Selection of Structure-Specific Inhibitors of the HIV Rev-Rev Response Element Complex

We devised a single-step selection method to identify short sequences within the folded Rev response element (RRE) of the human immunodeficiency virus (HIV) RNA genome that are proximal and able to bind short oligonucleotides. The method employed a library of partially randomized tethered oligonucleotide probes (TOPs) complementary to all regions of the RRE and an RNase H cleavage assay which identified those RRE regions preferred by the TOPs. Six short sequences were identified. Two TOPS synthesized on the basis of this selection, S1-542 and S1-1-S2, were potent, concentration-dependent inhibitors of a RRE function in vitro and abolished interaction of the RRE with the HIV regulatory protein Rev at nanomolar concentration (25 OC, 0.8 nM RRE, 45 nM Rev). TOPs S 1 5 4 2 and S 11 -S2 exhibited greater potency and faster association kinetics than traditional oligonucleotides targeted to the same regions. The random TOP selection procedure described here allows rational design of oligonucleotidebased ligands for RNA that exploit (rather than avoid) the complex structure of the R N A target. The manipulation of sequence-specific protein binding and gene expression using antigene or antisense technology requires oligonucleotides that bind DNA or R N A with high affinity and specificity.I4 Oligonucleotides capable of selective recognition of RNA must accommodate, in addition, both the kine ti^^,^ and the thermodynamic697 consequences of a nonuniform RNA conformation. Although formally single-stranded, RNA molecules base-pair intramolecularly and the resultant secondary structures fold to generate tertiary architecture (“self-structures”) of greater complexity than that found in DNA.8-11 The structural complexity inherent in R N A complicates recognition because it limits the accessibility of single-stranded regions that must be differentiated for specific binding. It is estimated that 11-1 5 nucleotides are necessary to define a unique mRNA sequence in a eukaryotic cell,l2 yet few well-characterized R N A secondary structures contain contiguous single-stranded regions of this size. Secondary structures, moreover, do not reveal which singlestranded regions in a folded RNA are accessible to oligonucleotides.l3J4 Antisense oligonucleotides that must disrupt structure to pair with their complements, if they bind, often do so with slow association kinetics, or low affinities, or b ~ t h . ~ , ~ We have described a class of synthetic molecules that bind RNA on the basis of both sequence and structure, thereby accommodating the structural complexity of RNA.ISJ6 Tethered oligonucleotide probes (TOPs) consist of two short oligonucleotides joined by a tether whose length and composition are varied * To whom correspondence should be addressed. (1) Crooke, S. T. BiolTechnology 1992, 10, 882. (2) Helene, C.; Toulme, J. Biochim. Biophys. Acta 1990, 1049, 99. (3) Uhlmann, E.; Peyman, A. Chem. Reu. 1990, 90, 543. (4) Weintraub, H. M. Sci. Am. 1990, 40. ( 5 ) Lima, W. F.; Monia, B. P.; Ecker, D. J.; Freier, S. M. Biochemistry 1992, 31, 12055. (6) Cload, S. T.; Richardson, P. L.; Huang, Y.