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. 1997 Feb 15;25(4):776–780. doi: 10.1093/nar/25.4.776

RNase H-independent antisense activity of oligonucleotide N3 '--> P5 ' phosphoramidates.

O Heidenreich 1, S Gryaznov 1, M Nerenberg 1
PMCID: PMC146512  PMID: 9016628

Abstract

Oligonucleotide N3'-->P5'phosphoramidates are a new and promising class of antisense agents. Here we report biological properties of phosphoramidate oligonucleotides targeted against the human T cell leukemia virus type-I Tax protein, the major transcriptional transactivator of this human retrovirus. Isosequential phosphorothioate oligodeoxynucleotides and uniformly modified and chimeric phosphoramidate oligodeoxynucleotides containing six central phosphodiester linkages are all quite stable in cell nuclei. The uniformly modified anti-tax phosphoramidate oligodeoxynucleotide does not activate nuclear RNase H, as was shown by RNase protection assay. In contrast, the chimeric phosphoramidate-phosphodiester oligodeoxynucleotide is an efficient activator of RNase H. The presence of one or two mismatched nucleotides in the phosphodiester portion of oligonucleotides affected this activation only negligibly. When introduced into tax-transformed fibroblasts ex vivo, only the uniformly modified anti-tax phosphoramidate oligodeoxynucleotide caused a sequence-dependent reduction in the Tax protein level. Neither the chimeric phosphoramidate nor the phosphorothioate oligodeoxynucleotides significantly reduced tax expression under similar experimental conditions.

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Selected References

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  1. Brown D. A., Kang S. H., Gryaznov S. M., DeDionisio L., Heidenreich O., Sullivan S., Xu X., Nerenberg M. I. Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding. J Biol Chem. 1994 Oct 28;269(43):26801–26805. [PubMed] [Google Scholar]
  2. Burgess T. L., Fisher E. F., Ross S. L., Bready J. V., Qian Y. X., Bayewitch L. A., Cohen A. M., Herrera C. J., Hu S. S., Kramer T. B. The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):4051–4055. doi: 10.1073/pnas.92.9.4051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen J. K., Schultz R. G., Lloyd D. H., Gryaznov S. M. Synthesis of oligodeoxyribonucleotide N3'-->P5' phosphoramidates. Nucleic Acids Res. 1995 Jul 25;23(14):2661–2668. doi: 10.1093/nar/23.14.2661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dagle J. M., Weeks D. L., Walder J. A. Pathways of degradation and mechanism of action of antisense oligonucleotides in Xenopus laevis embryos. Antisense Res Dev. 1991 Spring;1(1):11–20. doi: 10.1089/ard.1991.1.11. [DOI] [PubMed] [Google Scholar]
  5. DeDionisio L., Gryaznov S. M. Analysis of a ribonuclease H digestion of N3'-->P5' phosphoramidate-RNA duplexes by capillary gel electrophoresis. J Chromatogr B Biomed Appl. 1995 Jul 7;669(1):125–131. doi: 10.1016/0378-4347(95)00153-a. [DOI] [PubMed] [Google Scholar]
  6. Ding D., Grayaznov S. M., Lloyd D. H., Chandrasekaran S., Yao S., Ratmeyer L., Pan Y., Wilson W. D. An oligodeoxyribonucleotide N3'--> P5' phosphoramidate duplex forms an A-type helix in solution. Nucleic Acids Res. 1996 Jan 15;24(2):354–360. doi: 10.1093/nar/24.2.354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Giles R. V., Spiller D. G., Green J. A., Clark R. E., Tidd D. M. Optimization of antisense oligodeoxynucleotide structure for targeting bcr-abl mRNA. Blood. 1995 Jul 15;86(2):744–754. [PubMed] [Google Scholar]
  10. Gryaznov S. M., Lloyd D. H., Chen J. K., Schultz R. G., DeDionisio L. A., Ratmeyer L., Wilson W. D. Oligonucleotide N3'-->P5' phosphoramidates. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):5798–5802. doi: 10.1073/pnas.92.13.5798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gryaznov S., Skorski T., Cucco C., Nieborowska-Skorska M., Chiu C. Y., Lloyd D., Chen J. K., Koziolkiewicz M., Calabretta B. Oligonucleotide N3'-->P5' phosphoramidates as antisense agents. Nucleic Acids Res. 1996 Apr 15;24(8):1508–1514. doi: 10.1093/nar/24.8.1508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heidenreich O., Kang S. H., Brown D. A., Xu X., Swiderski P., Rossi J. J., Eckstein F., Nerenberg M. Ribozyme-mediated RNA degradation in nuclei suspension. Nucleic Acids Res. 1995 Jun 25;23(12):2223–2228. doi: 10.1093/nar/23.12.2223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heidenreich O., Kang S. H., Xu X., Nerenberg M. Application of antisense technology to therapeutics. Mol Med Today. 1995 Jun;1(3):128–133. doi: 10.1016/s1357-4310(95)80090-5. [DOI] [PubMed] [Google Scholar]
  14. Heidenreich O., Xu X., Swiderski P., Rossi J. J., Nerenberg M. Correlation of activity with stability of chemically modified ribozymes in nuclei suspension. Antisense Nucleic Acid Drug Dev. 1996 Summer;6(2):111–118. doi: 10.1089/oli.1.1996.6.111. [DOI] [PubMed] [Google Scholar]
  15. Herschlag D. Implications of ribozyme kinetics for targeting the cleavage of specific RNA molecules in vivo: more isn't always better. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6921–6925. doi: 10.1073/pnas.88.16.6921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kitajima I., Shinohara T., Minor T., Bibbs L., Bilakovics J., Nerenberg M. Human T-cell leukemia virus type I tax transformation is associated with increased uptake of oligodeoxynucleotides in vitro and in vivo. J Biol Chem. 1992 Dec 25;267(36):25881–25888. [PubMed] [Google Scholar]
  17. Knudsen H., Nielsen P. E. Antisense properties of duplex- and triplex-forming PNAs. Nucleic Acids Res. 1996 Feb 1;24(3):494–500. doi: 10.1093/nar/24.3.494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krieg A. M., Stein C. A. Phosphorothioate oligodeoxynucleotides: antisense or anti-protein? Antisense Res Dev. 1995 Winter;5(4):241–241. doi: 10.1089/ard.1995.5.241. [DOI] [PubMed] [Google Scholar]
  19. Larrouy B., Boiziau C., Sproat B., Toulmé J. J. RNase H is responsible for the non-specific inhibition of in vitro translation by 2'-O-alkyl chimeric oligonucleotides: high affinity or selectivity, a dilemma to design antisense oligomers. Nucleic Acids Res. 1995 Sep 11;23(17):3434–3440. doi: 10.1093/nar/23.17.3434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Monia B. P., Lesnik E. A., Gonzalez C., Lima W. F., McGee D., Guinosso C. J., Kawasaki A. M., Cook P. D., Freier S. M. Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense inhibitors of gene expression. J Biol Chem. 1993 Jul 5;268(19):14514–14522. [PubMed] [Google Scholar]
  22. Sands H., Gorey-Feret L. J., Ho S. P., Bao Y., Cocuzza A. J., Chidester D., Hobbs F. W. Biodistribution and metabolism of internally 3H-labeled oligonucleotides. II. 3',5'-blocked oligonucleotides. Mol Pharmacol. 1995 Mar;47(3):636–646. [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]

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