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. 1996 Feb 15;24(4):730–736. doi: 10.1093/nar/24.4.730

Triplex formation by a psoralen-conjugated oligodeoxyribonucleotide containing the base analog 8-oxo-adenine.

P S Miller 1, G Bi 1, S A Kipp 1, V Fok 1, R K DeLong 1
PMCID: PMC145696  PMID: 8604317

Abstract

Oligodeoxyribonucleotides containing thymidine and 8-oxo-2'-deoxyadenosine can form pyr.pur.pyr type triplexes with double-stranded DNA. Unlike triplexes whose third strands contain thymidine and deoxycytidine, the stability of these triplexes is independent of pH. We have prepared d-ps-TAAATAAATTTTTAT-L [I(A)], where A is 8-oxo-2'-deoxyadenosine, ps is 4'-hydroxymethyl-4,5',8- trimethylpsoralen and L is a 6-amino-2-(hydroxymethyl)hexyl linker. The oligomer is designed to interact with a homopurine sequence in the promoter region of the human gene coding for the 92 kDa form of collagenase type IV. Oligomer I(A) and oligomer I(C), which contains 2'-deoxycytidine in place of 8-oxo-2'-deoxycytidine, both form stable triplexes at pH 6.2, but only I(A) forms a stable triplex with a model duplex DNA target at pH 7.5, as determined by UV melting experiments. Triplex formation is stabilized by the presence of the psoralen group. Upon irradiation both I(A) and I(C) form photoadducts with the DNA target at pH 6.2, but only I(A) forms a photoadduct at pH 7.5. In these photoreactions oligomer I(A) appears to selectively form a photoadduct with a C in the purine-rich strand of the duplex target. Although a T residue is present in the pyrimidine-rich strand of the target at the duplex/triplex junction, essentially no adduct formation takes place with this strand, nor is interstrand cross-linking observed. The extent of photoadduct formation decreases with increasing temperature, behavior which is consistent with the UV melting curve of the triplex. A tetramethylrhodamine derivative of I(A) was prepared and found to cross-link less extensively than I(A) itself. Oligomer I(A) is completely resistant to hydrolysis when incubated for 24h in the presence of 10% fetal bovine serum at 37 degree C, although it is hydrolyzed by S1 nuclease. The properties of oligomer I(A) suggest that 8-oxo- containing oligomers may find utility as antigene oligonucleotide reagents.

