Phosphorothioate oligonucleotides are inhibitors of human DNA polymerases and RNase H: implications for antisense technology
Phosphorothioate oligodeoxycytidine (S-dCn) was used as a model compound to examine the impact of the number of phosphorothioate linkages and their position on the inhibition of human DNA polymerases and RNase H in vitro. S-dCn with a chain length longer than 15 could inhibit human DNA polymerases a...
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Veröffentlicht in: | Molecular pharmacology 1992-02, Vol.41 (2), p.223-229 |
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Zusammenfassung: | Phosphorothioate oligodeoxycytidine (S-dCn) was used as a model compound to examine the impact of the number of phosphorothioate
linkages and their position on the inhibition of human DNA polymerases and RNase H in vitro. S-dCn with a chain length longer
than 15 could inhibit human DNA polymerases and RNase H activities, in a linkage number-dependent manner. Longer oligomers
were more potent inhibitors than shorter ones. Kinetic studies indicated that S-dC28 was a competitive inhibitor of DNA polymerase
alpha and beta with respect to the DNA template, whereas it was a noncompetitive inhibitor of polymerases gamma and delta.
S-dC28 was also a competitive inhibitor of RNase H1 and H2 with respect to RNA-DNA duplex. Susceptibility of these enzymes
to inhibition by S-dC28 was in the order of delta approximately gamma greater than alpha greater than beta and RNase H1 greater
than RNase H2. Structural-activity relationships were explored with a group of S-dC28 analogs that have phosphorothioate internucleotide
linkages at various positions. The inhibitory effect depended on the total number of thioate linkages, rather than the position
of the linkages within the oligomer or the chain length itself. No sequence specificity was found. In the presence of the
complementary RNA, antisense phosphorothioates (S-oligos) exerted a biphasic effect on RNase H activity. At low concentrations
S-oligos could enhance the cleavage of the RNA portion of S-oligo-RNA duplex, whereas at high concentrations (in excess of
the complementary RNA) S-oligos could inhibit RNase H and protect the complementary RNA from degradation. Together, these
results suggest that the non-sequence-specific inhibitory effect of S-oligos should be taken into consideration in designing
antisense inhibitors. This inhibitory activity could be avoided by decreasing the number of phosphorothioate linkages at the
backbone, and S-oligos of 15-20 residues are preferable in antisense molecule design. |
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ISSN: | 0026-895X 1521-0111 |