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. 1996 Feb 1;24(3):494–500. doi: 10.1093/nar/24.3.494

Antisense properties of duplex- and triplex-forming PNAs.

H Knudsen 1, P E Nielsen 1
PMCID: PMC145651  PMID: 8602363

Abstract

The potential of peptide nucleic acids (PNAs) as specific inhibitors of translation has been studied. PNAs with a mixed purine/pyrimidine sequence form duplexes, while homopyrimidine PNAs form (PNA)2/RNA triplexes with complementary sequences on RNA. We show here that neither of these PNA/RNA structures are substrates for RNase H. Translation experiments in cell-free extracts showed that a 15mer duplex-forming PNA blocked translation in a dose-dependent manner when the target was 5'-proximal to the AUG start codon on the RNA, whereas similar 10-, 15- or 20mer PNAs had no effect when targeted towards sequences in the coding region. Triplex-forming 10mer PNAs were efficient and specific antisense agents with a target overlapping the AUG start codon and caused arrest of ribosome elongation with a target positioned in the coding region of the mRNA. Furthermore, translation could be blocked with a 6mer bisPNA or with a clamp PNA, forming partly a triplex, partly a duplex, with its target sequence in the coding region of the mRNA.

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Selected References

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  1. Agrawal S., Iyer R. P. Modified oligonucleotides as therapeutic and diagnostic agents. Curr Opin Biotechnol. 1995 Feb;6(1):12–19. doi: 10.1016/0958-1669(95)80003-4. [DOI] [PubMed] [Google Scholar]
  2. Bock L. C., Griffin L. C., Latham J. A., Vermaas E. H., Toole J. J. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature. 1992 Feb 6;355(6360):564–566. doi: 10.1038/355564a0. [DOI] [PubMed] [Google Scholar]
  3. Boiziau C., Kurfurst R., Cazenave C., Roig V., Thuong N. T., Toulmé J. J. Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism. Nucleic Acids Res. 1991 Mar 11;19(5):1113–1119. doi: 10.1093/nar/19.5.1113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bonham M. A., Brown S., Boyd A. L., Brown P. H., Bruckenstein D. A., Hanvey J. C., Thomson S. A., Pipe A., Hassman F., Bisi J. E. An assessment of the antisense properties of RNase H-competent and steric-blocking oligomers. Nucleic Acids Res. 1995 Apr 11;23(7):1197–1203. doi: 10.1093/nar/23.7.1197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boutorine A. S., Boiziau C., Le Doan T., Toulmé J. J., Hélène C. Effect of the terminal phosphate derivatization of beta- and alpha-oligodeoxynucleotides on their antisense activity in protein biosynthesis, stability and uptake by eucaryotic cells. Biochimie. 1992 May;74(5):485–489. doi: 10.1016/0300-9084(92)90089-w. [DOI] [PubMed] [Google Scholar]
  6. Brown S. C., Thomson S. A., Veal J. M., Davis D. G. NMR solution structure of a peptide nucleic acid complexed with RNA. Science. 1994 Aug 5;265(5173):777–780. doi: 10.1126/science.7519361. [DOI] [PubMed] [Google Scholar]
  7. Cazenave C., Frank P., Büsen W. Characterization of ribonuclease H activities present in two cell-free protein synthesizing systems, the wheat germ extract and the rabbit reticulocyte lysate. Biochimie. 1993;75(1-2):113–122. doi: 10.1016/0300-9084(93)90032-n. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Chang E. H., Miller P. S., Cushman C., Devadas K., Pirollo K. F., Ts'o P. O., Yu Z. P. Antisense inhibition of ras p21 expression that is sensitive to a point mutation. Biochemistry. 1991 Aug 27;30(34):8283–8286. doi: 10.1021/bi00098a001. [DOI] [PubMed] [Google Scholar]
  10. Christensen L., Fitzpatrick R., Gildea B., Petersen K. H., Hansen H. F., Koch T., Egholm M., Buchardt O., Nielsen P. E., Coull J. Solid-phase synthesis of peptide nucleic acids. J Pept Sci. 1995 May-Jun;1(3):175–183. doi: 10.1002/psc.310010304. [DOI] [PubMed] [Google Scholar]
  11. Clarenc J. P., Degols G., Leonetti J. P., Milhaud P., Lebleu B. Delivery of antisense oligonucleotides by poly(L-lysine) conjugation and liposome encapsulation. Anticancer Drug Des. 1993 Feb;8(1):81–94. [PubMed] [Google Scholar]
  12. Demidov V. V., Potaman V. N., Frank-Kamenetskii M. D., Egholm M., Buchard O., Sönnichsen S. H., Nielsen P. E. Stability of peptide nucleic acids in human serum and cellular extracts. Biochem Pharmacol. 1994 Sep 15;48(6):1310–1313. doi: 10.1016/0006-2952(94)90171-6. [DOI] [PubMed] [Google Scholar]
  13. Egholm M., Buchardt O., Christensen L., Behrens C., Freier S. M., Driver D. A., Berg R. H., Kim S. K., Norden B., Nielsen P. E. PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature. 1993 Oct 7;365(6446):566–568. doi: 10.1038/365566a0. [DOI] [PubMed] [Google Scholar]
