Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1992 Feb 25;20(4):763–770. doi: 10.1093/nar/20.4.763

Increased specificity for antisense oligodeoxynucleotide targeting of RNA cleavage by RNase H using chimeric methylphosphonodiester/phosphodiester structures.

R V Giles 1, D M Tidd 1
PMCID: PMC312015  PMID: 1371864

Abstract

One of the inherent problems in the use of antisense oligodeoxynucleotides to ablate gene expression in cell cultures is that the stringency of hybridization in vivo is not subject to control and may be sub-optimal. Consequently, phosphodiester or phosphorothioate antisense effectors and non-targeted cellular RNA may form partial hybrids which are substrates for RNase H. Such processes could promote the sequence dependent inappropriate effects recently reported in the literature. We have attempted to resolve this problem by using chimeric methylphosphonodiester/phosphodiester oligodeoxynucleotides. In contrast to the extensive RNA degradation observed with all-phosphodiester oligodeoxynucleotides, highly modified chimeric antisense effectors displayed negligible, or undetectable, cleavage at non-target sites without significantly impaired activity at the target site. We also note that all of the all-phosphodiester oligodeoxynucleotides tested demonstrated inappropriate effects, and that such undesirable activity could vary widely between different sequences.

Full text

PDF

Images in this article

765Image
on p.765
767Image
on p.767

Selected References

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

  1. Bacon T. A., Wickstrom E. Walking along human c-myc mRNA with antisense oligodeoxynucleotides: maximum efficacy at the 5' cap region. Oncogene Res. 1991;6(1):13–19. [PubMed] [Google Scholar]
  2. Blake K. R., Murakami A., Spitz S. A., Glave S. A., Reddy M. P., Ts'o P. O., Miller P. S. Hybridization arrest of globin synthesis in rabbit reticulocyte lysates and cells by oligodeoxyribonucleoside methylphosphonates. Biochemistry. 1985 Oct 22;24(22):6139–6145. doi: 10.1021/bi00343a016. [DOI] [PubMed] [Google Scholar]
  3. Calabretta B., Sims R. B., Valtieri M., Caracciolo D., Szczylik C., Venturelli D., Ratajczak M., Beran M., Gewirtz A. M. Normal and leukemic hematopoietic cells manifest differential sensitivity to inhibitory effects of c-myb antisense oligodeoxynucleotides: an in vitro study relevant to bone marrow purging. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2351–2355. doi: 10.1073/pnas.88.6.2351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cazenave C., Loreau N., Thuong N. T., Toulmé J. J., Hélène C. Enzymatic amplification of translation inhibition of rabbit beta-globin mRNA mediated by anti-messenger oligodeoxynucleotides covalently linked to intercalating agents. Nucleic Acids Res. 1987 Jun 25;15(12):4717–4736. doi: 10.1093/nar/15.12.4717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Furdon P. J., Dominski Z., Kole R. RNase H cleavage of RNA hybridized to oligonucleotides containing methylphosphonate, phosphorothioate and phosphodiester bonds. Nucleic Acids Res. 1989 Nov 25;17(22):9193–9204. doi: 10.1093/nar/17.22.9193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Harel-Bellan A., Ferris D. K., Vinocour M., Holt J. T., Farrar W. L. Specific inhibition of c-myc protein biosynthesis using an antisense synthetic deoxy-oligonucleotide in human T lymphocytes. J Immunol. 1988 Apr 1;140(7):2431–2435. [PubMed] [Google Scholar]
  8. Harlow E., Williamson N. M., Ralston R., Helfman D. M., Adams T. E. Molecular cloning and in vitro expression of a cDNA clone for human cellular tumor antigen p53. Mol Cell Biol. 1985 Jul;5(7):1601–1610. doi: 10.1128/mcb.5.7.1601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Heikkila R., Schwab G., Wickstrom E., Loke S. L., Pluznik D. H., Watt R., Neckers L. M. A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1. 1987 Jul 30-Aug 5Nature. 328(6129):445–449. doi: 10.1038/328445a0. [DOI] [PubMed] [Google Scholar]
