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

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.

SUBMITTER: Wahba AS

PROVIDER: S-EPMC2817455 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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

Wahba Alexander S AS Esmaeili Abbasali A Damha Masad J MJ Hudson Robert H E RH

Nucleic acids research 20091120 3

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 report ...[more]

PMID: 19933258

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