  14. Egholm M., Christensen L., Dueholm K. L., Buchardt O., Coull J., Nielsen P. E. Efficient pH-independent sequence-specific DNA binding by pseudoisocytosine-containing bis-PNA. Nucleic Acids Res. 1995 Jan 25;23(2):217–222. doi: 10.1093/nar/23.2.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gao W. Y., Han F. S., Storm C., Egan W., Cheng Y. C. Phosphorothioate oligonucleotides are inhibitors of human DNA polymerases and RNase H: implications for antisense technology. Mol Pharmacol. 1992 Feb;41(2):223–229. [PubMed] [Google Scholar]
  16. Giovannangeli C., Thuong N. T., Hélène C. Oligonucleotide clamps arrest DNA synthesis on a single-stranded DNA target. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10013–10017. doi: 10.1073/pnas.90.21.10013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hanvey J. C., Peffer N. J., Bisi J. E., Thomson S. A., Cadilla R., Josey J. A., Ricca D. J., Hassman C. F., Bonham M. A., Au K. G. Antisense and antigene properties of peptide nucleic acids. Science. 1992 Nov 27;258(5087):1481–1485. doi: 10.1126/science.1279811. [DOI] [PubMed] [Google Scholar]
  18. Johansson H. E., Belsham G. J., Sproat B. S., Hentze M. W. Target-specific arrest of mRNA translation by antisense 2'-O-alkyloligoribonucleotides. Nucleic Acids Res. 1994 Nov 11;22(22):4591–4598. doi: 10.1093/nar/22.22.4591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kean J. M., Murakami A., Blake K. R., Cushman C. D., Miller P. S. Photochemical cross-linking of psoralen-derivatized oligonucleoside methylphosphonates to rabbit globin messenger RNA. Biochemistry. 1988 Dec 27;27(26):9113–9121. doi: 10.1021/bi00426a008. [DOI] [PubMed] [Google Scholar]
  20. Leijon M., Gräslund A., Nielsen P. E., Buchardt O., Nordén B., Kristensen S. M., Eriksson M. Structural characterization of PNA-DNA duplexes by NMR. Evidence for DNA in a B-like conformation. Biochemistry. 1994 Aug 23;33(33):9820–9825. doi: 10.1021/bi00199a002. [DOI] [PubMed] [Google Scholar]
  21. Marcus-Sekura C. J., Woerner A. M., Shinozuka K., Zon G., Quinnan G. V., Jr Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages. Nucleic Acids Res. 1987 Jul 24;15(14):5749–5763. doi: 10.1093/nar/15.14.5749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Miller P. S., McParland K. B., Jayaraman K., Ts'o P. O. Biochemical and biological effects of nonionic nucleic acid methylphosphonates. Biochemistry. 1981 Mar 31;20(7):1874–1880. doi: 10.1021/bi00510a024. [DOI] [PubMed] [Google Scholar]
  23. Morvan F., Rayner B., Imbach J. L. Alpha-oligonucleotides: a unique class of modified chimeric nucleic acids. Anticancer Drug Des. 1991 Dec;6(6):521–529. [PubMed] [Google Scholar]
  24. Nielsen P. E., Egholm M., Berg R. H., Buchardt O. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science. 1991 Dec 6;254(5037):1497–1500. doi: 10.1126/science.1962210. [DOI] [PubMed] [Google Scholar]
  25. Nielsen P. E., Egholm M., Buchardt O. Peptide nucleic acid (PNA). A DNA mimic with a peptide backbone. Bioconjug Chem. 1994 Jan-Feb;5(1):3–7. doi: 10.1021/bc00025a001. [DOI] [PubMed] [Google Scholar]
  26. Shakin S. H., Liebhaber S. A. Destabilization of messenger RNA/complementary DNA duplexes by the elongating 80 S ribosome. J Biol Chem. 1986 Dec 5;261(34):16018–16025. [PubMed] [Google Scholar]
  27. Sproat B. S., Lamond A. I., Garcia R. G., Beijer B., Pieles U., Douglas M., Bohmann K., Carmo-Fonseco M., Weston S., O'Loughlin S. 2'-O-alkyloligoribonucleotides, synthesis and applications in molecular biology. Nucleic Acids Symp Ser. 1991;(24):59–62. [PubMed] [Google Scholar]
  28. Stein C. A., Cheng Y. C. Antisense oligonucleotides as therapeutic agents--is the bullet really magical? Science. 1993 Aug 20;261(5124):1004–1012. doi: 10.1126/science.8351515. [DOI] [PubMed] [Google Scholar]
  29. Volkmann S., Jendis J., Frauendorf A., Moelling K. Inhibition of HIV-1 reverse transcription by triple-helix forming oligonucleotides with viral RNA. Nucleic Acids Res. 1995 Apr 11;23(7):1204–1212. doi: 10.1093/nar/23.7.1204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wagner R. W. Gene inhibition using antisense oligodeoxynucleotides. Nature. 1994 Nov 24;372(6504):333–335. doi: 10.1038/372333a0. [DOI] [PubMed] [Google Scholar]
  31. Walder R. Y., Walder J. A. Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5011–5015. doi: 10.1073/pnas.85.14.5011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Woolf T. M., Melton D. A., Jennings C. G. Specificity of antisense oligonucleotides in vivo. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7305–7309. doi: 10.1073/pnas.89.16.7305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yakubov L., Khaled Z., Zhang L. M., Truneh A., Vlassov V., Stein C. A. Oligodeoxynucleotides interact with recombinant CD4 at multiple sites. J Biol Chem. 1993 Sep 5;268(25):18818–18823. [PubMed] [Google Scholar]

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