and J.A. Walder, Proc__ atl-~_ca_. Sci. USA~
85:5011-5015 (1988); Furdon, P.~. et al., Nucleic Acids Res., 17:9193 9204 (1989)~. Alternati~ely the sequence designated II can be a central RNase H sensitive section, ~ 091/12323 PCT/~S91/00968 ss described abo~e, flanked by two segments of ribonucleotides (subscript r), one on either side of the internal seg~ent. In this case, the oligomer of Figure 2(II) would be represented ~s follows:
05 ~-VAG-TAA-CCA-CCA-A. Hybridlzed ribonucleotide oligomers do not activate RNsse H; therefore, the sbove oligoocr ~ould also consist of ~Na~e H ~ensitive and reslstant ~egments.
As is described ~above ~lth reference to Figure l, the target segment i5 e~cised ln a site-spec~fic ~anner, resulting in an interrupted RNA ~olecule; in this case, AUC-GGU have been removed,;producin~ a 6-nucleotide gap.
For exa~ple, in an indivitual, the synthetic oligomer (II), dissolved ~n physiological saline solution (e.g., at a concentration of l x lO - l x lO M) is injected (e.g, intravenously) into an individual with cyst~c fibrosis. The oligomer ~ould become distributed lnto various organs and tissues of the individusl. HybrLdiza- ;
tion of the synthetic oligomer with pre-mRNA or mRNA at 20 the targetet site ~ould occur, activation of RNase H
would follow, and cleavage and excision of the six nucleotide segment of RNA w~uld result.
Subsequently, another construct, such as that designated IVa in Figure 2, is administered to the individual in such a manner that it enters cells (e.g., by Intravenous injection). This construct is a hybrid, partially double-stranded synthetic oligomer which consists of a r~bonucleo~ide unmodified nonamer ~ith a 5'-phosphate, hybridized to a 27-~er deoxyribonucleotide, in which all of the residues are modified so as to be nuclease resistant.
......
'' ' , 1 :' -' :' ' ' ' ': '' ' ", 2~76o37 As diagrammed in Figure 2, a hybridization exch~nge occurs, in ~hich the longer, double-stranded oligomer construction IV would replace the shortsr oligomer IIIb (A-IAG-T~A-CCA-CAA-A) in hybridizing ~ith the natursl RNA
05 (IIIa) at.the cr~tical, excised defect~Ye slte shown in Fi~ure 2(IIIa). The latter bears a 5iX nucleotide gap;
the tYo ~egments are held ~n place by the ol~gomer A-~AG-TAA-CCA-CAA-A. Due to the greHter hybridization length of the hybrld shown ln F~gure 2(IVb), a hybritiza-tion exchange occurs and the latter oligomer constructreplaces the A-TAG-TAA-CCA-CAA~A. The nonamer (IVa) fills in the gap left by the nucleotide excision. The complementary pattern of the oligomer construct with the cystic fibrosis R~A ~ill separate its two seg~ent.s sufficiently to permit a nine nucleotide segment to be inserted ~here a six segment nucleotide was removed. The 5' phosphate on this unmod~fied RNA nonamer will permit ATP and RNA ligase, both present endogenously, perhaps assisted by other intracellular factors, to make the two co~alent phosphodiester bonds necessary to repair the excision defect. The f~nal result is to insert a trinucleotide UUU, at the tesired location into the RNA
transcript of the cystic fibrosis patient's genome.
~ybridization exchange occurs because of the greater hybridization association constant and higher Tm of the 18 complementary nucleotides in the oligo~e-r construct as comparet ~ith that of the eight nucleotides in the A-TA~-TAA.-CCA-CAA-A. It is slso possible that a natural RNA repair process may fill in the gap, if the unmodified rebonucleotide nonamer is omitted, and just the oligomer : . .. : .~ . .
W091/l23~3 PCT/US91/00968 2076037 _ TTA-TAG-TAG-AAA-CCA-CAA-ACG-A~A ls used for the transhybridization and repair steps.
node of administration of ~odified backbone olI~onucleo-tide 05 The ~anner in ~hich ~odified backbone ollgonucleo-tides ~re provided will depend on the context ~n ~hlch they are used ~i.e., in vitro, in ~o).
