Skip to main content
PMC home page
RNA logoLink to RNA
. 1999 Dec;5(12):1605–1614. doi: 10.1017/s1355838299991318

In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1.

F Ducongé 1, J J Toulmé 1
PMCID: PMC1369882  PMID: 10606271

Abstract

We selected RNA aptamers specific for the trans-activation responsive (TAR) RNA, a stem-loop structure crucial for the transcription of the integrated genome of the human immunodeficiency virus. Most of the selected sequences could be folded as imperfect hairpins and displayed a 5'-GUCCCAGA-3' consensus motif constituting the apical loop. The six central bases of this consensus sequence are complementary to the entire TAR loop, leading to the formation of TAR RNA-aptamer "kissing" complexes. The consensus G and A residues closing the aptamer loop contributed to the high affinity (Kd = 30 nM at 23 degrees C) of the aptamers for the TAR RNA. This G A pair was shown to be crucial for binding to TAR at a low magnesium concentration. The selection also identified 5'-PuPy and 5'-PyPu base pairs at alpha and beta positions of the stem, next to the loop, respectively. This strategy offered a way to identify key determinants of loop-loop interactions and to generate high affinity ligands of TAR RNA structure.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bartel D. P., Szostak J. W. Isolation of new ribozymes from a large pool of random sequences [see comment]. Science. 1993 Sep 10;261(5127):1411–1418. doi: 10.1126/science.7690155. [DOI] [PubMed] [Google Scholar]
  2. Blommers M. J., Pieles U., De Mesmaeker A. An approach to the structure determination of nucleic acid analogues hybridized to RNA. NMR studies of a duplex between 2'-OMe RNA and an oligonucleotide containing a single amide backbone modification. Nucleic Acids Res. 1994 Oct 11;22(20):4187–4194. doi: 10.1093/nar/22.20.4187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bohjanen P. R., Liu Y., Garcia-Blanco M. A. TAR RNA decoys inhibit tat-activated HIV-1 transcription after preinitiation complex formation. Nucleic Acids Res. 1997 Nov 15;25(22):4481–4486. doi: 10.1093/nar/25.22.4481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boiziau C., Dausse E., Mishra R., Ducongé F., Toulmé J. J. Identification of aptamers against the DNA template for in vitro transcription of the HIV-1 TAR element. Antisense Nucleic Acid Drug Dev. 1997 Aug;7(4):369–380. doi: 10.1089/oli.1.1997.7.369. [DOI] [PubMed] [Google Scholar]
  5. Boiziau C., Dausse E., Yurchenko L., Toulmé J. J. DNA aptamers selected against the HIV-1 trans-activation-responsive RNA element form RNA-DNA kissing complexes. J Biol Chem. 1999 Apr 30;274(18):12730–12737. doi: 10.1074/jbc.274.18.12730. [DOI] [PubMed] [Google Scholar]
  6. Brierley I., Digard P., Inglis S. C. Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot. Cell. 1989 May 19;57(4):537–547. doi: 10.1016/0092-8674(89)90124-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cate J. H., Gooding A. R., Podell E., Zhou K., Golden B. L., Szewczak A. A., Kundrot C. E., Cech T. R., Doudna J. A. RNA tertiary structure mediation by adenosine platforms. Science. 1996 Sep 20;273(5282):1696–1699. doi: 10.1126/science.273.5282.1696. [DOI] [PubMed] [Google Scholar]
  8. Chang K. Y., Tinoco I., Jr Characterization of a "kissing" hairpin complex derived from the human immunodeficiency virus genome. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8705–8709. doi: 10.1073/pnas.91.18.8705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang K. Y., Tinoco I., Jr The structure of an RNA "kissing" hairpin complex of the HIV TAR hairpin loop and its complement. J Mol Biol. 1997 May 30;269(1):52–66. doi: 10.1006/jmbi.1997.1021. [DOI] [PubMed] [Google Scholar]
