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December 2001
Vol. 4, No. 12, pp 12.
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Directing RNase H
Ribonuclease H from E. coli
Ribonuclease H from E. coli.
DNA replication is initiated at regions containing short RNA primers, creating a DNA–RNA hybrid. After initiation, the RNA primers are removed by several enzymes, one being RNase H, which cleaves RNA found in DNA–RNA hybrids in a non-sequence-specific manner.

Several research groups have focused the enzymatic activity of RNase H at specific mRNA transcripts, such as the gag mRNA in HIV-1, which could provide insight into viral replication and a prospective new viral therapy. This enzymatic activity may be accomplished by adding a DNA oligonucleotide that is complementary to the target mRNA sequence, creating a DNA–RNA hybrid, which recruits RNase H, resulting in degradation of the target mRNA. But for this approach to work biologically, the enzyme and the DNA oligonucleotide must localize in the same cellular compartments as the target, at proper concentrations. To probe the effectiveness of this strategy, George Wu and colleagues in the Department of Medicine at the University of Connecticut Health Center in Farmington have coupled RNase H to a DNA oligonucleotide and assayed the conjugate’s enzymatic activity (Bioconjugate Chem. 2001, 12, 770–775). The researchers chemically cross-linked a 13-base oligonucleotide that is complementary to the hepatitis B viral (HBV) mRNA, to E. coli RNase H. They assayed the effect of the DNA oligonucleotide/RNase H conjugate on a 1200-base fragment of HBV mRNA. The HBV mRNA was incubated with the unconjugated RNase H, the unconjugated DNA oligonucleotide, or the DNA–RNase H conjugate. The HBV mRNA transcripts were degraded only in the presence of the conjugate. Also, the degradation was targeted specifically to the HBV mRNA, as non-HBV mRNA remained undegraded.

A quantitative assay revealed that the degradation of HBV mRNA was dose-dependent, as 68% of the mRNA was digested when incubated with 5 µmol of conjugate, in contrast to 24% degradation when only 0.625 µmol of conjugate was added.

This work shows that RNase H can be made to specifically degrade HBV mRNA. More studies of this type are needed to gain further insight into viral replication and therapy.

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