In this paper, we report a stepwise RNA-primed RNA polymerization method for the site-specific incorporation of multiple fluorescent moieties into RNA, mediated by an RNA polymerase. A screen of several RNA polymerases revealed that T7 RNA polymerase was the only one that functioned in the RNA-primed RNA polymerization. In the first fluorescence labeling step, a fluorescent rUthioTP residue was incorporated directly into the RNA using T7 RNA polymerase; the second fluorescence labeling step was performed using a post-labeling strategy: directly introducing an rUamiTP residue into RNA, using T7 RNA polymerase, and then reacting with ylidenemalononitrile enamine (P3). The whole process for the site-specific introduction of the multiple labeled moieties was performed through stepwise RNA-primed RNA polymerization. Interestingly, the resulting multiple-labeled RNA exhibited fluorescence resonance energy transfer (FRET) between the two fluorescent labels in the RNA. We optimized the FRET-breaking point in the RNA by changing of distance between the two fluorescent labels and then used this property for the detection of the structural change of the RNA. The FRET signal increased in intensity upon the transformation of the RNA from a single-strand structure to the G-quadruplex. This approach for site-specific FRET labeling into RNA using RNA polymerase suggests the possibility of performing other diverse site-specific modifications at predefined positions in RNA.
Keywords: FRET; RNA polymerase; enzymatic RNA labeling; fluorescent nucleotide; post labeling; primer extension.