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. 1996 Apr 1;24(7):1229–1237. doi: 10.1093/nar/24.7.1229

Triplex formation at physiological pH by 5-Me-dC-N4-(spermine) [X] oligodeoxynucleotides: non protonation of N3 in X of X*G:C triad and effect of base mismatch/ionic strength on triplex stabilities.

D A Barawkar 1, K G Rajeev 1, V A Kumar 1, K N Ganesh 1
PMCID: PMC145769  PMID: 8614624

Abstract

Oligodeoxynucleotide (ODN) directed triplex formation has therapeutic importance and depends on Hoogsteen hydrogen bonds between a duplex DNA and a third DNA strand. T*A:T triplets are formed at neutral pH and C+*G:C are favoured at acidic pH. It is demonstrated that spermine conjugation at N4 of 5-Me-dC in ODNs 1-5 (sp-ODNs) imparts zwitterionic character, thus reducing the net negative charge of ODNs 1-5. sp-ODNs form triplexes with complementary 24mer duplex 8:9 show foremost stability at neutral pH 7.3 and decrease in stability towards lower pH, unlike the normal ODNs where optimal stability is found at an acidic pH 5.5. At pH 7.3, control ODNs 6 and 7 carrying dC or 5-Me-dC, respectively, do not show any triple helix formation. The stability order of triplex containing 5-Me-dC-N4-(spermine) with normal and mismatched duplex was found to be X*G:C approximately X*A:T > X*C:G > X*T:A. The hysteresis curve of sp-ODN triplex 3*8:9 indicated a better association with complementary duplex 8:9 as compared to unmodified ODN 6 in triplex 6*8:9. pH-dependent UV difference spectra suggest that N3 protonation is not a requirement for triplex formation by sp-ODN and interstrand interaction of conjugated spermine more than compensates for loss in stability due to absence of a single Hoogsteen hydrogen bond. These results may have importance in designing oligonucleotides for antigene applications.

