Targeting nucleic acid secondary structures by antisense

Transcription

1 Pro. Natl. Aad. Si. USA Vol. 93, pp , Otober 1996 Biohemistry argeting nulei aid seondary strutures by antisense oligonuleotides designed through in vitro seletion (aptamer/hairpin struture/inhibition of translation) RAKESH K. MISHRA*, REJANE LE INEVEZ, AND JEAN-JACQUES OULME1t Institut National de la Sante et de la Reherhe Mediale, U 386, Laboratoire de Biophysique Moleulaire, Universite Bordeaux II, 146 rue L5o Saignat, Bordeaux, CEDEX, Frane Communiated by Marianne Grunberg-Manago, Institut de Biologie Physio-Chimique, Paris, Frane, May 21, 1996 (reeived for review Deember 1995) ABSRAC Using an in vitro seletion approah, we have isolated oligonuleotides that an bind to a DNA hairpin struture. Complex formation of these oligonuleotides with the target hairpin involves some type of triple-stranded struture with nonanonial interation, as indiated by bandshift assays and footprinting studies. he seleted oligomers an blok restrition endonulease leavage of the target hairpin in a sequene-speifi manner. We demonstrate that in vitro seletion an extend the antisense approah to funtional targeting of seondary struture motifs. his ould provide a basis for interfering with regulatory proesses mediated by a variety of nulei aid strutures. Regulation of gene expression by antisense oligonuleotides has provided a powerful approah for studying the funtion of a gene and for inhibiting genes responsible for undesirable traits. he binding of a syntheti oligomer to a omplementary site of a target RNA an prevent the maturation of the message, its transport from the nuleus to the ytoplasm, or its translation (for review, see ref. 1). Antisense oligonuleotides have also been shown to blok DNA synthesis by retroviral reverse transriptases (2). However, single-stranded target RNA may adopt seondary strutures that redue aessibility to a omplementary oligonuleotide, leading to a weaker antisense effet (if any) than expeted (3-5). One an avoid strutured target regions by a systemati sreening of the entire RNA length until an aessible target is reahed, i.e., umtil inhibition of gene expression is observed (6). However, several RNA strutures have been shown to play a role in regulatory proesses, in partiular in viruses (7-11). Binding an oligonuleotide to suh regions will likely interfere with these proesses. Complex formation an be ahieved by inreasing the affinity and/or the onentration of the antisense sequene to ompete out the intramoleular folding of the target, but this might be a soure of nonspeifi effets (1, 12). Alternatively, antisense oligomers ould be designed to bind to intat RNA strutures. his approah might be energetially favorable beause the seondary or tertiary interations within the targeted nulei aid bases are preserved. Moreover, antisense sequenes able to read the three-dimensional array of interating sites in the strutured RNA may provide a higher level of seletivity. It was previously demonstrated that a DNA hairpin an interat with a omplementary oligonuleotide, forming a triple helix-mediated double hairpin struture (13, 14). However, as only homopurine/homopyrimidine double strands an form triplexes (15), this approah is restrited to stem-loop strutures with appropriate sequenes. Combinatorial strategies have proved to be very powerful to identify RNA or DNA sequenes exhibiting high affinity and he publiation osts of this artile were defrayed in part by page harge payment. his artile must therefore be hereby marked "advertisement'? in aordane with 18 U.S.C solely to indiate this fat seletivity for small ligands or maromoleules (refs ; for review, see ref. 21). In vitro seletion has been used to generate oligonuleotides able to reognize a nulei'aid; numerous examples of RNA enzymes obtained from direted evolution are available (22-27). his strategy has also been applied against a double-stranded DNA, leading to the seletion of oligoribopyrimidines interating with the target through the formation of anonial.a- and C+.G-C triplets (20). Single-stranded regions of folded targets have been identified by in vitro seletion (19) or rational sreening-of