Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1995 Sep 11;23(17):3578–3584. doi: 10.1093/nar/23.17.3578

Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides.

E R Kandimalla 1, A Manning 1, C Lathan 1, R A Byrn 1, S Agrawal 1
PMCID: PMC307240  PMID: 7567472

Abstract

Short oligonucleotides that can bind to adjacent sites on target mRNA sequences are designed and evaluated for their binding affinity and biological activity. Sequence-specific binding of short tandem oligonucleotides is compared with a full-length single oligonucleotide (21mer) that binds to the same target sequence. Two short oligonucleotides that bind without a base separation between their binding sites on the target bind cooperatively, while oligonucleotides that have a one or two base separation between the binding oligonucleotides do not. The binding affinity of the tandem oligonucleotides is improved by extending the ends of the two oligonucleotides with complementary sequences. These extended sequences form a duplex stem when both oligonucleotides bind to the target, resulting in a stable ternary complex. RNase H studies reveal that the cooperative oligonucleotides bind to the target RNA with sequence specificity. A short oligonucleotide (9mer) with one or two mismatches does not bind at the intended site, while longer oligonucleotides (21mers) with one or two mismatches still bind to the same site, as does a perfectly matched 21mer, and evoke RNase H activity. HIV-1 inhibition studies reveal an increase in activity of the cooperative oligonucleotide combinations as the length of the dimerization domain increases.

