Biophysical Society 2022
MECHANISM-DRIVEN APPROACHES TOWARDS SYNTHESIS OF HIGHLY PURE RNA FOR A WIDE VARIETY OF APPLICATIONS
Authors: : Craig T. Martin, Ruptanu Banerjee, Amin Abek, Kithmie MalagodaPathiranage.
Chemistry, University of Massachusetts Amherst, Amherst, MA, USA.
Tuesday, Feb 22 1:45pm
The impact of RNA in science has grown exponentially in the past three decades. In biology, alternative splicing, long noncoding RNAs, RNA editing, and riboswitches complement translation and transcription. RNA is also increasingly used in nanotechnology and medicine. Biophysical studies of RNA require high purity, yet RNA is still synthesized today as it was 30 years ago and is similarly characterized with gel assays that cannot assess a wide diversity of impurities. Although RNA analytics are only starting to catch up, we know that in vitro transcription, as currently practiced, introduces a range of heterogeneous impurities. This arises largely due to the accumulation of product in traditional high yield reactions. The product RNA rebinds the polymerase to template extensions to the originally encoded RNA. Our lab is now developing greatly improved approaches for RNA syntheses. In one type of approach, high salt inhibits RNA product rebinding, while constructs that strengthen the polymerase-promoter interaction rescue salt’s inhibition of promoter binding (elongation is insensitive to salt). Relatively simple approaches can dramatically improve both the quality and yield of RNA, and the approaches apply equally to short and long RNAs. We are also developing flow synthesis approaches that remove RNA from the reaction as soon as it is formed, further preventing the conversion of correct product into (much) longer, partially double stranded RNA impurities. The end result is higher yields RNA with substantially higher purity.