modified oligonuleotide libraries (28). In these latter ases, onventional double-stranded strutures resulted from the assoiation between the target and the aptamers. We have applied an in vitro seletion approah to isolate "aptastrus," i.e., aptamer oligonuleotides able to interat with a struturednulei aid. MAERIALS AND MEHODS Materials. Oligodeoxynuleotides were obtained from Institut Pasteur (Paris, Frane). he library of random andidates was prepared by introduing simultaneously on the synthesizer, the four synthons in equimolar amounts at eah position from nuleotide 14 to nuleotide 29 (Fig. 1). Oligonuleotides were purified by reverse-phase HPLC, using an aetonitrile gradient (0-48%) in a 100 mm ammonium aetate (ph 7.0) buffer, and heked for purity by eletrophoresis on a 20% denaturing polyarylamide gel of 32P-5'-end-labeled produts. Chemial reagents were obtained from Aldrih, and [y_32p]ap was obtained from ICN. 4 polynuleotide kinase was from Boehringer Manheim, and aq polymerase was from Stehelin (Basel, Switzerland). Restrition endonuleases and DNase I were purhased from GIBCO. S1 nulease and 4 ligase were from Promega. Seletion. he seletion proedure shown in Fig. 1 was performed from a starting oligodeoxynuleotide repertoire of 416 = 4.29 x 109 unique speies, as desribed in a preliminary note (29). Briefly, target hairpin '(33 nm), seletor, and andidates (16.6,uM eah) were mixed in a 50 mm ris-aetate (ph 6.0) buffer ontaining 10 mm MgC92. his resulted in more than a 400-fold exess of andidate sequenes over the target, allowing strong ompetition for binding to the hairpin. After addition of 20 units of Sal, the samples were inubated for 20 h at 21 C, preipitated with ethanol and redissolved in sterile water. After denaturation (10 min at 95 C), DNA was amplified with aq polymerase (0.25 units), in the buffer supplied with the enzyme, in the presene of both primers for 30 yles (10 s at 95 C, 30 s of reannealing'at 40 C, and 30 s Abbreviation: DMS, dimethyl sulfate. *Present address: Department of Pathology, Shool of Mediine, St. Louis University, St. Louis, MO to whom reprint requests should be addressed.

2 10680 Biohemistry: Mishra et al. a n ji) RsaI C G A C C C C C 5' C A G A G G A G A G A A G A G G G A C C C 2C 10 G A Sal 5' G C C G C G A G C A G- N(16)- C C C C G A G C A C C g g g a t g a t P2 t t t t t t g a a Pi 5, u C1 ( A C G) A A C G A G G C A G C122 (222, 322) C2 (A C C A) G G G G G G G C C C C 4 C3 (G A A C) A A A C G G G C A CC116 (216, 316) 6CM 5 b Seletioni Amplifiation Cloning Sequening Aptastrus CM C C C C C C C C C C C C C 11 arget 5!..I MIL1 1L2 i C2i ----C Restrition /.0 5, Candidates '111 :.. 11 "1:1 L-Seletor FIG. 1. (a) Sequenes of the target, of the andidate library, and of the seleted oligomers (aptastrus). he RsaI restrition site on the target is indiated with dotted lines. he sequenes of the primers (P1 and P2) and of the seletor (S) are given in lower ase type. he Sad restrition site used for seletion is boxed. N16 indiates the 16-nt-long random motif in the andidates (C). he 3' ends of the aptastrus orrespond to the 16-nt-long random streth (C116, C216, and C316) derived from the seleted 26-mers, Cl, C2, and C3, respetively. Four bases (in parenthesis) have been deleted at the 3' end of Cl, C2, and C3 to generate the 22-mers, C122, C222, and C322. he 6-mer anhor motif is underlined both in the andidates and in the target sequenes. he 26-mer oligopyrimidine CM used in previous studies (13, 14) is shown. he hexamer 3'CCCC (6CM) orresponds to the anhor sequene. (b) Seletion sheme. () Eletrophoreti mobility shift assay, on a 15% nondenaturing polyarylamide gel, of the andidatetarget omplexes. he absene or the presene of the target hairpin is indiated by + or -, respetively. of synthesis at 72 C). After preipitation by ethanol and dissolution in water, a seond step of amplifiation was performed under the same onditions exept that only primer 1 was used, to obtain a seond generation of single-stranded andidates. Four idential rounds of seletion were performed, after whih andidates digested by SaI and BamHI were loned into puc19 linearized by SaI and BamHI. Sequening was performed using the