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Selected References

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  1. Bachellerie J. P., Thompson J. F., Wegnez M. R., Hearst J. E. Identification of the modified nucleotides produced by covalent photoaddition of hydroxymethyltrimethylpsoralen to RNA. Nucleic Acids Res. 1981 May 11;9(9):2207–2222. doi: 10.1093/nar/9.9.2207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bates P. J., Macaulay V. M., McLean M. J., Jenkins T. C., Reszka A. P., Laughton C. A., Neidle S. Characteristics of triplex-directed photoadduct formation by psoralen-linked oligodeoxynucleotides. Nucleic Acids Res. 1995 Nov 11;23(21):4283–4289. doi: 10.1093/nar/23.21.4283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cho B. P., Evans F. E. Structure of oxidatively damaged nucleic acid adducts. 3. Tautomerism, ionization and protonation of 8-hydroxyadenosine studied by 15N NMR spectroscopy. Nucleic Acids Res. 1991 Mar 11;19(5):1041–1047. doi: 10.1093/nar/19.5.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Degols G., Clarenc J. P., Lebleu B., Léonetti J. P. Reversible inhibition of gene expression by a psoralen functionalized triple helix forming oligonucleotide in intact cells. J Biol Chem. 1994 Jun 17;269(24):16933–16937. [PubMed] [Google Scholar]
  5. Frank-Kamenetskii M. D., Mirkin S. M. Triplex DNA structures. Annu Rev Biochem. 1995;64:65–95. doi: 10.1146/annurev.bi.64.070195.000433. [DOI] [PubMed] [Google Scholar]
  6. Gasparro F. P., Havre P. A., Olack G. A., Gunther E. J., Glazer P. M. Site-specific targeting of psoralen photoadducts with a triple helix-forming oligonucleotide: characterization of psoralen monoadduct and crosslink formation. Nucleic Acids Res. 1994 Jul 25;22(14):2845–2852. doi: 10.1093/nar/22.14.2845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Havre P. A., Glazer P. M. Targeted mutagenesis of simian virus 40 DNA mediated by a triple helix-forming oligonucleotide. J Virol. 1993 Dec;67(12):7324–7331. doi: 10.1128/jvi.67.12.7324-7331.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hobbs C. A., Yoon K. Differential regulation of gene expression in vivo by triple helix-forming oligonucleotides as detected by a reporter enzyme. Antisense Res Dev. 1994 Spring;4(1):1–8. doi: 10.1089/ard.1994.4.1. [DOI] [PubMed] [Google Scholar]
  10. Huhtala P., Tuuttila A., Chow L. T., Lohi J., Keski-Oja J., Tryggvason K. Complete structure of the human gene for 92-kDa type IV collagenase. Divergent regulation of expression for the 92- and 72-kilodalton enzyme genes in HT-1080 cells. J Biol Chem. 1991 Sep 5;266(25):16485–16490. [PubMed] [Google Scholar]
  11. Ing N. H., Beekman J. M., Kessler D. J., Murphy M., Jayaraman K., Zendegui J. G., Hogan M. E., O'Malley B. W., Tsai M. J. In vivo transcription of a progesterone-responsive gene is specifically inhibited by a triplex-forming oligonucleotide. Nucleic Acids Res. 1993 Jun 25;21(12):2789–2796. doi: 10.1093/nar/21.12.2789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Kean J. M., Miller P. S. Detection of psoralen cross-link sites in DNA modified by psoralen-conjugated oligodeoxyribonucleoside methylphosphonates. Bioconjug Chem. 1993 Mar-Apr;4(2):184–187. doi: 10.1021/bc00020a012. [DOI] [PubMed] [Google Scholar]
  14. Kean J. M., Miller P. S. Effect of target structure on cross-linking by psoralen-derivatized oligonucleoside methylphosphonates. Biochemistry. 1994 Aug 9;33(31):9178–9186. doi: 10.1021/bi00197a021. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. McShan W. M., Rossen R. D., Laughter A. H., Trial J., Kessler D. J., Zendegui J. G., Hogan M. E., Orson F. M. Inhibition of transcription of HIV-1 in infected human cells by oligodeoxynucleotides designed to form DNA triple helices. J Biol Chem. 1992 Mar 15;267(8):5712–5721. [PubMed] [Google Scholar]
  18. Miller P. S., Bhan P., Cushman C. D., Trapane T. L. Recognition of a guanine-cytosine base pair by 8-oxoadenine. Biochemistry. 1992 Jul 28;31(29):6788–6793. doi: 10.1021/bi00144a020. [DOI] [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. Orson F. M., Thomas D. W., McShan W. M., Kessler D. J., Hogan M. E. Oligonucleotide inhibition of IL2R alpha mRNA transcription by promoter region collinear triplex formation in lymphocytes. Nucleic Acids Res. 1991 Jun 25;19(12):3435–3441. doi: 10.1093/nar/19.12.3435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Porumb H., Dagneaux C., Letellier R., Malvy C., Taillandier E. Triple-helices targeted to the polypurine tract of a murine retrovirus. Gene. 1994 Nov 4;149(1):101–107. doi: 10.1016/0378-1119(94)90417-0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Roy C. Inhibition of gene transcription by purine rich triplex forming oligodeoxyribonucleotides. Nucleic Acids Res. 1993 Jun 25;21(12):2845–2852. doi: 10.1093/nar/21.12.2845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sage E., Moustacchi E. Sequence context effects on 8-methoxypsoralen photobinding to defined DNA fragments. Biochemistry. 1987 Jun 16;26(12):3307–3314. doi: 10.1021/bi00386a010. [DOI] [PubMed] [Google Scholar]
  26. Scaggiante B., Morassutti C., Tolazzi G., Michelutti A., Baccarani M., Quadrifoglio F. Effect of unmodified triple helix-forming oligodeoxyribonucleotide targeted to human multidrug-resistance gene mdr1 in MDR cancer cells. FEBS Lett. 1994 Oct 3;352(3):380–384. doi: 10.1016/0014-5793(94)00995-3. [DOI] [PubMed] [Google Scholar]
  27. Shi Y. B., Spielmann H. P., Hearst J. E. Base-catalyzed reversal of a psoralen-DNA cross-link. Biochemistry. 1988 Jul 12;27(14):5174–5178. doi: 10.1021/bi00414a034. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Takasugi M., Guendouz A., Chassignol M., Decout J. L., Lhomme J., Thuong N. T., Hélène C. Sequence-specific photo-induced cross-linking of the two strands of double-helical DNA by a psoralen covalently linked to a triple helix-forming oligonucleotide. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5602–5606. doi: 10.1073/pnas.88.13.5602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thaden J., Miller P. S. Photoaffinity behavior of a conjugate of oligonucleoside methylphosphonate, rhodamine, and psoralen in the presence of complementary oligonucleotides. Bioconjug Chem. 1993 Sep-Oct;4(5):386–394. doi: 10.1021/bc00023a014. [DOI] [PubMed] [Google Scholar]
  31. Wang Q., Tsukahara S., Yamakawa H., Takai K., Takaku H. pH-independent inhibition of restriction endonuclease cleavage via triple helix formation by oligonucleotides containing 8-oxo-2'-deoxyadenosine. FEBS Lett. 1994 Nov 21;355(1):11–14. doi: 10.1016/0014-5793(94)01139-7. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Xodo L., Alunni-Fabbroni M., Manzini G., Quadrifoglio F. Pyrimidine phosphorothioate oligonucleotides form triple-stranded helices and promote transcription inhibition. Nucleic Acids Res. 1994 Aug 25;22(16):3322–3330. doi: 10.1093/nar/22.16.3322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yeung A. T., Dinehart W. J., Jones B. K. Alkali reversal of psoralen cross-link for the targeted delivery of psoralen monoadduct lesion. Biochemistry. 1988 Aug 23;27(17):6332–6338. doi: 10.1021/bi00417a020. [DOI] [PubMed] [Google Scholar]
  35. Young S. L., Krawczyk S. H., Matteucci M. D., Toole J. J. Triple helix formation inhibits transcription elongation in vitro. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10023–10026. doi: 10.1073/pnas.88.22.10023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zendegui J. G., Vasquez K. M., Tinsley J. H., Kessler D. J., Hogan M. E. In vivo stability and kinetics of absorption and disposition of 3' phosphopropyl amine oligonucleotides. Nucleic Acids Res. 1992 Jan 25;20(2):307–314. doi: 10.1093/nar/20.2.307. [DOI] [PMC free article] [PubMed] [Google Scholar]

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