Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Oct 1;24(19):3858–3865. doi: 10.1093/nar/24.19.3858

A new approach to overcome potassium-mediated inhibition of triplex formation.

F Svinarchuk 1, D Cherny 1, A Debin 1, E Delain 1, C Malvy 1
PMCID: PMC146176  PMID: 8871568

Abstract

G,A-containing purine oligonucleotides of various lengths form extremely stable and specific triplexes with the purine-pyrimidine stretch of the vpx gene [Svinarchuk,F., Monnot,M., Merle,A., Malvy,C. and Fermandjian,S. (1995) Nucleic Acids Res., 22, 3742--3747]. The potential application of triple-helix-forming oligonucleotides (TFO) in gene-targeted therapy has prompted us to study triplex formation mimicking potassium concentrations and temperatures in cells. Triplex formation was tested by dimethyl sulphate (DMS) footprinting, gel-retardation, UV melting studies and electron microscopy. In the presence of 10 mM MgCl2, KCl concentrations up to 150 mM significantly lowered both efficiency (triplex : initial duplex) and rate constants of triplex formation. The KCl effect was more pronounced for 11mer and 20mer TFOs than for 14mer TFO. Since the dissociation half-life for the 11mer TFO decreases from 420 min in the absence of monovalent cations to 40 min in the presence of 150 mM KCI, we suggest that the negative effect could be explained by a decrease in triplex stability. In contrast, for the 20mer TFO no dissociation of the triplex was observed during 24 h of incubation either in the absence of monovalent cations or in the presence of 150 mM KCl. We suppose that in the case of the 20mer TFO the negative effect of KCI on triplex formation is probably due to the self-association of the oligonucleotide in competitive structures such as parallel duplexes and/or tetraplexes. This negative effect may be overcome by the prior formation of a short duplex either on the 3'- or 5'-end of the 20mer TFO. We refer to these partial duplexes as 'zipper' TFOs. It was demonstrated that a 'zipper' TFO can form a triplex over the full length of the target, thus unzipping the short complementary strand. The minimal single-stranded part of the 'zipper' oligonucleotide which is sufficient to initiate triplex formation can be as short as three nucleotides at the 3'-end and six nucleotides at the 5'-end. We suggest that this type of structure may prove useful for in vivo applications.

Full Text

The Full Text of this article is available as a PDF (175.7 KB).

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. Behe M. J. An overabundance of long oligopurine tracts occurs in the genome of simple and complex eukaryotes. Nucleic Acids Res. 1995 Feb 25;23(4):689–695. doi: 10.1093/nar/23.4.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cheng A. J., Van Dyke M. W. Monovalent cation effects on intermolecular purine-purine-pyrimidine triple-helix formation. Nucleic Acids Res. 1993 Dec 11;21(24):5630–5635. doi: 10.1093/nar/21.24.5630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cherney D. I., Kurakin A. V., Lyamichev V. I., Frank-Kamenetskii M. D., Zinkevich V. E., Firman K., Nielsen P. E. Electron microscopic studies of sequence-specific recognition of duplex DNA by different ligands. J Mol Recognit. 1994 Sep;7(3):171–176. doi: 10.1002/jmr.300070304. [DOI] [PubMed] [Google Scholar]
  5. Cherny D. I., Malkov V. A., Volodin A. A., Frank-Kamenetskii M. D. Electron microscopy visualization of oligonucleotide binding to duplex DNA via triplex formation. J Mol Biol. 1993 Mar 20;230(2):379–383. doi: 10.1006/jmbi.1993.1154. [DOI] [PubMed] [Google Scholar]
  6. Demidov V. V., Yavnilovich M. V., Belotserkovskii B. P., Frank-Kamenetskii M. D., Nielsen P. E. Kinetics and mechanism of polyamide ("peptide") nucleic acid binding to duplex DNA. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2637–2641. doi: 10.1073/pnas.92.7.2637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dubochet J., Ducommun M., Zollinger M., Kellenberger E. A new preparation method for dark-field electron microscopy of biomacromolecules. J Ultrastruct Res. 1971 Apr;35(1):147–167. doi: 10.1016/s0022-5320(71)80148-x. [DOI] [PubMed] [Google Scholar]
  8. Gee J. E., Revankar G. R., Rao T. S., Hogan M. E. Triplex formation at the rat neu gene utilizing imidazole and 2'-deoxy-6-thioguanosine base substitutions. Biochemistry. 1995 Feb 14;34(6):2042–2048. doi: 10.1021/bi00006a026. [DOI] [PubMed] [Google Scholar]
  9. Grigoriev M., Praseuth D., Guieysse A. L., Robin P., Thuong N. T., Hélène C., Harel-Bellan A. Inhibition of gene expression by triple helix-directed DNA cross-linking at specific sites. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3501–3505. doi: 10.1073/pnas.90.8.3501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Noonberg S. B., François J. C., Garestier T., Hélène C. Effect of competing self-structure on triplex formation with purine-rich oligodeoxynucleotides containing GA repeats. Nucleic Acids Res. 1995 Jun 11;23(11):1956–1963. doi: 10.1093/nar/23.11.1956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. O'Neill D., Bornschlegel K., Flamm M., Castle M., Bank A. A DNA-binding factor in adult hematopoietic cells interacts with a pyrimidine-rich domain upstream from the human delta-globin gene. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8953–8957. doi: 10.1073/pnas.88.20.8953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Olivas W. M., Maher L. J., 3rd Competitive triplex/quadruplex equilibria involving guanine-rich oligonucleotides. Biochemistry. 1995 Jan 10;34(1):278–284. doi: 10.1021/bi00001a034. [DOI] [PubMed] [Google Scholar]
  14. Olivas W. M., Maher L. J., 3rd Overcoming potassium-mediated triplex inhibition. Nucleic Acids Res. 1995 Jun 11;23(11):1936–1941. doi: 10.1093/nar/23.11.1936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Puglisi J. D., Tinoco I., Jr Absorbance melting curves of RNA. Methods Enzymol. 1989;180:304–325. doi: 10.1016/0076-6879(89)80108-9. [DOI] [PubMed] [Google Scholar]
  16. Suda T., Mishima Y., Asakura H., Kominami R. Formation of a parallel-stranded DNA homoduplex by d(GGA) repeat oligonucleotides. Nucleic Acids Res. 1995 Sep 25;23(18):3771–3777. doi: 10.1093/nar/23.18.3771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Svinarchuk F., Bertrand J. R., Malvy C. A short purine oligonucleotide forms a highly stable triple helix with the promoter of the murine c-pim-1 proto-oncogene. Nucleic Acids Res. 1994 Sep 11;22(18):3742–3747. doi: 10.1093/nar/22.18.3742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Svinarchuk F., Debin A., Bertrand J. R., Malvy C. Investigation of the intracellular stability and formation of a triple helix formed with a short purine oligonucleotide targeted to the murine c-pim-1 proto-oncogene promotor. Nucleic Acids Res. 1996 Jan 15;24(2):295–302. doi: 10.1093/nar/24.2.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Svinarchuk F., Monnot M., Merle A., Malvy C., Fermandjian S. The high stability of the triple helices formed between short purine oligonucleotides and SIV/HIV-2 vpx genes is determined by the targeted DNA structure. Nucleic Acids Res. 1995 Oct 11;23(19):3831–3836. doi: 10.1093/nar/23.19.3831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Svinarchuk F., Paoletti J., Malvy C. An unusually stable purine(purine-pyrimidine) short triplex. The third strand stabilizes double-stranded DNA. J Biol Chem. 1995 Jun 9;270(23):14068–14071. doi: 10.1074/jbc.270.23.14068. [DOI] [PubMed] [Google Scholar]
  21. Voloshin O. N., Mirkin S. M., Lyamichev V. I., Belotserkovskii B. P., Frank-Kamenetskii M. D. Chemical probing of homopurine-homopyrimidine mirror repeats in supercoiled DNA. Nature. 1988 Jun 2;333(6172):475–476. doi: 10.1038/333475a0. [DOI] [PubMed] [Google Scholar]
  22. Wang G., Seidman M. M., Glazer P. M. Mutagenesis in mammalian cells induced by triple helix formation and transcription-coupled repair. Science. 1996 Feb 9;271(5250):802–805. doi: 10.1126/science.271.5250.802. [DOI] [PubMed] [Google Scholar]

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

RESOURCES