dideoxynuleotide terminatiop method (30). :l Pro. Natl. Aad. Si. USA 93 (1996) Analysis of Complexes. Before analysis, unless otherwise indiated, the oligonuleotide mixture (target plus andidate) was heated for 5 min at 75 C in 50 mm ris-aetate buffer, ontaining 10 mm MgCl2 (buffer A), and then allowed to ome to room temperature in about 45 min, after whih it was stored at 4 C for at least 3 h. For eletrophoreti mobility-shift assays, 32P-5'-end-labeled andidates (0.6,uM) were mixed with old target (24,uM) in buffer A (ph 7.5) and loaded immediately onto a 15% nondenaturing polyarylamide gel. For ligation experiments, the mix ontaining stoihiometri amounts of target and andidates in buffer A (ph 6.0) was inubated at 15 C for 16 h with 10 mm AP and 10 units of 4 ligase. he produts were then analyzed on a 20% denaturing polyarylamide gel. Si mapping and DNase I footprinting experiments were performed on preformed omplexes in buffer A at ph 6.0. S1 nulease (15 units) was added to the omplex (1.2,uM 5'-endlabeled andidate and 24,tM target), and the digestion was arried out for 15 min at 20 C and stopped by the addition of 10 pkg trna, 1,ul of 3 M sodium aetate (ph 5.6), and 100,ul of ethanol (95%). For DNase I footprinting, samples ontaining 1.2,uM 5'-end-labeled target and 24,uM andidate were inubated for 5 min at 20'C with 0.6 units of DNase I. he reation was stopped as indiated above. Chemial [dimethyl sulfate (DMS), KMnO4 and EDA- Fe2+] footprinting was performed in buffer A at ph 6.0 as desribed (31) on a mixture ontaining 1.2,tM 5'-end-labeled target and 24,uM andidate (DMS), 0.3 pm 32p-5'-end-labeled andidate, and 15,uM target (KMnO4) or 0.3 pm 5'-endlabeled target and 60,uM andidate. Samples were reated with 5% DMS (final onentration) for 4 min at 85 C and preipitated with ethanol after addition of 15,ug of trna and sodium aetate up to a onentration of 0.5 M. For permanganate footprinting, omplexes were inubated in the presene of 22.8 mg/ml KMnO4 for 30 min at 4 C and preipitated with ethanol. After dissolution in water, samples were treated for 30 min at 90 C by 1 M piperidine and preipitated twie before eletrophoresis. For OH' footprinting, oligonuleotides were inubated for 3 min at 18 C with 13 mm EDA and 6.7 mm ferrous ammonium sulfate in the presene of 1% H202 and 100 mm sodium asorbate. he reation was stopped as indiated above for DMS footprinting using a mix supplemented with 33 mm thiourea. Samples were then preipitated by ethanol and analyzed by eletrophoresis. Funtional Studies of Complexes. RsaI ativity was evaluated on omplexes (1.2,tM target plus 12 jkm andidates) preformed in buffer A (ph 7.5) and inubated for 3 h at 20 C with 2 units of RsaI. Produts were analyzed by eletrophoresis on a 20% denaturing polyarylamide gel. RESULS AND DISCUSSION Seletion of Anti-Hairpin Candidates. A pool of 45-nt-long oligodeoxynuleotides, ontaining a random streth of 16 nuleotides, onstituted the population of andidates against a DNA hairpin used in previous work (13, 14). he random streth was flanked on the 5' side by a multifuntional sequene: 3'CCCCGAGCAC that, in addition to being used for amplifiation and loning, ontained the binding site of the seletor oligonuleotide and a six residue anhor 3'CCCC, omplementary to the 5' single-stranded part of the target (Fig. la). Our in vitro seletion approah has the distintion of being a "single-pot" series of reations that does not require a purifiation step before amplifiation and is appliable to DNA as well as RNA targets. his approah involves a "seletor" oligonuleotide (5'GGGAGCCG) that, upon binding to the 5' end of a andidate, generates a Sal site, leading to the loss of one of the PCR primer binding sites after restrition digestion (Fig. lb). he restrition site

