Polymerase-Mediated Site-Specific Incorporation of a Synthetic Fluorescent Isomorphic G Surrogate into RNA

An enzyme-mediated approach for the assembly of singly modified RNA constructs in which specific G residues are replaced with (th)G, an emissive isomorphic G surrogate, is reported. Transcription in the presence of thG and native nucleoside triphosphates enforces initiation with the unnatural analogue, yielding 5'-end modified transcripts that can be mono-phosphorylated and ligated to provide longer site specifically modified RNA constructs. The scope of this unprecedented enzymatic approach to non-canonical purine-containing RNAs is explored via the assembly of several altered hammerhead (HH) ribozymes and a singly modified HH substrate. By strategically modifying key positions, a mechanistic insight into the ribozyme-mediated cleavage is gained. Additionally, the emissive features of the modified nucleoside and its responsiveness to environmental changes can be used to monitor cleavage in real time by steady state fluorescence spectroscopy.