biostudies-literatureUnknown38(3)Wahba AS6-Phenylpyrrolocytidine (PhpC), a structurally conservative and highly fluorescent cytidine analog, was incorporated into oligoribonucleotides. The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group being non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for HIV-1 RT Ribonuclease H and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional molecular beacon approach in terms of responsiveness, rapidity and ease (single label versus dual). Furthermore, the PhpC-based assay is amenable to high-throughput microplate assay format and may form the basis for a new screen for inhibitors of HIV-RT RNase H.Nucleic acids research1048-56https://www.ebi.ac.uk/biostudies/studies/S-EPMC2817455biostudies-literatureA single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity.PMC2817455Damha MJHudson RHWahba ASEsmaeili AfalseA single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity.6-Phenylpyrrolocytidine (PhpC), a structurally conservative and highly fluorescent cytidine analog, was incorporated into oligoribonucleotides. The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group being non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for HIV-1 RT Ribonuclease H and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional molecular beacon approach in terms of responsiveness, rapidity and ease (single label versus dual). Furthermore, the PhpC-based assay is amenable to high-throughput microplate assay format and may form the basis for a new screen for inhibitors of HIV-RT RNase H.2010-01-01T00:00:00Z2010 Jan2025-04-03T22:24:19.988Z2019-03-27T00:28:22ZS-EPMC28174551993325810.1093/nar/gkp1022