3 Biohemistry: Mishra et al. (5'GAGCC) was derived from the 5' end of the anhor region, leading to the hoie of Sal, whih is sensitive to DNA onformation (more ative on linear DNA), has no star ativity, and works well at low ph and under a broad range of salt onditions. he andidates interating with the target struture are not aessible to seletor binding and hene esape restrition digestion. After four rounds of seletion/ amplifiation, the andidate population was loned, and we arbitrarily piked three of them for further studies. Charaterization of Hairpin-Candidate Complexes. We synthesized the three 26-mer andidates, Cl, C2, and C3 (Fig. la), omposed of the 16-base streth (C116, C216, C316) linked to the 10-base ommon sequene (the four linker and the anhor). Based on sequene analysis, none of these andidates displayed any obvious way to interat with either the folded or the unfolded target DNA, exept the expeted formation of 6 bp with the anhor motif. Sequenes of the three andidates were different from the triplex-forming oligopyrimidine CM used in previous work to aommodate the DNA hairpin (13, 14) (Fig. la). We analyzed the interations of these three seleted sequenes with the target hairpin. As shown by eletrophoreti mobility-shift assay, the oligomers Cl, C2, and C3 form one major type of omplex with the target DNA hairpin at ph values of both 7.5 (Fig. l) and 6.0 (not shown). Additional minor omplexes of redued mobility an be deteted on overexposed gels under these experimental onditions (not shown). he mobility of omplex also varied with the target/andidate ratio. Potential intermoleular multimeri omplexes due to the self-omplementarity in parts of the target sequene may explain these observations. his proposition is substantiated by the fat that the mobility of these omplexes is sensitive to the target onentration (not shown). he extent of shift and the ratio of different kinds of omplexes with Cl or C3 are slightly different from that with C2 (Fig. l), indiating subtle variation in the shape of the omplex as both the size and the harge are idential. However, the three types of omplexes were indistinguishable by their irular dihroi spetra (not shown). An exess (about 100-fold) of the hexamer 5'CCCC (6CM), was needed to ompete out the C2/target omplex (not shown). his indiates that the 3' end of C2, and not only its 6-nt anhor, ontributes to its binding to the DNA hairpin. Similar results were obtained with Cl and C3 Ẇhen 32P-5'-end-labeled andidates were used, the resulting omplexes with the target were enzymatially ligated (Fig. 2a), demonstrating that the 5' ends of the andidates are adjaent to the 3' end of the target, as expeted from the design of the anhor region. S1 nulease (Fig. 2b), KMnO4 (Fig. 2), or OS04 (not shown) footprinting assays showed that the aessibility of the thymine residues in the region between nuleotides 7 and 10 in andidates is inreased in the presene of the target. his is onsistent with a folded onformation of the andidates in the omplex suh that the "-region" forms a loop. he hypersensitivity of C3 to S1 nulease, ompared with the two other andidates, denotes weaker interation of the 3' end, as leavage extends beyond the "-loop." DMS and 0S04 footprinting of the target revealed only minor hanges in the presene of either andidate; a slight protetion of the Gs in the stem was seen (Fig. 3a). In ontrast, the DNase I leavage pattern of the target DNA was signifiantly hanged; all three andidates yielded a similar pattern, onsistent with the formation of a double-stranded struture with the 5' part of the target, i.e., with the anhor region (Fig. 3b). herefore, although the 3' part of the andidate interats with the target hairpin, that binding does not prevent DNase I ativity. his result ontrasts to that observed in the presene of CM, the 26-mer oligopyrimidine able to form a loal triple helix; in this ase, no DNase I leavage was seen in the stem region, whereas a hypersensitive site appeared at the double helix/triple helix juntion, in good agreement with DMS footprinting (13). 