Nonradioactive detection of retroviral-associated RNase H activity in a microplate-based, high-throughput format

Biotechniques. 2002 Aug;33(2):424-9. doi: 10.2144/02332ht03.


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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Bacteriological Techniques / methods*
  • Enzyme-Linked Immunosorbent Assay / instrumentation*
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Escherichia coli / enzymology
  • HIV Reverse Transcriptase / chemistry
  • HIV Reverse Transcriptase / metabolism
  • Isotope Labeling
  • Quality Control
  • Ribonuclease H / analysis*
  • Ribonuclease H / chemistry
  • Ribonuclease H / metabolism
  • Sensitivity and Specificity


  • HIV Reverse Transcriptase
  • Ribonuclease H