Department of Chemistry, McGill University, Montreal, QC, H3A 2K6 Canada.
Nucleic Acids Res. 2010 Jan;38(3):1048-56. doi: 10.1093/nar/gkp1022. Epub 2009 Nov 20.
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.
6-苯基吡咯胞苷（PhpC）是一种结构保守且具有强荧光的胞苷类似物，被整合到寡核苷酸中。含有 PhpC 的 RNA 与互补的 DNA 或 RNA 形成类似天然的双链结构。PhpC 修饰被发现是一种灵敏的报告基团，对结构和 RNase H 的酶活性没有破坏作用。具有单个 PhpC 插入的 RNA/DNA 杂交物是 HIV-1 RT 核糖核酸酶 H 的极好底物，其 RNA 链的切割迅速，荧光强度增加 14 倍。基于 PhpC 的 RNase H 测定法在响应性、速度和易用性（单标记与双标记）方面优于传统的分子信标方法。此外，基于 PhpC 的测定法适用于高通量微孔板测定格式，并且可能成为 HIV-RT RNase H 抑制剂新筛选的基础。