  10. Colvin R. A., Garcia-Blanco M. A. Unusual structure of the human immunodeficiency virus type 1 trans-activation response element. J Virol. 1992 Feb;66(2):930–935. doi: 10.1128/jvi.66.2.930-935.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dassonneville L., Hamy F., Colson P., Houssier C., Bailly C. Binding of Hoechst 33258 to the TAR RNA of HIV-1. Recognition of a pyrimidine bulge-dependent structure. Nucleic Acids Res. 1997 Nov 15;25(22):4487–4492. doi: 10.1093/nar/25.22.4487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Davanloo P., Rosenberg A. H., Dunn J. J., Studier F. W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2035–2039. doi: 10.1073/pnas.81.7.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ecker D. J., Vickers T. A., Bruice T. W., Freier S. M., Jenison R. D., Manoharan M., Zounes M. Pseudo--half-knot formation with RNA. Science. 1992 Aug 14;257(5072):958–961. doi: 10.1126/science.1502560. [DOI] [PubMed] [Google Scholar]
  14. Eguchi Y., Tomizawa J. Complex formed by complementary RNA stem-loops and its stabilization by a protein: function of CoIE1 Rom protein. Cell. 1990 Jan 26;60(2):199–209. doi: 10.1016/0092-8674(90)90736-x. [DOI] [PubMed] [Google Scholar]
  15. Eguchi Y., Tomizawa J. Complexes formed by complementary RNA stem-loops. Their formations, structures and interaction with ColE1 Rom protein. J Mol Biol. 1991 Aug 20;220(4):831–842. doi: 10.1016/0022-2836(91)90356-b. [DOI] [PubMed] [Google Scholar]
  16. Ellington A. D., Conrad R. Aptamers as potential nucleic acid pharmaceuticals. Biotechnol Annu Rev. 1995;1:185–214. doi: 10.1016/s1387-2656(08)70052-8. [DOI] [PubMed] [Google Scholar]
  17. Gait M. J., Karn J. RNA recognition by the human immunodeficiency virus Tat and Rev proteins. Trends Biochem Sci. 1993 Jul;18(7):255–259. doi: 10.1016/0968-0004(93)90176-n. [DOI] [PubMed] [Google Scholar]
  18. Gold L., Polisky B., Uhlenbeck O., Yarus M. Diversity of oligonucleotide functions. Annu Rev Biochem. 1995;64:763–797. doi: 10.1146/annurev.bi.64.070195.003555. [DOI] [PubMed] [Google Scholar]
  19. Gregorian R. S., Jr, Crothers D. M. Determinants of RNA hairpin loop-loop complex stability. J Mol Biol. 1995 May 19;248(5):968–984. doi: 10.1006/jmbi.1995.0275. [DOI] [PubMed] [Google Scholar]
  20. Grosjean H., Söll D. G., Crothers D. M. Studies of the complex between transfer RNAs with complementary anticodons. I. Origins of enhanced affinity between complementary triplets. J Mol Biol. 1976 May 25;103(3):499–519. doi: 10.1016/0022-2836(76)90214-x. [DOI] [PubMed] [Google Scholar]
  21. Haasnoot C. A., Hilbers C. W., van der Marel G. A., van Boom J. H., Singh U. C., Pattabiraman N., Kollman P. A. On loop folding in nucleic acid hairpin-type structures. J Biomol Struct Dyn. 1986 Apr;3(5):843–857. doi: 10.1080/07391102.1986.10508468. [DOI] [PubMed] [Google Scholar]
  22. Hamy F., Felder E. R., Heizmann G., Lazdins J., Aboul-ela F., Varani G., Karn J., Klimkait T. An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3548–3553. doi: 10.1073/pnas.94.8.3548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Harper J. W., Logsdon N. J. Refolded HIV-1 tat protein protects both bulge and loop nucleotides in TAR RNA from ribonucleolytic cleavage. Biochemistry. 1991 Aug 13;30(32):8060–8066. doi: 10.1021/bi00246a026. [DOI] [PubMed] [Google Scholar]
  24. Jaeger J. A., Tinoco I., Jr An NMR study of the HIV-1 TAR element hairpin. Biochemistry. 1993 Nov 23;32(46):12522–12530. doi: 10.1021/bi00097a032. [DOI] [PubMed] [Google Scholar]
  25. Le Tinévez R., Mishra R. K., Toulmé J. J. Selective inhibition of cell-free translation by oligonucleotides targeted to a mRNA hairpin structure. Nucleic Acids Res. 1998 May 15;26(10):2273–2278. doi: 10.1093/nar/26.10.2273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lee A. J., Crothers D. M. The solution structure of an RNA loop-loop complex: the ColE1 inverted loop sequence. Structure. 1998 Aug 15;6(8):993–1005. doi: 10.1016/s0969-2126(98)00101-4. [DOI] [PubMed] [Google Scholar]