70 -: Pro. Natl. Aad. Si. USA 93 (1996) a M b.. Cl C2 C3 C1 C2 C3 S L... FIG. 2. (a) Eletrophoreti analysis of ligation produts of 32P-5'- end-labeled andidates with the target. Oligomers CM (lanes 1 and 2), Cl (lanes 3 and 4), C2 (lanes 5 and 6), or C3 (lanes 7 and 8) were inubated with the ligase mix, in the absene (odd lanes) or in the presene (even lanes) of the target. Lane M ontains size markers: labeled target (44 nt) and CM oligomer (26 nt). he expeted size of the ligation produt (70 nt) is also marked to the left. Si mapping (b) and KMnO4 footprinting analysis () of 32P-5'-end-labeled andidates Cl, C2, or C3 in the absene (-) or in the presene (+) of the target. he sequene ommon to the three andidates is indiated to the right. Samples were analyzed on a 20% polyarylamide/7 M urea gel. hese results strongly suggest that the omplexes formed by the andidates do not orrespond to anonial triple helies. his hypothesis was indeed onfirmed by experiments performed at neutral ph; andidates did bind to the target hairpin, whereas CM did not, underlining the role played by C-G.C+ triplets in the latter, but not the former, omplex. Indeed, C-G.C+-ontaining triplexes are muh less stable at neutral ph than at aidi ph (32, 33). Hene, our seleted andidates are viewed as aptastru oligomers, i.e., aptamers able to interat with the target hairpin by some unonventional triple-stranded motif. Seleted Oligonuleotides Prevent Restrition of the arget Hairpin. We designed a restrition leavage assay to monitor the interation of the target with aptastru sequenes, taking advantage of an RsaI site loated at the top of the stem. CM I C

4 ~~~~~~~~~~~~~~~. ::,W: Biohemistry: Mishra et al Pro. Natl. Aad. Si. USA 93 (1996) a b C v~ CN N N es U ", V" %o u uu DNasel -> Oligo -> A - AC FIG. 3. (a) Chemial footprinting of the DNA target in the absene (-) or in the presene of andidates Cl, C2, or C3, by DMS or OSO4. he target sequene is indiated to the left. (b) DNase I footprinting of the target in the absene (oligo -) or in the presene of oligomers Cl, C2, C3, or CM. he far left lane is a mok-inubated target. () EDA-Fe2+ footprint of the target in the absene (-) or in the presene of the oligonuleotides indiated at the top of the lanes. Lane G shows DMS reation of Maxam-Gilbert sequening. Sequene is indiated to the right. Hypersensitive sites with 26-mers (Cl, C2, or C3) or with 22-mers (C122, C222, or C322) are indiated by arrows. proteted the DNA hairpin from RsaI digestion at ph 6.0 (14), of the aptastrus was used (not shown). he onneting loop but not at ph 7.5 (Fig. 4, lane 1). Unrelated oligonuleotides turned out to be important; the 16-mers C116 and C316, (Fig. 4, lanes 17 and 18) also did not show a notieable effet, orresponding to the "third strand" of andidates Cl and C3, but all three aptastrus redued the leavage of the target showed only a slight inhibitory effet when ombined with the hairpin by RsaI at ph 7.5, when present in 10-fold exess, with hexamer 6CM (Fig. 4, lanes 14 and 16). inreasing effiieny in the order Cl < C2 < C3 (Fig. 4, lanes It is likely that base pairing of the first six nuleotides of the 4-6). he inhibition was nearly omplete when a 20-fold exess andidates followed by folding through the loop sequene is ritial for final omplex formation. As bases in the loop are probably not engaged in the omplex (the residues remain aessible to strutural probes), it is tempting to suggest that this region may ontribute to kinetially promote the assoiation, as demonstrated for lamps or irular oligonuleotides (34, 35). he moleular analysis presented here does not take into aount possible multimeri omplexes that orrespond to low migrating speies on nondenaturing gels (see above). In suh supramoleular strutures, the loop region of the andidates, along with the self-omplementary regions of the target, also plays the anhoring role. his networking may ontribute to the higher order organization of the resulting omplex, whereas the interation of the third strand alone may not be as FIG. 4. Inhibition of RsaI digestion of the target by andidate stable as a anonial triple helix. his might have been an oligonuleotides. arget alone was nondigested (lane 3) or digested by the overriding fator restrition during the seletion enzyme (lane 2). In all yles, hene other lanes, the resulting in target was inubated with RsaI in the presene of various oligonuleotides: lane the isolation of Cl, C2, and C3 sequenes. 1, CM; lane 4, Cl; lane 5, C2; lane 6, C3; lane 7, C122; lane 8, C222; In ontrast, the homologous oligonuleotide C216 derived lane 9, C322; lane 10, 6CM; lane 11, C116; lane 12, C216; lane 13, C316; from C2 indues a very strong inhibition of RsaI leavage, even lane 14, 6CM + C116; lane 15, 6CM + C216; lane 16, 6CM + C316; in the absene of the hexamer 6CM (Fig. 4, lanes 12 and 15). lanes 17 and 18, unrelated oligonuleotides. his behavior might be due to the fat that G,-ontaining

5 oligomers an bind with rather weak affinity to a target homopurine strand of a duplex to form triple-stranded helies (36); however, it is unlikely that a regular triple helix with a G,-third strand is formed in this ase beause it would ontain more than 50% of nonanonial triplets (15). he inhibition of RsaI digestion of the DNA hairpin by aptastrus Cl, C2, or C3 was sequene-speifi; none had any effet on the digestion of plasmid DNA byrsal, demonstrating that the protetion of the DNA hairpin was not due to a diret interation of the oligomers with the enzyme. Also noteworthy, the seleted andidates had no diret inhibitory effet on Sal, the enzyme used for in vitro seletion. Protetion of the hairpin against leavage byrsal an be due either to a steri hindrane of the restrition site by the oligomer or/and a onformational hange of the target DNA upon andidate binding, whih makes the site less suseptible to RsaI ativity. We favor the latter hypothesis as, first, the four nuleotide-shorter version of the aptastrus (C122, C222, and C322; see Fig. la) bloked RsaI with an even greater effiieny than the parent aptastrus (Fig. 4, lanes 7-9). Seond, OH footprinting showed that, while the sugar-phosphate bakbone in the stem region of the hairpin beomes less aessible in the presene of Cl, C2, or C3, a hyperative site is formed at nuleotide 26 of the target (Fig. 3). his hyperative site moves 4 nuleotides loser to the 5' end of the target when the shortened andidates, C122, C222, or C322, are used for omplex formation. In all these ases, the hyperative site is about 6 nuleotides away from the 3' end of the andidate bound to the target, assuming an extended onformation of the oligonuleotide along the hairpin, with a 3- to 4-base loop. his indiates that the influene of Cl, C2, or C3 on the sugarphosphate bakbone onformation of the target is of similar kind, even though the sequene of the 3' end of the andidate is varied. CONCLUSION Using an in vitro seletion approah, we have extrated aptastrus i.e. oligonuleotides interating with a DNA struture, from a library of random oligonuleotides. he omplexes formed by three seleted andidates with their stem-loop target an be viewed as a double-hairpin wherein the 5' end of the aptastru oligomer is adjaent to the 3' end of the target sequene. Aording to our anhor design, the first 6 nuleotides on the 5' side of the aptastrus are base paired with the target and extend the stem region of the hairpin (Fig. 5). he next few residues provide the loop of the hairpin turn as that the remaining sequene an be loated in the major groove of the extended double helix, whih bends at the double strand- "triple strand" juntion, toward the 3' terminus of the andidate, thereby exposing the other side of the helix. Despite differenes in the aessibility of some bases to hemial and enzymati probes, the three omplexes adopt a similar overall struture. his may reflet a low seletivity of binding. As only four rounds of seletion were performed, we likely seleted a sublass of andidates that organize themselves on the folded hairpin with limited interations with the target. We ould likely obtain andidates that bind tighter and arget Biohemistry: Mishra et al. C G A C 5' G A C C C C C C C C C C A G A G G A G A G A A G A G G G A C C 5 A C C A G G G G G G G FIG. 5. Overall organization of the hairpin-c2 omplex dedued from footprinting experiments. he sequene of C2 is italiized and underlined. he 3' end of the aptastru is aligned along the hairpin stem. Pro. Natl. Aad. Si. USA 93 (1996) more speifially to the target after additional rounds of seletion under more stringent onditions. he initial base pairing of the anhor region is ritial in the formation of the aptastru/target omplex. Indeed, oligonuleotides derived from the three aptastrus by deleting the anhor did not display detetable affinity for the target with use of an eletrophoreti mobility-shift assay. Conversely, the properties exhibited by the oligomers