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Selected References

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  1. Barawkar D. A., Kumar V. A., Ganesh K. N. Triplex formation at physiological pH by oligonucleotides incorporating 5-Me-dC-(N4-spermine). Biochem Biophys Res Commun. 1994 Dec 30;205(3):1665–1670. doi: 10.1006/bbrc.1994.2859. [DOI] [PubMed] [Google Scholar]
  2. Bergeron R. J., Weimar W. R., Wu Q., Austin J. K., Jr, McManis J. S. Impact of polyamine analogues on the NMDA receptor. J Med Chem. 1995 Feb 3;38(3):425–428. doi: 10.1021/jm00003a004. [DOI] [PubMed] [Google Scholar]
  3. Colige A., Sokolov B. P., Nugent P., Baserga R., Prockop D. J. Use of an antisense oligonucleotide to inhibit expression of a mutated human procollagen gene (COL1A1) in transfected mouse 3T3 cells. Biochemistry. 1993 Jan 12;32(1):7–11. doi: 10.1021/bi00052a002. [DOI] [PubMed] [Google Scholar]
  4. Cooney M., Czernuszewicz G., Postel E. H., Flint S. J., Hogan M. E. Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro. Science. 1988 Jul 22;241(4864):456–459. doi: 10.1126/science.3293213. [DOI] [PubMed] [Google Scholar]
  5. Feuerstein B. G., Pattabiraman N., Marton L. J. Molecular mechanics of the interactions of spermine with DNA: DNA bending as a result of ligand binding. Nucleic Acids Res. 1990 Mar 11;18(5):1271–1282. doi: 10.1093/nar/18.5.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fox K. R., Polucci P., Jenkins T. C., Neidle S. A molecular anchor for stabilizing triple-helical DNA. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7887–7891. doi: 10.1073/pnas.92.17.7887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hampel K. J., Crosson P., Lee J. S. Polyamines favor DNA triplex formation at neutral pH. Biochemistry. 1991 May 7;30(18):4455–4459. doi: 10.1021/bi00232a012. [DOI] [PubMed] [Google Scholar]
  8. Haworth I. S., Rodger A., Richards W. G. A molecular mechanics study of spermine complexation to DNA: a new model for spermine-poly(dG-dC) binding. Proc Biol Sci. 1991 May 22;244(1310):107–116. doi: 10.1098/rspb.1991.0058. [DOI] [PubMed] [Google Scholar]
  9. INMAN R. B. TRANSITIONS OF DNA HOMOPOLYMERS. J Mol Biol. 1964 Sep;9:624–637. doi: 10.1016/s0022-2836(64)80171-6. [DOI] [PubMed] [Google Scholar]
  10. Jain S., Zon G., Sundaralingam M. Base only binding of spermine in the deep groove of the A-DNA octamer d(GTGTACAC). Biochemistry. 1989 Mar 21;28(6):2360–2364. doi: 10.1021/bi00432a002. [DOI] [PubMed] [Google Scholar]
  11. Jetter M. C., Hobbs F. W. 7,8-Dihydro-8-oxoadenine as a replacement for cytosine in the third strand of triple helices. Triplex formation without hypochromicity. Biochemistry. 1993 Apr 6;32(13):3249–3254. doi: 10.1021/bi00064a006. [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. Lavelle L., Fresco J. R. UV spectroscopic identification and thermodynamic analysis of protonated third strand deoxycytidine residues at neutrality in the triplex d(C(+)-T)6:[d(A-G)6.d(C-T)6]; evidence for a proton switch. Nucleic Acids Res. 1995 Jul 25;23(14):2692–2705. doi: 10.1093/nar/23.14.2692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lee J. S., Woodsworth M. L., Latimer L. J., Morgan A. R. Poly(pyrimidine) . poly(purine) synthetic DNAs containing 5-methylcytosine form stable triplexes at neutral pH. Nucleic Acids Res. 1984 Aug 24;12(16):6603–6614. doi: 10.1093/nar/12.16.6603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ma C., Bloomfield V. A. Gel electrophoresis measurement of counterion condensation on DNA. Biopolymers. 1995 Feb;35(2):211–216. doi: 10.1002/bip.360350209. [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. Mergny J. L., Sun J. S., Rougée M., Montenay-Garestier T., Barcelo F., Chomilier J., Hélène C. Sequence specificity in triple-helix formation: experimental and theoretical studies of the effect of mismatches on triplex stability. Biochemistry. 1991 Oct 8;30(40):9791–9798. doi: 10.1021/bi00104a031. [DOI] [PubMed] [Google Scholar]
  19. Milligan J. F., Matteucci M. D., Martin J. C. Current concepts in antisense drug design. J Med Chem. 1993 Jul 9;36(14):1923–1937. doi: 10.1021/jm00066a001. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Perales J. C., Ferkol T., Molas M., Hanson R. W. An evaluation of receptor-mediated gene transfer using synthetic DNA-ligand complexes. Eur J Biochem. 1994 Dec 1;226(2):255–266. doi: 10.1111/j.1432-1033.1994.tb20049.x. [DOI] [PubMed] [Google Scholar]
  22. Plum G. E., Park Y. W., Singleton S. F., Dervan P. B., Breslauer K. J. Thermodynamic characterization of the stability and the melting behavior of a DNA triplex: a spectroscopic and calorimetric study. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9436–9440. doi: 10.1073/pnas.87.23.9436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Plum G. E., Pilch D. S., Singleton S. F., Breslauer K. J. Nucleic acid hybridization: triplex stability and energetics. Annu Rev Biophys Biomol Struct. 1995;24:319–350. doi: 10.1146/annurev.bb.24.060195.001535. [DOI] [PubMed] [Google Scholar]
  24. Radhakrishnan I., Patel D. J. DNA triplexes: solution structures, hydration sites, energetics, interactions, and function. Biochemistry. 1994 Sep 27;33(38):11405–11416. doi: 10.1021/bi00204a001. [DOI] [PubMed] [Google Scholar]
  25. Rougée M., Faucon B., Mergny J. L., Barcelo F., Giovannangeli C., Garestier T., Hélène C. Kinetics and thermodynamics of triple-helix formation: effects of ionic strength and mismatches. Biochemistry. 1992 Sep 29;31(38):9269–9278. doi: 10.1021/bi00153a021. [DOI] [PubMed] [Google Scholar]
  26. Singleton S. F., Dervan P. B. Influence of pH on the equilibrium association constants for oligodeoxyribonucleotide-directed triple helix formation at single DNA sites. Biochemistry. 1992 Nov 17;31(45):10995–11003. doi: 10.1021/bi00160a008. [DOI] [PubMed] [Google Scholar]
  27. Tabor C. W., Tabor H. Polyamines. Annu Rev Biochem. 1984;53:749–790. doi: 10.1146/annurev.bi.53.070184.003533. [DOI] [PubMed] [Google Scholar]
  28. Takeda Y., Samejima K., Nagano K., Watanabe M., Sugeta H., Kyogoku Y. Determination of protonation sites in thermospermine and in some other polyamines by 15N and 13C nuclear magnetic resonance spectroscopy. Eur J Biochem. 1983 Feb 1;130(2):383–389. doi: 10.1111/j.1432-1033.1983.tb07164.x. [DOI] [PubMed] [Google Scholar]
  29. Thomas T., Thomas T. J. Selectivity of polyamines in triplex DNA stabilization. Biochemistry. 1993 Dec 21;32(50):14068–14074. doi: 10.1021/bi00213a041. [DOI] [PubMed] [Google Scholar]
  30. Tung C. H., Breslauer K. J., Stein S. Polyamine-linked oligonucleotides for DNA triple helix formation. Nucleic Acids Res. 1993 Nov 25;21(23):5489–5494. doi: 10.1093/nar/21.23.5489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Völker J., Klump H. H. Electrostatic effects in DNA triple helices. Biochemistry. 1994 Nov 15;33(45):13502–13508. doi: 10.1021/bi00249a039. [DOI] [PubMed] [Google Scholar]
  32. Wagner R. W., Matteucci M. D., Lewis J. G., Gutierrez A. J., Moulds C., Froehler B. C. Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines. Science. 1993 Jun 4;260(5113):1510–1513. doi: 10.1126/science.7684856. [DOI] [PubMed] [Google Scholar]
  33. 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]

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