Title

A Single-label Phenylpyrrolocytidine Provides a Molecular Beacon-like Response Reporting HIV-1 RT RNase H Activity

Document Type

Article

Publication Date

1-2010

Journal

Nucleic Acids Research

Volume

38

Issue

3

First Page

1048

Last Page

1056

URL with Digital Object Identifier

http://dx.doi.org/10.1093/nar/gkp1022

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.