The modified backb~one oligonucleotide is generally dissolved in water or'a suitable buffèred medium, such as Dulbecco's medium, Eagle's medium, or a similar physio-logical saline ~edium, typically at a concentr~tion of 10 5 to 10 8 mol~r. In the case of a tissue cultuxe system, the dissolved oligomer is ster~lized by filtra-tion through a bacterial filter, nnd is added to the other components of the tissue culture incubation medium.
In the case of a seed, the oligomer is d~ssolved in ~ater and added to the seeds spread on sterile filter paper iDside a sterile covered glass or plastlc dish. In the case of a plant, dissolved oligomèr, in aqueous media, is added to the soil or other nutrient material in which the plant is gro~ing. In the case of an animal or man, the oligomer, dissolved ln physiological saline, may be iniected subcutaneously, intraper~toneally, intra-muscularly, intravenously, or possible by capsule orally.
It has been shown that oligomers such as the above-described enter living cells, and are found in signifi-cant concentrations both in the cytoplasm and in the nucleus ~ithin mi~utes after administration (Zamecnik,' P.C. et al., Proc._Natl. Acad._Sci._USA, 83:4143-4146 (1986); Coodchild, J- et_al., C rre_t_Co m__ica ions in , :
.;
- ::
WO 91/123'3 PCI/l!S91/00968 .
Molecular Biolo~v Aneisense RNA and DNA, Cold Spring Harbor, pp. 135 139 (1988~; Wickstrom, E.L. et al., Proc.
Natl._Acad. Sci. USA, 85:1028-1032 (1988)).
The follo~ing exempllfication demonstrates that S site-spec~f$c excision of nucleotides ~t the RNA le~el ha~e been carried out, us~n~ the present ~ethod ~nd ~odified bac~bone oligonuclsotides ~s described herein.
E~EMPLIFICASION
Materials and Methods _____________________ Oli~odeoxynucleotide Synthesis ___ _____ ____________ _______ Oligodeoxynucleotides were synthesized on an auto-matet instrument (model 8700, Milligen, MA~. Nor~al phosphodiester (PO) oligodeoxynucleotides and the analogous phosphorothioate (PS) or phosphoramidate oligodeoxynucleotides were synthesized using H-phos-phonate chemistry (Agrawal, S. et_al., Proc Nael. Acad.
Sci _~SQ, 86:7790-7794 (1989); and Agrawal, S. et al., Proc Natl. Acad. Sci. USA, 85:7079-7083 (1988)).
01igodeoxynucleos~de methylphosphonate (PC) ~nalogues uere a~sembled by using nucleoside methylphosphonamidites (Agrawal. S. snd J. Goodchild, Tetra_edron Lett., 28:
3539-3542 (1987~). Oligodeoxynucleotides containing both PO and PC internucleoside linkages were assembled by using nucleoside-~-cyanoethylphosphoramidites and nucleo-side methylphosphonamidites, and oligodeoxynucleot-ides containing both PC and PS linkages were synthesized from nucleoside methylphosphonamidites (Agrawal, S. and J.
Goodchild, Tetrahe_ro__Lett., 28:3539-3542 (19B7)~ and , WO 91/12323 PC~/l,'S9t/00968 nucleoside H-phosphonates. Oligodeoxynucleotides con-taining both P0 and phosphor~midate linka~es were syn-thes~zed by us~ng nucleoside-B-cyanoethylphosphora~idites and nucleoside H-phosphonates.
05 RNase H Assays __ _ _ The 13-~er oli~odeoxynucl~otides complement ry to nucleotides 2-14 of hunan Ul s~all nuclear RNA ~count~ng the G cap as nucleotide (0)) ~ere stded at 100 ~g/~l to a HeLa cell nuciear extract (Dignam, J.D. et_al., Nucleic Acids_Res., 11:1475 1479 (1983)~ coneaining 0.5 mM ATP, 20 mM crestine phosphate, 3.2 ~M MgC12, ant 1000 units of RNase per ml and ~ere incubated under the cond~tions specified. RNA was isolated from the nucle~r extract by phenol~chloroform extraction ~nd ethanol precipitation, followed by electrophoresis In a 10~ polyacrylamid~ ~et con~aining 8.3 M urea. The RNAs were visualized by ethidiu~ bromide staining.
A second RNase H assay was also carried out in nuclear extracts by using an exogenous P-labeled RNA.
A 5}4-nucleotide test RNA (here~fter ter~ed ~514 RNA~ for convenience) was generated by SP6 RNA polymerase trans-cription of a HindIII-linearized pGEM-2 clone, pT7H~6.
514 RNA is antisense to the first two exons and intron of human ~-globin pre-mRNA ~nd was chosen for the reasons described below. 514 RNA labeled with a ¦~- 2P~GTP, l~-32PlCTP, snd l~-32P]UTP was added to the nuclear extract containing the specified oligodeoxynucleotide at an oligo~er-to-514 RNA ~olar ratio of 3000:1, unless other~ise noted. After incubation as specified, RNA was extracted and the P-labeled 514 RNA or its clea~age :' , ~ : '~
U'091~1232~ PCT/~;S91/~0968 2076~37 -l9-products were visualized by electrophore~is and auto-radiography.
~esules Act~on of RNase H on Oli~odeoxynucleotide Ul Small 05 Nuclear RNA Hybrids HeLa cell ~uclear e~tracts cont~in RNase H sctivity that can ~ct on DNA-RNA hybrids that form after addit-on of oligodeoxynucleotides complementary to rertain endo-genous nuclear RNAs (Krainer, A.R. and T. ~aniatis, Cell, 42:725-736 (1985)). The ability of this RNase H activity __ to cleave the'5'-terminal nucleotides of endo~enous Ul small nuclear RNA, after incubation of the various ~odi-fied oligomers ~n the nuclear extract, uas investigated.
Incubation of the P0 oligomer $n the nuclear extract led to cleavage of a large proportion of the Ul RNA to a product (Ul~) hav~ng the mobility expected for removal of the first 15 nucleotides (cap, nucleotide l, and nucleotides 2-14). There was a lack of effect on thP
mobility of any other RNAs present, demonstrating the high ~equence specificity of the Ul oligomer-directed RNase H cleavage. Ul cleavage ~as also observed with the PS oligomer, although to a consistently lesser extent than with the P0 oligomer. In contrast, no cleavage was obser~ed ~ith the PG, phosphoro~-morpholidate (PM), or phosphoro-N-butylam~dAte (PB) oligomers. Reducing the temperature of ~ncubation to 20-C and extending the time to 60 minutes did ~ot increase the extent of cleavage observed with the PS oligomer, nor did it reveal cleavages with the PC, PM or PB oligomers. The pattern .
:-- . :
WOgl/12323 PCT/US91/00968 of UI cleavages seen with the vario~s oligomers w~s also not aleered by adding E._coli RNase H (Yharmacia, final concentrstion 8 units/ml~ to the nuclear ~xtract at the outset of incubation.
S RNase H Action at an Internal RNA Site Hybridized with __ ___ ____ _________ __________ _____ _______ or~al and Modified Oligodeo~ynucleotides She foregoing Ul RNA cloavsges lnvolve the 5' estFe~ity of a s~all RNA. Indeed, the 5' end of Ul RNA
is sn exceptionally fsvored target for ollgodeoxy-Ducleotide-directed RN~se H cleavage aince al~ost all the other regions of this RNA molecule are tightly complexed vith proteins in the Ul small nuclear ribonucleoprotein particle (Patton, J.R. et al., Mol. Cell. Biol., 7:
4030-6037 (1987); Patton, J.R. et al., Proc. Natl. Acad.
lS Sci. USA, 85:747-751 (1988); Patton, J.R. et al., Mol.
Cell. Biol., 9:3360-3368 (1989)). Therefore, the action of RNase H on hybrids formed by normal or modified oligomers at internal sites in a longer RNA was also in-~estigated.
For this purpose a test RNA which is 514 nucleotides long (termed 514 RNA) ~as uged. 514 RNA is antisense to the first t~o exons and intron of human ~-glo~in pre-mRNA. The underlying reasoning was that, because it is antisense to a pre-mRNA, 514 RNA ~ould not undergo splicing when ~dded to nuciear extract (the cleava~e-ligation steps of whlch ~ould otherwise complicate analysis of oligodeoxynucleotide-directed RNase H clea~-ages).
A series of normal and modified lS-~er oligodeoxy-nucleotides complementary to nucleotides 349-363 of 514 ': . . , ' `'i:
. .
WO91~12323 PCT/US91/00968 RNA vas synthesized chemically (Table 1, oligomers A-E).
