Extending the RNA PPT primer by more than 18 DNA residues sensitizes the NNTIs while decreasing the potency of the NNRTIs. A, diagram of the chimeric primer/template substrates. B, time course of the different chimeric primer/template substrates. C, compound potency was established for each chimeric primer using the optimal reaction time derived from the time-course experiment showed in B (DNA PPT and RNA PPT+24D = 5 min; RNA PPT, RNA PPT+12D, and RNA PPT+18D = 30 min). D, -fold change in potency of RT inhibitors (IC50 using the chimeric primer/IC50 using the pure RNA PPT primer) upon addition of DNA residues at the 3′-end of the RNA PPT primer.