| Assay Method Information | |
| | HIV-1 RT-RNase H MLSCN MH077605 Probe Assessment: Dose response Assay |
| Description: | A fluorescence resonance energy transfer assay is developed in 96-well and 384-well microplate formats with robotic manipulation to enable high-throughput screening for inhibitors of HIV-1 RT-RNH. The substrate is an 18 nucleotide 3 - fluorescein labeled RNA annealed to a complementary 18 nucleotide 5 - DABCYL modified DNA. The intact duplex has an extremely low background fluorescent signal and provides up to 50-fold fluorescent signal enhancement following hydrolysis. The size and sequence of the duplex are such that RT-RNH cuts the RNA strand four nucleotides from the 3-end. The labeled tetraribonucleotide readily dissociates from the complementary DNA at ambient temperature with immediate generation of a fluorescent signal. The assay is rapid, inexpensive and robust, providing Z factors of 0.8 and coefficients of variation of about 5%. The assay requires only two addition steps with no washing and is thus suitable for robotic operation. Plate 10 uL of 100 uM compound in 10% DMSO into 320 wells of a 384-well black polystyrene plate (VWR catalog 82051-272). Use the PMLSC Rapid 320 plate map. Actives identified in the HIV RNase H primary HTS, were confirmed by re-screening at 10 uM in duplicate wells, AID (pending). If the compound was active in the primary HTS, and the % inhibition in both of the confirmation duplicate tests was > 50%, it was followed up in 10-point IC50 dose response assays. |
| Affinity data for this assay | |
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