This particular 514 RNA site ~as selected because oligomer-directed RNase H cleavage would generate two frdgments of readlly distinguishable lengths (lS0 and 348 05 nucleotides) ~nd also because this 15-nucleotide ~equence does ~ot occur else~here in 514 RNA (Lawn, R.M. et al., Cell, 21:647-651 (1980)). ~elting curves ln 0.16 M Na f~r four of these oli~odeo~yDucleotides ~fter duplex formstion with the co~plementnry (P0) oligomer reve~led that the P0, PC, PS and P~ oligomer-containing duplexes had tms of 53-C, 46-C, 43-C and 38-C, respectively, indicating a lower duplex ~tability for the modified oligomers, in part confir~lng previous reports (Stein, C.A. et al., Nucleic_Acids Res., 16:3209 3221 (1988);
lS Froehler, B. et_al., Nucleic_Ac~ds Res., 16:4831 4839 (1988); Quartin, R.S. ~nd J.G. Wetmur, Biochemistry, 2B:1040-1047 (1989); Agrawal, S. et_al., Nucleosides Nucleot~_es, 8:819-823 (1989)), . , . :
:: . .
. :- - . . :
, .
, ~'091/12323 PCT/US9l/0096~
2~7 ~ ~ I 22-___ ___________ _____ ___ ___ _ ___ __ _____ _ __ _ .
Ineernucleos~de Oli~omer Sequence Linka~e ___ __ _ _ _ __ ____ _ _ _ _ __ __ _ _ _ AGTA TCA ~GG TTA CAA PO
05 ~ GIA TCA AGG TTA CAA PS
CGTA TCA AGG TTA CAA PM
DGTA TCA ~GG TTA CAA PB
EGTA TCA AGG TTA CAA PC
F*GTA TCA TAT GAG ACA PO
G* GTA GCA AGG CTA CAA PO
H*GTA TGA GAC ATA TAC PO
__ _ _____ ___________________ ________________ Underlined nucle~tides indicate base pairing mismatches; PB is pbospho-N-butylamidate.
The beries of normal snd modified ol$godeoxy-nucleotides shown in Table 1 were incubated with P-labeled 514 RNA in nuclear extracts, under the conditions specified tn the ~aterisls and ~ethods section, for either 30 minutes or 3.5 hours.
The results of incubating these oligomers with 514 RNA in HeLa nuclear extract ~re as follows: After 30 minutes of incubation without any oligomer, intact input 514 RNA was the only labeled ~pecies v~sualized. After 3.5 hours of incubation without any oligomer, the 514 RNA
~as completely degraded by the action of ribonuclease kno~n to be present ~n the extract. Incubation of 514 RNA in the extract for 30 minutes together ~ith either the PO or PS oligomers resulted in precise cleavage of the substrate RNA into two fragments of the sizes . .
, ... . ~ ,.
., , . ~ , . .
:
W~91/123'~ PCT/~IS91/0096~
2~76037 23- , expected fro~ ehe location of the oligomer-complementary sequence. Surprisingly, in the case of the PS oligomer, these two fragments vere still present, albeit ln sli~htly degrnded form, after 3.5 hours of incubatlon.
05 Incubation of 514 RNA with PM ol~omer for 30 minutes resulted in partial cleavage (see the figure legend for additional details). No cleavage was observed with the PB or PC oligomers under these conditions.
PO oligomers that vere only pareially complementary eo 514 RNA (oligomers F, G and H ~n Table 1) were also tested. None of these oligomers, containing 4, 5 or 6 uninterrupted complementary nucleotides out of the 15 (Table 1), elicited RNase H cleavage of 514 RNA.
The finding that the PS oligomer was less effective than the P0 oligomer in eliciting RNase H cleavage in the Ul RNA assay raised the possibility that the more complete RNase H cleavages observed with both P0 and PS
oligomers in the S14 RNA assay might reflect the particular reaction conditions employed. Therefore, a ran~e of oligomer-to-514 RNA molar ratios tests (0.1:1-1000:1), all below that used in the above-described (3000:1), was investigated. Results showed that virtually complete 514 RNA cleavage occurred with the PO oligomer at an oligomer-to-~NA ratio of lO0:1, whereas a comparabl~ extent of 514 cleavage with the PS oli~omer occurred at a oligomer-to-RNA ratio of 1000:1. A very similar, ~ncomplete extent of cleavage was obser~ed with the P0 and PS oligomers at ratios of 10:1 and 100:1, respectively. The possibility that these results might reflect a preferential instability of the PS oligomer turing incubation in ~he nuclear extracts was ', .
