RT Journal Article
SR Electronic
T1 Deep sequencing of nonenzymatic RNA primer extension
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.02.18.955120
DO 10.1101/2020.02.18.955120
A1 Daniel Duzdevich
A1 Christopher E. Carr
A1 Jack W. Szostak
YR 2020
UL http://biorxiv.org/content/early/2020/02/19/2020.02.18.955120.abstract
AB Life emerging in an RNA world is expected to propagate RNA as hereditary information, requiring some form of primitive replication without enzymes. Nonenzymatic template-directed RNA primer extension is a model of the polymerisation step in this posited form of replication. The sequence space accessed by primer extension dictates potential pathways to self-replication and, eventually, ribozymes. Which sequences can be accessed? What is the fidelity of the reaction? Does the recently-illuminated mechanism of primer extension affect the distribution of sequences that can be copied? How do sequence features respond to experimental conditions and prebiotically relevant contexts? To help answer these and related questions, we here introduce a deep-sequencing methodology for studying RNA primer extension. We have designed and vetted special RNA constructs for this purpose, honed a protocol for sample preparation and developed custom software that sorts and analyses raw sequencing data. We apply this new methodology to proof-of-concept controls, and demonstrate that it works as expected and reports on key features of the sequences accessed by primer extension.