Nonradioactive Detection of Retroviral- Associated RNase H Activity in a Microplate-Based, High-Throughput Format
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.003 | 0.001 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.002 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.003 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.003 | 0.003 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".