WO91~1232~ PCT/US91/00968 2076~'~7 examined by experiments in which either the P0 or the PS
oligomer ~as preincubated in nuclear extract or 30 minutes, followed by addition of P-labeled 514 RNA and incubation for an atditional 30 min. This revealed the 05 same e~tent of difference in RNase H cleavage as described above.
~Restriction Endonuclease.Like~ Cleava~e with Oli~omers Containin~ RNase H Sensitive and -Resistant Internucleo-s~de_Linka~es-The extreme differences be~ween the R~ase H sensi-tivity of DNA-RNA hybrids containing PO or PS oligodeoxy-nucleotides, contrasted with ones with PC, PM or PB
oligomers, led to an investlgat~on of how RNase H acts on a DNA-RNA hybrid in which only a small proportion of lS internucleoside linkages in the DNA strand were RNase H-sensitive. Table 2 shows the series of oligomers that were synthesized to address this issue,.
WO91t12323 PCT/US91/00968 2076~37 _________ ____ _ _____ ___ ______ Oli~omer Sequence __ ___ _ _ _ ____________ AGG T
J A AGG TT
L GTA ICA ~GG TTA CAA
~ GTA TCA AGG TTA CAA
N GTA TCA AGG TTA CAA
O GTA TCA AGG TTA CAA
p GTA TCA AGG TTA CAA
___ _ __ Q GTA TCA AGG TTA CAA
R GTA TCA AGG TTA CAA
__ _____________ _ _ _ _ ____ _ _ Vnderlined nucleotides are PC; dashed nucleotides are PS; Boxed nucleotides are phosphoromorpholidates;
lS Double-underlined nucleotites are PM (oligomer Q) or PB;
the remaining nucleotides are PO.
The oligomers listed yere tested for their capacity to elicit RNase H action after hybridization to 514 RNA, as in the preced1ng experiments.
20 Results demonstrated that neither a tetramer nor a hexa~er (all PO) oligodeoxynucleoeide co~plementary to ~14 R~A was able to induce RNase H cleavage in this nuclear extract system. ~hen PO/PC-containing penta-decamers containing either two or four coDsecutive PO
linkages were used, a ~ow but readily detectable level of RNase H cleavage occurred. In contrast, a pentadecamer containing six consecutive PO linkages elicited complete RNase H cleavage of the substrate RNA. Note thst the six WO91/123~3 PCT/~'S91/00968 207~37 PO nucleotides in this oligomer (oligomer M in Table 2) are identical in sequence to the RNase H-inactive hexamer (oligomer J), from which it is inferred that the potency of the pentadec~mer reflects its increased hybrid ~5 stability with 514 RNA owing to the additional nine complenentary nucleotides.
Similar tests ~ere performed ~ith mixed PO~PC
pent-dec-mers co~pl~mentsry to a different site ~n 514 RNA (i,e,, nucleotides 463-477). These tests revealed an effect of the number of PO linksges on RNase H cleavage similar to that described above.
Additional variants of mixed PO/PC oligomers were also tested. Pentadecamer PO/PC oligomers with five or six consecutive PO linkages at either the extreme 5' or 3' end were highly effective in eliciting RNase H cleav-age, A PS/PC pentadecamer with six consecutive PS
linkages at the extreme 5' end ~oligomer P in Table 2) was only partially active. Comparison of these results with lanes 2 and 3 in Figure 4B confirms the above-described sesults showing that all-PS oligomers are less effective than all-P0 oligomers in eliciting RNase H
cleavage. RNase H cleavage~of 514 RNA was also observed with a 15-mer containing nine consecutive PM or PB
linkages followed by six PO-linked nucleotides (oli~omers Q and R in Table 2).
. . .
' ' ' : . '"' ~ ~ . ,. `
.: '' ' ' . , , ~ ' :
... . .. . .
