α-Anomeric DNA: β-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase

Abstract

Nuclease-resistant α-anomeric DNA: β-rna hybrids are inhibitors of Escherichia coli RNase H, and Drosophila embryo RNase H. RNase H activities were measured by polyacrylamide gel electrophoresis, employing a short substrate, (A) 12: d[G-G-(T) 12-G-G], or by acid-solubility techniques, using a long substrate, poly(A): poly(dT). Strand exchanges which could be responsible for the observed inhibition have been ruled out by S1 nuclease experiments and by using inhibitors which do not allow strand exchange. Our results suggest that RNase H, for which DNA: RNA duplexes are the natural substrates, binds to non-physiological α-DNA: RNA hybrids and is consequently inhibited. These hybrids also inhibit the RNA-dependent DNA polymerase activity of M-MLV reverse transcriptase, therefore appearing as potential inhibitors of at least two reverse transcriptase activities. However, the inhibitory effect of these hybrids with respect to M-MLV reverse transcriptase is also observed with the single-stranded α-DNA itself. Unexpectedly, polymerase activity is highly stimulated by α-oligos, analogous in their sequence to the β primer used at a concentration unable to generate a detectable synthesis. These results suggest that the inhibition of reverse transcriptase activity with the α: β may occur at different levels.