Cl, C2, and C3 are not only aounted for by the 6-nt-long anhor, as indiated by the ompetition experiment between the hexamer and eah of the three aptastrus for binding to the DNA target. Moreover, no retarded band was seen when the anhor hexamer was used in native gel assays. Last, the oligopyrimidine CM that ontains the same anhor as the three seleted aptastrus did not bind to the target at neutral ph, in ontrast to what was observed with Cl, C2, and C3. his indiates that the omplex formed by the aptastrus and the target involves different types of third-strand binding interation than the one in the pyrimidine motif triple-stranded omplex. It is worth mentioning that this anhor-driven seletion undoubtedly restrited the repertoire of seletable andidates. Seletion of andidates from an anhor-free library would likely provide different aptastru sequenes. herefore, we have demonstrated that in vitro seletion extends antisense strategy to a new lass of targets. But this study also points out the limitation of the antisense approah; oligonuleotides an bind to unintended sites that annot be predited on the basis of primary sequenes, possibly giving rise to unexpeted effets. We thank Mrs. Chantal Bourget for skillful tehnial support. his work was supported in part by the Conseil Regional d'aquitaine, by the P6le Mediament Aquitaine, and by the Agene Nationale de Reherhe sur le SIDA. 1. Hel&ne, C. & oulme, J. J. (1990) Biohim. Biophys. Ata 1049, Boiziau, C., huong, N.. & oulme, J. J. (1992) Pro. Natl. Aad. Si. USA 89, Verspieren, P., Loreau, N., huong, N.., Shire, D. & oulme, J. J. (1990) Nulei Aids Res. 18, Pasolo, E., Hudrisier, D., Sproat, B., huong, N.. & oulme, J.J. (1994) Biohim. Biophys. Ata 1219, Vikers,. & Eker, D. J. (1992) Nulei Aids Res. 20, Peyman, A., Helsberg, M., Kretzshmar, G., Mag, M., Grabley, S. & Uhlmann, E. (1995) Biol. Chem. Hoppe-Seyler 376, Pollak, J. R. & Ganem, D. (1993) J. Virol. 67, Wang, C., Le, S. Y., Ali, N. & Siddiqui, A. (1995) RNA 1, Churher, M. J., Lamont, C., Hamy, F., Dingwall, C., Green, S. M., Lowe, A. D., Butler, J. G., Gait, M. & Karn, J. (1993) J. Mol. Biol. 230, Baudin, F., Marquet, R., Isel, C., Darlix, J. L., Ehresmann, B. & Ehresmann, C. (1993) J. Mol. Biol. 229, Iwai, S., Prithard, C., Mann, D. A., Karn, J. & Gait, M. (1992) Nulei Aids Res. 20, Vikers,., Baker, F., Cook, P. D., Zounes, M., Bukheit, R. W., Germany, J. & Eker, D. J. (1991) Nulei Aids Res. 19, Brossalina, E. & oulme, J. J. (1993) J. Am. Chem. So. 115, Brossalina, E., Pasolo, E. & oulme, J. J. (1993) Nulei Aids Res. 21, huong, N.. & Helene, C. (1993) Angew. Chem. Int. Ed. Engl. 32, Blakwell,. K., Kretzner, L., Blakwood, E. M., Eisenman, R. N. & Weintraub, H. (1990) Siene 250, Ellington, A. D. & Szostak, J. W. (1990) Nature (London) 346, uerk, C. & Gold, L. (1990) Siene 249, Cload, S.. & Shepartz, A. (1994) J. Am. Chem. So. 116,

6 10684 Biohemistry: Mishra et al. 20. Pei, D., Ulrih, H. D. & Shultz, P. G. (1991) Siene 253, Gold, L., Polisky, B., Uhlenbek, 0. & Yarus, M. (1995) Annu. Rev. Biohem. 64, Bartel, D. P. & Szostak, J. W. (1993) Siene 261, Green, R. & Szostak, J. W. (1992) Siene 258, Joseph, S. & Burke, J. M. (1993).J. Bio. Chem. 268, Lorsh, J. R. & Szostak, J. W. (1994) Nature (London) 371, Pan,. & Uhlenbek, 0. C. (1992) Nature (London) 358, Wilson, C. & Szostak, J. W. (1995) Nature (London) 374, Eker, D. J., Vikers,. A., Haneak, R., Driver, V. & Anderson, K. (1993) Nulei Aids Res. 21, Mishra, R. K. & oulme, J. J. (1994) C. R. Aad. Si. Paris 317, Pro. Natl. Aad. Si. USA 93 (1996) 30. Sambrook, J., Fritsh, E. F. & Maniatis,. (1989) Moleular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY), 2nd Ed. 31. Lilley, D. M. J. & Dahlberg, J. E. (1992) Methods Enzymol. 212, Moser, H. E. & Dervan, P. B. (1987) Siene 238, Xodo, L. E., Manzini, G., Quadrifoglio, F., Vandermarel, G. A. & Vanboom, J. H. (1991) Nulei Aids Res. 19, Wang, S. & Kool, E.. (1994)J. Am. Chem. So. 116, Giovannangeli, C., Montenay-Garestier,., Rougee, M., Chassignol, M., huong, N.. & Helene, C. (1991) J. Am. Chem. So. 113, Mergny, J. L., Sun, J. S., Rougee, M., Montenay-Garestier,., Barelo, F., Chomilier, J. & H6lene, C. (1991) Biohemistry 30,