Novel improvements to RNA synthesis for biophysical studies.

Abstract

RNA plays a central role in biology and is emerging as a powerful tool in nanotechnology, diagnostics, and therapeutics/vaccines. Biophysical characterizations of RNA in those roles and in complex processes like splicing, long noncoding RNA, and much more requires the preparation of homogeneous samples. While the T7 family of RNA polymerases is agile at synthesizing very large quantities of RNA, the pursuit of those high yields often drives off-pathway reactions that generate a wide range of contaminant RNAs. Building on a mechanistic understanding of the process, we are developing approaches for researchers to use that can net substantial improvements in RNA quality and yield. The first approach exploits high ionic strength to limit the RNA rebinding that drives off-pathway reactions, while strengthening of overall promoter binding allows the maintenance of high RNA production levels. A second approach tethers promoter DNA near its binding site to increase relative local concentrations. This allows promoter binding to better compete with RNA rebinding, and also allows for salt challenges for the latter. Finally, we are developing deep sequencing of in vitro transcription products to characterize polymerase fidelity and to compare fidelity under conditions widely used in RNA synthesis with more physiologically relevant conditions.