Full text

PDF
3578

Images in this article

3582Image
on p.3582
3583Image
on p.3583

Selected References

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

  1. Agrawal S., Tang J. Y. GEM 91--an antisense oligonucleotide phosphorothioate as a therapeutic agent for AIDS. Antisense Res Dev. 1992 Winter;2(4):261–266. doi: 10.1089/ard.1992.2.261. [DOI] [PubMed] [Google Scholar]
  2. Asseline U., Delarue M., Lancelot G., Toulmé F., Thuong N. T., Montenay-Garestier T., Hélène C. Nucleic acid-binding molecules with high affinity and base sequence specificity: intercalating agents covalently linked to oligodeoxynucleotides. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3297–3301. doi: 10.1073/pnas.81.11.3297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Awang G., Sen D. Mode of dimerization of HIV-1 genomic RNA. Biochemistry. 1993 Oct 26;32(42):11453–11457. doi: 10.1021/bi00093a024. [DOI] [PubMed] [Google Scholar]
  4. Baudin F., Marquet R., Isel C., Darlix J. L., Ehresmann B., Ehresmann C. Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains. J Mol Biol. 1993 Jan 20;229(2):382–397. doi: 10.1006/jmbi.1993.1041. [DOI] [PubMed] [Google Scholar]
  5. Bayever E., Iversen P. L., Bishop M. R., Sharp J. G., Tewary H. K., Arneson M. A., Pirruccello S. J., Ruddon R. W., Kessinger A., Zon G. Systemic administration of a phosphorothioate oligonucleotide with a sequence complementary to p53 for acute myelogenous leukemia and myelodysplastic syndrome: initial results of a phase I trial. Antisense Res Dev. 1993 Winter;3(4):383–390. doi: 10.1089/ard.1993.3.383. [DOI] [PubMed] [Google Scholar]
  6. Cedergren R., Grosjean H. RNA design by in vitro RNA recombination and synthesis. Biochem Cell Biol. 1987 Aug;65(8):677–692. doi: 10.1139/o87-090. [DOI] [PubMed] [Google Scholar]
  7. Clever J., Sassetti C., Parslow T. G. RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1. J Virol. 1995 Apr;69(4):2101–2109. doi: 10.1128/jvi.69.4.2101-2109.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crooke S. T., Grillone L. R., Tendolkar A., Garrett A., Fratkin M. J., Leeds J., Barr W. H. A pharmacokinetic evaluation of 14C-labeled afovirsen sodium in patients with genital warts. Clin Pharmacol Ther. 1994 Dec;56(6 Pt 1):641–646. doi: 10.1038/clpt.1994.189. [DOI] [PubMed] [Google Scholar]
  9. Crum C., Johnson J. D., Nelson A., Roth D. Complementary oligodeoxynucleotide mediated inhibition of tobacco mosaic virus RNA translation in vitro. Nucleic Acids Res. 1988 May 25;16(10):4569–4581. doi: 10.1093/nar/16.10.4569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Galbraith W. M., Hobson W. C., Giclas P. C., Schechter P. J., Agrawal S. Complement activation and hemodynamic changes following intravenous administration of phosphorothioate oligonucleotides in the monkey. Antisense Res Dev. 1994 Fall;4(3):201–206. doi: 10.1089/ard.1994.4.201. [DOI] [PubMed] [Google Scholar]
  11. Gazdar A. F., Carney D. N., Bunn P. A., Russell E. K., Jaffe E. S., Schechter G. P., Guccion J. G. Mitogen requirements for the in vitro propagation of cutaneous T-cell lymphomas. Blood. 1980 Mar;55(3):409–417. [PubMed] [Google Scholar]
  12. Gryaznov S. M., Lloyd D. H. Modulation of oligonucleotide duplex and triplex stability via hydrophobic interactions. Nucleic Acids Res. 1993 Dec 25;21(25):5909–5915. doi: 10.1093/nar/21.25.5909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. HERSKOVITS T. T. Nonaqueous solutions of DNA; denaturation by urea and its methyl derivatives. Biochemistry. 1963 Mar-Apr;2:335–340. doi: 10.1021/bi00902a027. [DOI] [PubMed] [Google Scholar]
  14. Harada S., Koyanagi Y., Yamamoto N. Infection of HTLV-III/LAV in HTLV-I-carrying cells MT-2 and MT-4 and application in a plaque assay. Science. 1985 Aug 9;229(4713):563–566. doi: 10.1126/science.2992081. [DOI] [PubMed] [Google Scholar]
  15. Herschlag D. Implications of ribozyme kinetics for targeting the cleavage of specific RNA molecules in vivo: more isn't always better. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6921–6925. doi: 10.1073/pnas.88.16.6921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Krämer A., Keller W., Appel B., Lührmann R. The 5' terminus of the RNA moiety of U1 small nuclear ribonucleoprotein particles is required for the splicing of messenger RNA precursors. Cell. 1984 Aug;38(1):299–307. doi: 10.1016/0092-8674(84)90551-8. [DOI] [PubMed] [Google Scholar]
  17. Maher L. J., 3rd, Dolnick B. J. Comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxyribonucleoside methylphosphonates in a cell-free system. Nucleic Acids Res. 1988 Apr 25;16(8):3341–3358. doi: 10.1093/nar/16.8.3341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maher L. J., 3rd, Dolnick B. J. Specific hybridization arrest of dihydrofolate reductase mRNA in vitro using anti-sense RNA or anti-sense oligonucleotides. Arch Biochem Biophys. 1987 Feb 15;253(1):214–220. doi: 10.1016/0003-9861(87)90654-0. [DOI] [PubMed] [Google Scholar]
  19. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  20. Padmapriya A. A., Tang J., Agrawal S. Large-scale synthesis, purification, and analysis of oligodeoxynucleotide phosphorothioates. Antisense Res Dev. 1994 Fall;4(3):185–199. doi: 10.1089/ard.1994.4.185. [DOI] [PubMed] [Google Scholar]
  21. Pauwels R., Balzarini J., Baba M., Snoeck R., Schols D., Herdewijn P., Desmyter J., De Clercq E. Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. J Virol Methods. 1988 Aug;20(4):309–321. doi: 10.1016/0166-0934(88)90134-6. [DOI] [PubMed] [Google Scholar]
  22. Popovic M., Sarngadharan M. G., Read E., Gallo R. C. Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science. 1984 May 4;224(4648):497–500. doi: 10.1126/science.6200935. [DOI] [PubMed] [Google Scholar]
  23. Posner M. R., Hideshima T., Cannon T., Mukherjee M., Mayer K. H., Byrn R. A. An IgG human monoclonal antibody that reacts with HIV-1/GP120, inhibits virus binding to cells, and neutralizes infection. J Immunol. 1991 Jun 15;146(12):4325–4332. [PubMed] [Google Scholar]
  24. Stein C. A., Cheng Y. C. Antisense oligonucleotides as therapeutic agents--is the bullet really magical? Science. 1993 Aug 20;261(5124):1004–1012. doi: 10.1126/science.8351515. [DOI] [PubMed] [Google Scholar]
  25. Stephenson M. L., Zamecnik P. C. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc Natl Acad Sci U S A. 1978 Jan;75(1):285–288. doi: 10.1073/pnas.75.1.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sundquist W. I., Heaphy S. Evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic RNA. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3393–3397. doi: 10.1073/pnas.90.8.3393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Vujcic L. K., Shepp D. H., Klutch M., Wells M. A., Hendry R. M., Wittek A. E., Krilov L., Quinnan G. V., Jr Use of a sensitive neutralization assay to measure the prevalence of antibodies to the human immunodeficiency virus. J Infect Dis. 1988 May;157(5):1047–1050. doi: 10.1093/infdis/157.5.1047. [DOI] [PubMed] [Google Scholar]
  28. Woolf T. M., Melton D. A., Jennings C. G. Specificity of antisense oligonucleotides in vivo. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7305–7309. doi: 10.1073/pnas.89.16.7305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Young S., Wagner R. W. Hybridization and dissociation rates of phosphodiester or modified oligodeoxynucleotides with RNA at near-physiological conditions. Nucleic Acids Res. 1991 May 11;19(9):2463–2470. doi: 10.1093/nar/19.9.2463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zhang R., Yan J., Shahinian H. K., Shahinian H., Amin G., Lu Z., Liu T., Saag M. S., Jiang Z., Temsamani J. Pharmacokinetics of an anti-human immunodeficiency virus antisense oligodeoxynucleotide phosphorothioate (GEM 91) in HIV-infected subjects. Clin Pharmacol Ther. 1995 Jul;58(1):44–53. doi: 10.1016/0009-9236(95)90071-3. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES