Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Oct 1;25(19):3787–3794. doi: 10.1093/nar/25.19.3787

DNA triple helix formation at oligopurine sites containing multiple contiguous pyrimidines.

D M Gowers 1, K R Fox 1
PMCID: PMC146974  PMID: 9380499

Abstract

We have used DNase I footprinting to assess the formation of triple helices at 15mer oligopurine target sites which are interrupted by several (up to four) adjacent central pyrimidine residues. Third strand oligonucleotides were designed to generate complexes containing central (X.TA)nor (X.CG)n triplets (X = each base in turn) surrounded by C+.GC and T.AT triplets. It has previously been shown that G.TA and T.CG are the most stable triplets for recognition of single TA and CG interruptions. We show that these triplets are the most useful for recognizing consecutive pyrimidine interruptions and find that addition of each pyrimidine residue leads to a 30-fold decrease in third strand affinity. The addition of 10 microM naphthylquinoline triplex-binding ligand stabilizes each complex so that all the oligonucleotides produce footprints at similar concentrations (0.3 microM). Targets containing two pyrimidines are only bound by oligonucleotides generating (G.TA)2 and (T.CG)2 with a further 30-fold decrease in affinity. (G.TA)2 is slightly more stable than (T.CG)2. In the presence of the triplex-binding ligand the order of stability is (G.TA)2 > (C.TA)2 > (T.TA)2 > (A.TA)2 and (T.CG)2 > (C.CG)2 > (G.CG)2 = (A.CG)2. No oligonucleotide footprints are generated at target sites containing three consecutive pyrimidines, though addition of 10 microM triplex-binding ligand produces stable complexes with oligonucleotides generating (G.TA)3, (T.CG)3 and (C.CG)3, with a further 30-fold reduction in affinity. No footprints are generated at targets containing four Ts, though the ligand induces a weak interaction with the oligonucleotide generating (T.CG)4.

Full Text

The Full Text of this article is available as a PDF (629.2 KB).

Selected References

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

  1. Alunni-Fabbroni M., Manfioletti G., Manzini G., Xodo L. E. Inhibition of T7 RNA polymerase transcription by phosphate and phosphorothioate triplex-forming oligonucleotides targeted to a R.Y site downstream from the promoter. Eur J Biochem. 1994 Dec 15;226(3):831–839. doi: 10.1111/j.1432-1033.1994.00831.x. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Belotserkovskii B. P., Veselkov A. G., Filippov S. A., Dobrynin V. N., Mirkin S. M., Frank-Kamenetskii M. D. Formation of intramolecular triplex in homopurine-homopyrimidine mirror repeats with point substitutions. Nucleic Acids Res. 1990 Nov 25;18(22):6621–6624. doi: 10.1093/nar/18.22.6621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Broitman S. L., Im D. D., Fresco J. R. Formation of the triple-stranded polynucleotide helix, poly(A.A.U). Proc Natl Acad Sci U S A. 1987 Aug;84(15):5120–5124. doi: 10.1073/pnas.84.15.5120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown P. M., Drabble A., Fox K. R. Effect of a triplex-binding ligand on triple helix formation at a site within a natural DNA fragment. Biochem J. 1996 Mar 1;314(Pt 2):427–432. doi: 10.1042/bj3140427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cassidy S. A., Strekowski L., Fox K. R. DNA sequence specificity of a naphthylquinoline triple helix-binding ligand. Nucleic Acids Res. 1996 Nov 1;24(21):4133–4138. doi: 10.1093/nar/24.21.4133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cassidy S. A., Strekowski L., Wilson W. D., Fox K. R. Effect of a triplex-binding ligand on parallel and antiparallel DNA triple helices using short unmodified and acridine-linked oligonucleotides. Biochemistry. 1994 Dec 27;33(51):15338–15347. doi: 10.1021/bi00255a015. [DOI] [PubMed] [Google Scholar]
  8. Chandler S. P., Fox K. R. Extension of DNA triple helix formation to a neighbouring (AT)n site. FEBS Lett. 1995 Feb 20;360(1):21–25. doi: 10.1016/0014-5793(95)00069-l. [DOI] [PubMed] [Google Scholar]
  9. Chandler S. P., Fox K. R. Triple helix formation at A8XA8.T8YT8. FEBS Lett. 1993 Oct 11;332(1-2):189–192. doi: 10.1016/0014-5793(93)80510-2. [DOI] [PubMed] [Google Scholar]
  10. Chandler S. P., Strekowski L., Wilson W. D., Fox K. R. Footprinting studies on ligands which stabilize DNA triplexes: effects on stringency within a parallel triple helix. Biochemistry. 1995 May 30;34(21):7234–7242. doi: 10.1021/bi00021a039. [DOI] [PubMed] [Google Scholar]
  11. FELSENFELD G., RICH A. Studies on the formation of two- and three-stranded polyribonucleotides. Biochim Biophys Acta. 1957 Dec;26(3):457–468. doi: 10.1016/0006-3002(57)90091-4. [DOI] [PubMed] [Google Scholar]
  12. Griffin L. C., Dervan P. B. Recognition of thymine adenine.base pairs by guanine in a pyrimidine triple helix motif. Science. 1989 Sep 1;245(4921):967–971. doi: 10.1126/science.2549639. [DOI] [PubMed] [Google Scholar]
  13. Ji H., Francisco T., Smith L. M., Guilfoyle R. A. Rapid restriction mapping of cosmids by sequence-specific triple-helix-mediated affinity capture. Genomics. 1996 Jan 15;31(2):185–192. doi: 10.1006/geno.1996.0030. [DOI] [PubMed] [Google Scholar]
  14. Johnson A. F., Wang R., Ji H., Chen D., Guilfoyle R. A., Smith L. M. Purification of single-stranded M13 DNA by cooperative triple-helix-mediated affinity capture. Anal Biochem. 1996 Feb 1;234(1):83–95. doi: 10.1006/abio.1996.0053. [DOI] [PubMed] [Google Scholar]
  15. Kiessling L. L., Griffin L. C., Dervan P. B. Flanking sequence effects within the pyrimidine triple-helix motif characterized by affinity cleaving. Biochemistry. 1992 Mar 17;31(10):2829–2834. doi: 10.1021/bi00125a026. [DOI] [PubMed] [Google Scholar]
  16. Kiyama R., Nishikawa N., Oishi M. Enrichment of human DNAs that flank poly(dA).poly(dT) tracts by triplex DNA formation. J Mol Biol. 1994 Mar 25;237(2):193–200. doi: 10.1006/jmbi.1994.1221. [DOI] [PubMed] [Google Scholar]
  17. Lavrovsky Y., Mastyugin V., Stoltz R. A., Abraham N. G. Specific inhibition of c-fos proto-oncogene expression by triple-helix-forming oligonucleotides. J Cell Biochem. 1996 May;61(2):301–309. doi: 10.1002/(SICI)1097-4644(19960501)61:2%3C301::AID-JCB13%3E3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Nishikawa N., Kanda N., Oishi M., Kiyama R. Enrichment of oligo(dG).oligo(dC)-containing fragments from human genomic DNA by Mg 2+-dependent triplex affinity capture. Nucleic Acids Res. 1997 May 1;25(9):1701–1708. doi: 10.1093/nar/25.9.1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pfannschmidt C., Schaper A., Heim G., Jovin T. M., Langowski J. Sequence-specific labeling of superhelical DNA by triple helix formation and psoralen crosslinking. Nucleic Acids Res. 1996 May 1;24(9):1702–1709. doi: 10.1093/nar/24.9.1702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Radhakrishnan I., Gao X., de los Santos C., Live D., Patel D. J. NMR structural studies of intramolecular (Y+)n.(R+)n(Y-)nDNA triplexes in solution: imino and amino proton and nitrogen markers of G.TA base triple formation. Biochemistry. 1991 Sep 17;30(37):9022–9030. doi: 10.1021/bi00101a016. [DOI] [PubMed] [Google Scholar]
  24. Radhakrishnan I., Patel D. J., Gao X. Three-dimensional homonuclear NOESY-TOCSY of an intramolecular pyrimidine.purine.pyrimidine DNA triplex containing a central G.TA triple: nonexchangeable proton assignments and structural implications. Biochemistry. 1992 Mar 10;31(9):2514–2523. doi: 10.1021/bi00124a011. [DOI] [PubMed] [Google Scholar]
  25. Radhakrishnan I., Patel D. J. Solution structure and hydration patterns of a pyrimidine.purine.pyrimidine DNA triplex containing a novel T.CG base-triple. J Mol Biol. 1994 Aug 26;241(4):600–619. doi: 10.1006/jmbi.1994.1534. [DOI] [PubMed] [Google Scholar]
  26. Radhakrishnan I., Patel D. J. Solution structure of a purine.purine.pyrimidine DNA triplex containing G.GC and T.AT triples. Structure. 1993 Oct 15;1(2):135–152. doi: 10.1016/0969-2126(93)90028-f. [DOI] [PubMed] [Google Scholar]
  27. Radhakrishnan I., Patel D. J. Solution structure of a pyrimidine.purine.pyrimidine DNA triplex containing T.AT, C+.GC and G.TA triples. Structure. 1994 Jan 15;2(1):17–32. doi: 10.1016/s0969-2126(00)00005-8. [DOI] [PubMed] [Google Scholar]
  28. Radhakrishnan I., Patel D. J. Solution structure of a pyrimidine.purine.pyrimidine DNA triplex containing T.AT, C+.GC and G.TA triples. Structure. 1994 Jan 15;2(1):17–32. doi: 10.1016/s0969-2126(00)00005-8. [DOI] [PubMed] [Google Scholar]
  29. Wang E., Malek S., Feigon J. Structure of a G.T.A triplet in an intramolecular DNA triplex. Biochemistry. 1992 May 26;31(20):4838–4846. doi: 10.1021/bi00135a015. [DOI] [PubMed] [Google Scholar]
  30. Wilson W. D., Tanious F. A., Mizan S., Yao S., Kiselyov A. S., Zon G., Strekowski L. DNA triple-helix specific intercalators as antigene enhancers: unfused aromatic cations. Biochemistry. 1993 Oct 12;32(40):10614–10621. doi: 10.1021/bi00091a011. [DOI] [PubMed] [Google Scholar]
  31. Yoon K., Hobbs C. A., Koch J., Sardaro M., Kutny R., Weis A. L. Elucidation of the sequence-specific third-strand recognition of four Watson-Crick base pairs in a pyrimidine triple-helix motif: T.AT, C.GC, T.CG, and G.TA. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3840–3844. doi: 10.1073/pnas.89.9.3840. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES