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

Authors

Alexander S. Wahba, McGill University
Abbasali Esmaeili, University of Birjand, Birjand, Iran
Masad J. Damha, McGill University
Robert H. E. Hudson, University of Western Ontario

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.