-H.; Schepartz, A. J . Am. Chem. SOC. 1993, 115, 5005. (7) Ecker, D. J.; Vickers, T. A.; Bruice, T. W.; Freier, S. M.; Jenison, R. D.; Manoharan, M.; Zounes, M. Science 1992, 257, 958. (8) Draper, D. E. Acc. Chem. Res. 1992, 25, 201. (9) Tinoco, I., Jr.; Puglisi, J. D.; Wyatt, J. R. Nucleic Acids Mol. Biol. 1990, 4, 205. (10) Tinoco, I., Jr.; Davis, P. W.; Hardin, C. C.; Puglisi, J. D.; Walker, G. T.; Wyatt, J. Cold Spring Harbor Symp. Quant. Biol. 1987, 52, 135. (11) Wyatt, J. R.; Puglisi, J. D.; Tinoco, I., Jr. BioEssuys 1989, 11 , 100. (12) Woolf, T. M.; Melton, D. A,; Jennings, C. G. B. Proc. Natl. Acad. Sci. U.S,A. 1992, 89, 7305. (13) Latham, J. A.; Cech, T. R. Science 1989, 245, 276. (14) Weller, J . W.; Hill, W. E. Biochemistry 1992, 31, 2748. Abstract published in Aduance ACS Abstracts, December 15, 1993. 0002-7863/94/ 1516-0437$04.50/0 using chemical synthesis (Figure 1 B). In contrast with traditional antisense oligonucleotides that recognize a single, contiguous RNA sequence, TOPs recognize two short, noncontiguous sequences that are proximal in the folded RNA.6J5.16 Because TOPs have the potential to bind simultaneously to two accessible sequences, rather than one long sequence that may not be fully accessible, we predicted that TOPs could exhibit very high affinities for structured RNA targets.6 We also predicted that TOPs might equilibrate more rapidly with their targets than oligonucleotides that must disrupt a stable conformation in order to bind. The human immunodeficiency virus (HIV) contains a t least two structured RNAs that interact withviral proteins and mediate novel genetic regulatory pathways. One such R N A is a 234 nucleotide sequence located within the env coding region called the Rev response element (RRE). Interaction of the RRE with the viral protein RevI7J8 regulates the appearance of unspliced or singly spliced viral mRNAs in the cytoplasm of infected ~ells.Ig-2~ Without these mRNAs, structural proteins do not accumulate and the virus cannot The primary binding region for Rev is located between RRE nucleotides G39 and C104 (Figure lA).20JOJ* The secondary structure proposed for the RRE in the absence of Rev is based on free energy (15) Richardson,P. L.; Schepartz,A.J. Am. Chem.Soc. 1991,113,5109. (16) Cload, S. T.; Schepartz, A. J . Am. Chem. SOC. 1991, 113, 6324. (17) Zapp, M. A,; Green, M. R. Nature (London) 1989, 342,714. (18) Daly, T. J.; Cook, K. S.; Gray, G. S.; Maione, T. E.; Rusche, J. R. (19) Rosen, C. A.; Terwilliger, E.; Dayton, A.; Sodroski, J. G.; Haseltine, (20) Malim, M. H.; Hauber, J.; Le, S. Y.; Maizel, J. V.; Cullen, B. R. (21) Felber, B. K.; Hadzopoulou-Cladaras, M.; Cladaras, C.; Copeland, (22) Emerman, M.; Vazeux, R.; Peden, K. Cell 1989, 57, 1155. (23) Dayton, E. T.; Powell, D. M.; Dayton, A. I. Science 1989,246, 1625. (24) Hammarskjold, M.-L.; Heimer, J.; Hammarskjold, B.; Sangwan, I.; (25) Chang, D. D.; Sharp, P. A. Cell 1989, 59, 789. (26) Lu, X.; Heimer, J.; Rekosh, D.; Hammarskjold, M.-L. Proc. Narl. (27) Hadzopoulou, C. M.; Felber, B. R.; Claderas, C.; Athanassaopoulos, (28) Sodroski, J.; Goh, W. C.; Rosen, C.; Dayton, A.; Terwilliger, E.; (29) Feinberg, M. B.; Jarrett, R. F.; Aldovini, A.; Gallo, R. C.; Wong(30) Malim, M. H.; Bohnlein, S.; Hauber, J.; Cullen, B. R. Cell 1989,58, Nature (London) 1989, 342, 816. W. A. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 2017. Nature (London) 1989, 338,254. T.; Pavlakis, G. N. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 1495. Albert, L.; Rekosh, D. J. Virol. 1989, 63, 1969. Acad. Sci. U.S.A. 1990, 87, 7598. A,; Tse, A,; Pavlakis, G. N. J . Virol. 1989, 63, 1265. Haseltine, W. Nature (London) 1986, 321, 412. Staal, F. Cell 1986, 46, 807.