Claims (12)
1. A method of site-specific alteration of a target RNA
segment of an RNA molecule, comprising the steps of:
a) combining the RNA molecule with a fixed phosphate backbone oligonucleotide which is complementary to all or a portion of the RNA
molecule, in the presence of RNase H, the mixed phosphate backbone oligonucleotide including an internal segment which activates RNase H and two flanking nucleotide sequences which are unable to activate RNase H, one of the two flanking nucleotide sequences being present on either side of the internal segment, under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby excising the target RNA segment, creating a gap in the RNA
molecule and producing an interrupted RNA
molecule which has two segments; and b) contacting the interrupted RNA molecule with an appropriate ligase, under conditions appropri-ate for joining the two segments of the interrupted RNA molecule by the ligase, thereby producing an altered RNA molecule.
segment of an RNA molecule, comprising the steps of:
a) combining the RNA molecule with a fixed phosphate backbone oligonucleotide which is complementary to all or a portion of the RNA
molecule, in the presence of RNase H, the mixed phosphate backbone oligonucleotide including an internal segment which activates RNase H and two flanking nucleotide sequences which are unable to activate RNase H, one of the two flanking nucleotide sequences being present on either side of the internal segment, under conditions appropriate for hybridization of complementary nucleotide sequences and activation of RNase H, thereby excising the target RNA segment, creating a gap in the RNA
molecule and producing an interrupted RNA
molecule which has two segments; and b) contacting the interrupted RNA molecule with an appropriate ligase, under conditions appropri-ate for joining the two segments of the interrupted RNA molecule by the ligase, thereby producing an altered RNA molecule.
2. The method of Claim 1 wherein the mixed phosphate backbone oligonucleotide is a mixed phosphate backbone oligodeoxynucleotide; the internucleoside bridging phosphate residues of the internal segment are unmodified phosphates and the internucleoside bridging phosphate residues of the flanking sequences are modified phosphate residues.
3. The method of Claim 2 wherein the bridging phosphate residues of the flanking sequences are modified phosphates selected from the group consisting of:
methyl phosphonates, phosphoromorpholidates, phosphoropiperazidates, and phosphoramidates.
methyl phosphonates, phosphoromorpholidates, phosphoropiperazidates, and phosphoramidates.
4. The method of Claim 1 wherein the mixed phosphate backbone oligonucleotide is a mixed phosphate backbone oligodeoxynucleotide; the internucleoside bridging phosphate residues of the internal segment are modified phosphates and the internucleoside bridging phosphate residues of the flanking sequences are modified phosphates.
5. The method of Claim 4 wherein the internucleoside bridging phosphate residues of the internal segment are modified phosphates which are phosphorothioates and the internucleoside bridging phosphate residues of the flanking sequences are modified phosphates selected from the group consisting of: methyl phosphonates, phosphoromorpholidates, phosphoro-piperazidates, and phosphoramidates.
6. A method of producing a selected polypeptide com-prising expressing an altered RNA molecule produced by the method of Claim 1.
7. A method of producing a selected polypeptide encoded by an altered RNA molecule, comprising the steps of:
a) providing an altered RNA molecule produced by a method of site-specific alteration of a target RNA segment of an RNA molecule; and b) expressing the altered RNA modlecule provided in step a).
a) providing an altered RNA molecule produced by a method of site-specific alteration of a target RNA segment of an RNA molecule; and b) expressing the altered RNA modlecule provided in step a).
8. A method of site-specific alteration of Claim 1, further comprising replacing the target RNA segment excised in step a) by introducing a segment of replacement nucleotides into the gap in the RNA
molecule and b) linking the nucleotide present at each end of the segment of replacement nucleotides to the respective nucleotide present at the gap created in the RNA molecule.
molecule and b) linking the nucleotide present at each end of the segment of replacement nucleotides to the respective nucleotide present at the gap created in the RNA molecule.
9. A mixed phosphate backbone oligonucleotide useful in the method of Claim 1.
10. A mixed phosphate backbone oligonucleotide consisting essentially of an internal segment of deoxynucleotides which activates RNase H and two modified nucleotide sequences which do not activate RNase H, in which the two modified nucleotide sequences flank the internal segment, one on each side of the internal segment.
11. The mixed phosphate backbone oligonucleotide of Claim 10 wherein the internucleoside bridging phosphate residues of the internal segment are unmodified phosphates and the internucleoside bridging phosphate residues of the two modified nucleotide sequences are modified phosphates selected from the group consisting of: methyl phosphonates, phosphoromorpholidates, phosphoropiperazitates, and phosphoramidates.
