<br/><br/><b>T</b>he recent explosion in RNA research has largely been driven by the discovery of non-coding RNAs (ncRNA) which have a wide range of important functions. This has led to the development of novel methods to explore RNA structure, dynamics, and functions; many of which are based on fluorescence. Presently, the methods employed to incorporate fluorophores to the RNA of interest (ROI) have a wide range of limitations in labelling long and structured RNA. We have therefore proposed the development of a novel, site-specific, chemo-enzymatic method of fluorophore incorporation to RNA. This novel method is based on employing Klenow fragment of DNA polymerase I to incorporate modified nucleotides to RNA in a templated manner. We have achieved efficient 3’ terminal end labelling using this method. However, utilizing wild type Klenow in elongating the 3’ terminally modified RNA has not been successful. To achieve Klenow mediated RNA extension, we are designing a new variant of Klenow enzyme to improve its utility as an RNA polymerase. This new variant will have two baseline mutations in the sugar gate and second gate residues that control the substrate selectivity of a DNA polymerase.This will allow us to achieve internal modifications in the ROI which would allow for subsequent labelling based on NHS ester chemistry.

Designing RNA with site-specific fluorophores using a chemo-enzymatic approach

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.