Nonradioactive detection of retroviral-associated RNase H activity in a microplate-based, high-throughput format.
None of the available antiretroviral drugs that are currently used in the clinic to treat infection with HIV-1 is directed against the RNase H active site of the reverse transcriptase. Here we developed a nonradioactive, 96-well plate assay designed to be used for high-throughput screening of compounds capable of inhibiting the RNase H activity of HIV-1 reverse transcriptase. We employed a tRNA as substrate that was labeled with digoxygenin-modified reporter residues. The labeled tRNA was prehybridized with a DNA oligonucleotide that contained a single biotinylated residue at its 5'-terminus to ensure its attachment to streptavidin-coated microplates. The uncleaved, immobilized DNA/tRNA substrate was detected through the use of established ELISA protocols. Incubation with purified HIV-1 reverse transcriptase initiated RNase H degradation and caused a signal reduction to negligible background levels. In contrast, the signal intensity remained unaffected when using an RNase H deficient mutant enzyme. The assay was validated using the hydrazone derivative BBNH that was previously shown to inhibit RNase H degradation below concentrations of 10 microM.
McGill University, Montréal, Québec, Canada.
N
N
McLellan
X
X
Wei
B
B
Marchand
M A
MA
Wainberg
M
M
Götte
D001431:Bacteriological Techniques / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation* / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling; D011786:Quality Control; D016914:Ribonuclease H / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity
D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H