  10. Holt J. T., Redner R. L., Nienhuis A. W. An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation. Mol Cell Biol. 1988 Feb;8(2):963–973. doi: 10.1128/mcb.8.2.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kroczek R. A. Immediate visualization of blotted RNA in northern analysis. Nucleic Acids Res. 1989 Nov 25;17(22):9497–9497. doi: 10.1093/nar/17.22.9497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Laurence J., Sikder S. K., Kulkosky J., Miller P., Ts'o P. O. Induction of chronic human immunodeficiency virus infection is blocked in vitro by a methylphosphonate oligodeoxynucleoside targeted to a U3 enhancer element. J Virol. 1991 Jan;65(1):213–219. doi: 10.1128/jvi.65.1.213-219.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Levy Y., Tsapis A., Brouet J. C. Interleukin-6 antisense oligonucleotides inhibit the growth of human myeloma cell lines. J Clin Invest. 1991 Aug;88(2):696–699. doi: 10.1172/JCI115355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Miller P. S., Ts'o P. O. A new approach to chemotherapy based on molecular biology and nucleic acid chemistry: Matagen (masking tape for gene expression). Anticancer Drug Des. 1987 Oct;2(2):117–128. [PubMed] [Google Scholar]
  16. Shuttleworth J., Colman A. Antisense oligonucleotide-directed cleavage of mRNA in Xenopus oocytes and eggs. EMBO J. 1988 Feb;7(2):427–434. doi: 10.1002/j.1460-2075.1988.tb02830.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shuttleworth J., Matthews G., Dale L., Baker C., Colman A. Antisense oligodeoxyribonucleotide-directed cleavage of maternal mRNA in Xenopus oocytes and embryos. Gene. 1988 Dec 10;72(1-2):267–275. doi: 10.1016/0378-1119(88)90152-7. [DOI] [PubMed] [Google Scholar]
  18. Smith R. C., Bement W. M., Dersch M. A., Dworkin-Rastl E., Dworkin M. B., Capco D. G. Nonspecific effects of oligodeoxynucleotide injection in Xenopus oocytes: a reevaluation of previous D7 mRNA ablation experiments. Development. 1990 Nov;110(3):769–779. doi: 10.1242/dev.110.3.769. [DOI] [PubMed] [Google Scholar]
  19. Stein C. A., Subasinghe C., Shinozuka K., Cohen J. S. Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res. 1988 Apr 25;16(8):3209–3221. doi: 10.1093/nar/16.8.3209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Storey A., Oates D., Banks L., Crawford L., Crook T. Anti-sense phosphorothioate oligonucleotides have both specific and non-specific effects on cells containing human papillomavirus type 16. Nucleic Acids Res. 1991 Aug 11;19(15):4109–4114. doi: 10.1093/nar/19.15.4109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tidd D. M. A potential role for antisense oligonucleotide analogues in the development of oncogene targeted cancer chemotherapy. Anticancer Res. 1990 Sep-Oct;10(5A):1169–1182. [PubMed] [Google Scholar]
  22. Tidd D. M., Hawley P., Warenius H. M., Gibson I. Evaluation of N-ras oncogene anti-sense, sense and nonsense sequence methylphosphonate oligonucleotide analogues. Anticancer Drug Des. 1988 Aug;3(2):117–127. [PubMed] [Google Scholar]
  23. Tidd D. M., Warenius H. M. Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups. Br J Cancer. 1989 Sep;60(3):343–350. doi: 10.1038/bjc.1989.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Watt R., Stanton L. W., Marcu K. B., Gallo R. C., Croce C. M., Rovera G. Nucleotide sequence of cloned cDNA of human c-myc oncogene. Nature. 1983 Jun 23;303(5919):725–728. doi: 10.1038/303725a0. [DOI] [PubMed] [Google Scholar]
  25. Wickstrom E. L., Bacon T. A., Gonzalez A., Freeman D. L., Lyman G. H., Wickstrom E. Human promyelocytic leukemia HL-60 cell proliferation and c-myc protein expression are inhibited by an antisense pentadecadeoxynucleotide targeted against c-myc mRNA. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1028–1032. doi: 10.1073/pnas.85.4.1028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Woolf T. M., Jennings C. G., Rebagliati M., Melton D. A. The stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos. Nucleic Acids Res. 1990 Apr 11;18(7):1763–1769. doi: 10.1093/nar/18.7.1763. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES