A 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.
Wahba
Alexander S
AS
Department of Chemistry, McGill University, Montreal, QC, H3A 2K6 Canada.
Esmaeili
Abbasali
A
Damha
Masad J
MJ
Hudson
Robert H E
RH
D003562:Cytidine / Q000031:analogs & derivatives* / Q000737:chemistry; D057075:Enzyme Assays / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry*; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000737:chemistry; D011758:Pyrroles / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D016914:Ribonuclease H / Q000378:metabolism*
C541628:6-phenylpyrrolocytidine; D005456:Fluorescent Dyes; D009843:Oligoribonucleotides; D011758:Pyrroles; D003562:Cytidine; D012313:RNA; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H