Antisense Gene Inhibition by C-5-Substituted Deoxyuridine-Containing Oligodeoxynucleotides

Antisense oligodeoxynucleotides (ODNs) are capable of inhibiting gene expression via a RNase H mechanism in which the complementary RNA is degraded by RNase H. C-5 propyne dU phosphorothioate ODNs bind selectively and with high affinity to RNA within cells leading to potent antisense inhibition of R...

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Bibliographic Details
Published in:Biochemistry (Easton) 1997-01, Vol.36 (4), p.743-748
Main Authors: Gutierrez, Arnold J, Matteucci, Mark D, Grant, Deborah, Matsumura, Sandra, Wagner, Richard W, Froehler, Brian C
Format: Article
Language:English
Subjects:
Animals
Antigens, Polyomavirus Transforming - genetics
Cell Line
Cell Nucleus - drug effects
Cell Nucleus - genetics
Cell Nucleus - metabolism
Gene Expression - drug effects
Kinetics
Microinjections
Molecular Structure
Oligonucleotides, Antisense - chemical synthesis
Oligonucleotides, Antisense - chemistry
Oligonucleotides, Antisense - pharmacology
Ribonuclease H - metabolism
RNA, Complementary - genetics
RNA, Complementary - metabolism
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Summary:Antisense oligodeoxynucleotides (ODNs) are capable of inhibiting gene expression via a RNase H mechanism in which the complementary RNA is degraded by RNase H. C-5 propyne dU phosphorothioate ODNs bind selectively and with high affinity to RNA within cells leading to potent antisense inhibition of RNA translation. The effect that increasing steric bulk of C-5-substituted deoxyuridine analogs has on affinity for RNA and ability to inhibit gene expression is discussed. The relative binding affinity was measured by thermal denaturation (Tm) analysis, and antisense activity was determined by inhibition of SV40 T-antigen (TAg) expression in CV1 cells. The results show that antisense activity is not directly correlated to Tm measurements. In vitro analysis (RNase H cleavage, on-rates, and off-rates) and pre-formed ODN/RNA experiments indicate that RNase H activity and intracellular dissociation appear to be major determinants of the antisense potency of the various substituted ODNs. The results of our analysis point to the unique ability of C-5 propyne dU ODNs to selectively bind to RNA within cells and activate cleavage of RNA by RNase H leading to potent inhibition of gene expression.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi9620971