Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1993 Dec 11;21(24):5616–5622. doi: 10.1093/nar/21.24.5616

The binding of an antisense oligonucleotide to a hairpin structure via triplex formation inhibits chemical and biological reactions.

E Brossalina 1, E Pascolo 1, J J Toulmé 1
PMCID: PMC310525  PMID: 8284206

Abstract

We have investigated the binding of a 26-mer antisense oligodeoxynucleotide to a 69-mer DNA hairpin with a 13 base pair stem, bearing an Rsa1 restriction site. The 5' part of the 26-mer annealed to a stretch of six purines at the bottom of the hairpin. The 3' part was designed to fold back to form a triplex with both the stem of the hairpin and with the sequence paired to its own 5' region. Using non-denaturing polyacrylamide gel electrophoresis, melting curves (Tm) and chemical footprinting, we were able to show the formation of a 'double-hairpin' complex between the 69-mer and the 26-mer antisense oligopyrimidines. The association was both sequence and pH-dependent. The formation of a double hairpin complex was shown to prevent the alkylation of the 69-mer DNA target by an oligonucleotide-nitrogen mustard conjugate and to selectively inhibit the action of Rsa1.

Full text

PDF
5616

Images in this article

5619Image
on p.5619
5620Image
on p.5620
5620Image
on p.5620
5621Image
on p.5621

Selected References

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

  1. Beal P. A., Dervan P. B. Second structural motif for recognition of DNA by oligonucleotide-directed triple-helix formation. Science. 1991 Mar 15;251(4999):1360–1363. doi: 10.1126/science.2003222. [DOI] [PubMed] [Google Scholar]
  2. Blake K. R., Murakami A., Spitz S. A., Glave S. A., Reddy M. P., Ts'o P. O., Miller P. S. Hybridization arrest of globin synthesis in rabbit reticulocyte lysates and cells by oligodeoxyribonucleoside methylphosphonates. Biochemistry. 1985 Oct 22;24(22):6139–6145. doi: 10.1021/bi00343a016. [DOI] [PubMed] [Google Scholar]
  3. Boiziau C., Blonski C., Thuong N. T., Shire D., Toulme J. J. Inhibition of reverse transcription by unmodified and modified antisense oligodeoxynucleotides. Nucleic Acids Symp Ser. 1991;(24):121–125. [PubMed] [Google Scholar]
  4. Bordier B., Hélène C., Barr P. J., Litvak S., Sarih-Cottin L. In vitro effect of antisense oligonucleotides on human immunodeficiency virus type 1 reverse transcription. Nucleic Acids Res. 1992 Nov 25;20(22):5999–6006. doi: 10.1093/nar/20.22.5999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cazenave C., Stein C. A., Loreau N., Thuong N. T., Neckers L. M., Subasinghe C., Hélène C., Cohen J. S., Toulmé J. J. Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodeoxynucleotides. Nucleic Acids Res. 1989 Jun 12;17(11):4255–4273. doi: 10.1093/nar/17.11.4255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. François J. C., Saison-Behmoaras T., Thuong N. T., Hélène C. Inhibition of restriction endonuclease cleavage via triple helix formation by homopyrimidine oligonucleotides. Biochemistry. 1989 Dec 12;28(25):9617–9619. doi: 10.1021/bi00451a011. [DOI] [PubMed] [Google Scholar]
  7. Gagnor C., Bertrand J. R., Thenet S., Lemaître M., Morvan F., Rayner B., Malvy C., Lebleu B., Imbach J. L., Paoletti C. alpha-DNA. VI: Comparative study of alpha- and beta-anomeric oligodeoxyribonucleotides in hybridization to mRNA and in cell free translation inhibition. Nucleic Acids Res. 1987 Dec 23;15(24):10419–10436. doi: 10.1093/nar/15.24.10419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Giovannangéli C., Rougée M., Garestier T., Thuong N. T., Hélène C. Triple-helix formation by oligonucleotides containing the three bases thymine, cytosine, and guanine. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8631–8635. doi: 10.1073/pnas.89.18.8631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grigoriev M., Praseuth D., Robin P., Hemar A., Saison-Behmoaras T., Dautry-Varsat A., Thuong N. T., Hélène C., Harel-Bellan A. A triple helix-forming oligonucleotide-intercalator conjugate acts as a transcriptional repressor via inhibition of NF kappa B binding to interleukin-2 receptor alpha-regulatory sequence. J Biol Chem. 1992 Feb 15;267(5):3389–3395. [PubMed] [Google Scholar]
