Assay for RNase H activity

A method for measuring a RNase H activity of an agent, which method comprises:

• (a) hybridizing a target RNA to a fluorescently labeled oligodesoxyribonucleotide probe complementary to the target RNA and containing a flourophor at one terminus and a quenching at the other terminus, wherein (i) when the probe is unhybridized to the target RNA, the probe adopts a conformation that places the flourophor and quencher in such proximity that the quencher quenches the flourescent signal of the flourophor and (ii) formation of the probe-target hybrid causes sufficient separation of the flourophor and quencher to reduce quenching of the flourescent signal of the flourophor;
• (b) contacting the probe-target hybrid with the agent in an amount sufficient to selectively cleave the target RNA and thereby release the intact probe;
• and
• (c) measuring a decrease in the flourescent signal of the flourophor as compared to the signal of the probe-target hybrid.

A method of screening for a modulator of the nuclease activity of an agent, which method comprises:

• (a) hybridizing a target nucleic acid to a fluorescently labeled oligonucleotide probe complementary to the target nucleic acid and containing a flourophor at one terminus and a quenching group at the other terminus, wherein (i) when the probe is unhybridized to the target nucleic acid, the probe adopts a conformation that places the flourophor and quencher in such proximity that the quencher quenches the flourescent signal of the flourophor and (ii) formation of the probe-target hybrid causes sufficient separation of the flourophor and quencher to reduce quenching of the flourescent signal of the flourophor;
• (b) preparing two samples containing the probe-target hybrid;
• (c) contacting the probe-target hybrid of a first sample with the agent in an amount sufficient to selectively cleave the target nucleic acid and thereby release the intact probe;
• (d) contacting the probe-target hybrid of a second sample with the agent in an amount sufficient to selectively cleave the target nucleic acid and thereby release the intact probe in the presence of a candidate compound, which is being tested for its ability to modulate the nuclease activity of the agent;
• (e) detecting the release of the probe in each sample by measuring a decrease in the flourescent signal of the flourophor as compared to the signal of the probe-target hybrid;
• and
• (f) comparing the rate of the decrease in the flourescent signal of the flourophor in the two samples, wherein a difference in the rate of the decrease in the flourescent signal of the flourophor during the nuclease reaction in the two samples is indicative of the ability of the compound to either inhibit or enhance the nuclease activity of the agent.