12. The mixed phosphate backbone oligonucleotide of Claim 10 wherein the internucleoside bridging phosphate residues of the internal segment are modified phosphates which are phosphorothioates and the internucleoside bridging phosphate residues of the two modified nucleotide sequences are modified phosphates selected from the group consisting of:
methyl phosphonates, phosphoromorpholidates, phos-phoropiperazidates, and phosphoramidates.
methyl phosphonates, phosphoromorpholidates, phos-phoropiperazidates, and phosphoramidates.
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US480,269 | 1990-02-15 | ||
US07/480,269 US5149797A (en) | 1990-02-15 | 1990-02-15 | Method of site-specific alteration of rna and production of encoded polypeptides |
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CA2076037A1 true CA2076037A1 (en) | 1991-08-16 |
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CA002076037A Abandoned CA2076037A1 (en) | 1990-02-15 | 1991-02-13 | Method of site-specific alteration of rna and production of encoded polypeptides |
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US (3) | US5149797A (en) |
EP (1) | EP0515511B1 (en) |
JP (1) | JPH05506355A (en) |
AT (1) | ATE141326T1 (en) |
CA (1) | CA2076037A1 (en) |
DE (1) | DE69121391T2 (en) |
DK (1) | DK0515511T3 (en) |
ES (1) | ES2090314T3 (en) |
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US4661450A (en) * | 1983-05-03 | 1987-04-28 | Molecular Genetics Research And Development Limited Partnership | Molecular cloning of RNA using RNA ligase and synthetic oligonucleotides |
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ATE151467T1 (en) * | 1987-11-30 | 1997-04-15 | Univ Iowa Res Found | DNA MOLECULES STABILIZED BY MODIFICATIONS TO THE 3'-TERMINAL PHOSPHODIESTER BOND, THEIR USE AS NUCLEIC ACID PROBE AND AS THERAPEUTIC AGENTS FOR INHIBITING THE EXPRESSION OF SPECIFIC TARGET GENES |
US5098890A (en) * | 1988-11-07 | 1992-03-24 | Temple University-Of The Commonwealth System Of Higher Education | Antisence oligonucleotides to c-myb proto-oncogene and uses thereof |
US5176996A (en) * | 1988-12-20 | 1993-01-05 | Baylor College Of Medicine | Method for making synthetic oligonucleotides which bind specifically to target sites on duplex DNA molecules, by forming a colinear triplex, the synthetic oligonucleotides and methods of use |
US5149798A (en) * | 1989-04-06 | 1992-09-22 | Worcester Foundation For Experimental Biology | Process for synthesizing oligonucleotides and their analogs adaptable to large scale syntheses |
KR920701230A (en) * | 1989-06-05 | 1992-08-11 | 원본미기재 | Exonuclease-resistant oligonucleotides and methods for preparing the same |
US5220007A (en) * | 1990-02-15 | 1993-06-15 | The Worcester Foundation For Experimental Biology | Method of site-specific alteration of RNA and production of encoded polypeptides |
US5149797A (en) * | 1990-02-15 | 1992-09-22 | The Worcester Foundation For Experimental Biology | Method of site-specific alteration of rna and production of encoded polypeptides |
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1991
- 1991-02-13 AT AT91904770T patent/ATE141326T1/en not_active IP Right Cessation
- 1991-02-13 DK DK91904770.4T patent/DK0515511T3/en active
- 1991-02-13 ES ES91904770T patent/ES2090314T3/en not_active Expired - Lifetime
- 1991-02-13 JP JP91505563A patent/JPH05506355A/en active Pending
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- 1991-02-13 DE DE69121391T patent/DE69121391T2/en not_active Expired - Fee Related
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1993
- 1993-03-24 US US08/036,503 patent/US5366878A/en not_active Expired - Fee Related
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1994
- 1994-11-18 US US08/342,302 patent/US5635377A/en not_active Expired - Fee Related
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1996
- 1996-10-07 GR GR960402639T patent/GR3021278T3/en unknown
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US5149797A (en) | 1992-09-22 |
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GR3021278T3 (en) | 1997-01-31 |
ES2090314T3 (en) | 1996-10-16 |
WO1991012323A1 (en) | 1991-08-22 |
EP0515511A1 (en) | 1992-12-02 |
JPH05506355A (en) | 1993-09-22 |
US5366878A (en) | 1994-11-22 |
US5635377A (en) | 1997-06-03 |
DE69121391T2 (en) | 1997-03-06 |
DE69121391D1 (en) | 1996-09-19 |
DK0515511T3 (en) | 1996-11-18 |
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