  10. Hanvey J. C., Shimizu M., Wells R. D. Site-specific inhibition of EcoRI restriction/modification enzymes by a DNA triple helix. Nucleic Acids Res. 1990 Jan 11;18(1):157–161. doi: 10.1093/nar/18.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Heikkila R., Schwab G., Wickstrom E., Loke S. L., Pluznik D. H., Watt R., Neckers L. M. A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1. 1987 Jul 30-Aug 5Nature. 328(6129):445–449. doi: 10.1038/328445a0. [DOI] [PubMed] [Google Scholar]
  12. Krawczyk S. H., Milligan J. F., Wadwani S., Moulds C., Froehler B. C., Matteucci M. D. Oligonucleotide-mediated triple helix formation using an N3-protonated deoxycytidine analog exhibiting pH-independent binding within the physiological range. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3761–3764. doi: 10.1073/pnas.89.9.3761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kulka M., Smith C. C., Aurelian L., Fishelevich R., Meade K., Miller P., Ts'o P. O. Site specificity of the inhibitory effects of oligo(nucleoside methylphosphonate)s complementary to the acceptor splice junction of herpes simplex virus type 1 immediate early mRNA 4. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6868–6872. doi: 10.1073/pnas.86.18.6868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Le Doan T., Perrouault L., Praseuth D., Habhoub N., Decout J. L., Thuong N. T., Lhomme J., Hélène C. Sequence-specific recognition, photocrosslinking and cleavage of the DNA double helix by an oligo-[alpha]-thymidylate covalently linked to an azidoproflavine derivative. Nucleic Acids Res. 1987 Oct 12;15(19):7749–7760. doi: 10.1093/nar/15.19.7749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Loreau N., Boiziau C., Verspieren P., Shire D., Toulmé J. J. Blockage of AMV reverse transcriptase by antisense oligodeoxynucleotides. FEBS Lett. 1990 Nov 12;274(1-2):53–56. doi: 10.1016/0014-5793(90)81327-k. [DOI] [PubMed] [Google Scholar]
  16. Maher L. J., 3rd, Wold B., Dervan P. B. Inhibition of DNA binding proteins by oligonucleotide-directed triple helix formation. Science. 1989 Aug 18;245(4919):725–730. doi: 10.1126/science.2549631. [DOI] [PubMed] [Google Scholar]
  17. Manzini G., Xodo L. E., Gasparotto D., Quadrifoglio F., van der Marel G. A., van Boom J. H. Triple helix formation by oligopurine-oligopyrimidine DNA fragments. Electrophoretic and thermodynamic behavior. J Mol Biol. 1990 Jun 20;213(4):833–843. doi: 10.1016/S0022-2836(05)80267-0. [DOI] [PubMed] [Google Scholar]
  18. Moser H. E., Dervan P. B. Sequence-specific cleavage of double helical DNA by triple helix formation. Science. 1987 Oct 30;238(4827):645–650. doi: 10.1126/science.3118463. [DOI] [PubMed] [Google Scholar]
  19. Postel E. H., Flint S. J., Kessler D. J., Hogan M. E. Evidence that a triplex-forming oligodeoxyribonucleotide binds to the c-myc promoter in HeLa cells, thereby reducing c-myc mRNA levels. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8227–8231. doi: 10.1073/pnas.88.18.8227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Roberts R. W., Crothers D. M. Stability and properties of double and triple helices: dramatic effects of RNA or DNA backbone composition. Science. 1992 Nov 27;258(5087):1463–1466. doi: 10.1126/science.1279808. [DOI] [PubMed] [Google Scholar]
  21. Verspieren P., Loreau N., Thuong N. T., Shire D., Toulmé J. J. Effect of RNA secondary structure and modified bases on the inhibition of trypanosomatid protein synthesis in cell free extracts by antisense oligodeoxynucleotides. Nucleic Acids Res. 1990 Aug 25;18(16):4711–4717. doi: 10.1093/nar/18.16.4711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wells R. D., Collier D. A., Hanvey J. C., Shimizu M., Wohlrab F. The chemistry and biology of unusual DNA structures adopted by oligopurine.oligopyrimidine sequences. FASEB J. 1988 Nov;2(14):2939–2949. [PubMed] [Google Scholar]
  23. Xodo L. E., Manzini G., Quadrifoglio F., van der Marel G. A., van Boom J. H. Effect of 5-methylcytosine on the stability of triple-stranded DNA--a thermodynamic study. Nucleic Acids Res. 1991 Oct 25;19(20):5625–5631. doi: 10.1093/nar/19.20.5625. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES