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Nucleic Acids Res. 2010 Jan;38(3):1048-56. doi: 10.1093/nar/gkp1022. Epub 2009 Nov 20.

A single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity.

Author information

1
Department of Chemistry, McGill University, Montreal, QC, H3A 2K6 Canada.

Abstract

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.

PMID:
19933258
PMCID:
PMC2817455
DOI:
10.1093/nar/gkp1022
[Indexed for MEDLINE]
Free PMC Article

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