  27. Marino J. P., Gregorian R. S., Jr, Csankovszki G., Crothers D. M. Bent helix formation between RNA hairpins with complementary loops. Science. 1995 Jun 9;268(5216):1448–1454. doi: 10.1126/science.7539549. [DOI] [PubMed] [Google Scholar]
  28. Mhashilkar A. M., Biswas D. K., LaVecchio J., Pardee A. B., Marasco W. A. Inhibition of human immunodeficiency virus type 1 replication in vitro by a novel combination of anti-Tat single-chain intrabodies and NF-kappa B antagonists. J Virol. 1997 Sep;71(9):6486–6494. doi: 10.1128/jvi.71.9.6486-6494.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mishra R. K., Le Tinévez R., Toulmé J. J. Targeting nucleic acid secondary structures by antisense oligonucleotides designed through in vitro selection. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10679–10684. doi: 10.1073/pnas.93.20.10679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mishra R. K., Toulmé J. J. In vitro selection of antisense oligonucleotides targeted to a hairpin structure. C R Acad Sci III. 1994 Nov;317(11):977–982. [PubMed] [Google Scholar]
  32. Muriaux D., Fossé P., Paoletti J. A kissing complex together with a stable dimer is involved in the HIV-1Lai RNA dimerization process in vitro. Biochemistry. 1996 Apr 16;35(15):5075–5082. doi: 10.1021/bi952822s. [DOI] [PubMed] [Google Scholar]
  33. Paillart J. C., Westhof E., Ehresmann C., Ehresmann B., Marquet R. Non-canonical interactions in a kissing loop complex: the dimerization initiation site of HIV-1 genomic RNA. J Mol Biol. 1997 Jul 4;270(1):36–49. doi: 10.1006/jmbi.1997.1096. [DOI] [PubMed] [Google Scholar]
  34. SantaLucia J., Jr, Kierzek R., Turner D. H. Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs. Biochemistry. 1991 Aug 20;30(33):8242–8251. doi: 10.1021/bi00247a021. [DOI] [PubMed] [Google Scholar]
  35. Serra M. J., Lyttle M. H., Axenson T. J., Schadt C. A., Turner D. H. RNA hairpin loop stability depends on closing base pair. Nucleic Acids Res. 1993 Aug 11;21(16):3845–3849. doi: 10.1093/nar/21.16.3845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Skripkin E., Paillart J. C., Marquet R., Ehresmann B., Ehresmann C. Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4945–4949. doi: 10.1073/pnas.91.11.4945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sullenger B. A., Gallardo H. F., Ungers G. E., Gilboa E. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell. 1990 Nov 2;63(3):601–608. doi: 10.1016/0092-8674(90)90455-n. [DOI] [PubMed] [Google Scholar]
  38. Tomizawa J. Control of ColE1 plasmid replication. Intermediates in the binding of RNA I and RNA II. J Mol Biol. 1990 Apr 20;212(4):683–694. doi: 10.1016/0022-2836(90)90230-j. [DOI] [PubMed] [Google Scholar]
  39. Wagner R. W., Matteucci M. D., Grant D., Huang T., Froehler B. C. Potent and selective inhibition of gene expression by an antisense heptanucleotide. Nat Biotechnol. 1996 Jul;14(7):840–844. doi: 10.1038/nbt0796-840. [DOI] [PubMed] [Google Scholar]
  40. Walczak R., Carbon P., Krol A. An essential non-Watson-Crick base pair motif in 3'UTR to mediate selenoprotein translation. RNA. 1998 Jan;4(1):74–84. [PMC free article] [PubMed] [Google Scholar]
  41. Wu-Baer F., Lane W. S., Gaynor R. B. The cellular factor TRP-185 regulates RNA polymerase II binding to HIV-1 TAR RNA. EMBO J. 1995 Dec 1;14(23):5995–6009. doi: 10.1002/j.1460-2075.1995.tb00288.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zhou Q., Sharp P. A. Tat-SF1: cofactor for stimulation of transcriptional elongation by HIV-1 Tat. Science. 1996 Oct 25;274(5287):605–610. doi: 10.1126/science.274.5287.605. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES