Quantification of induced RNA mutation by nucleoside analogs in the repRNA-v4 EGFP region

REPLACE was engineered from an orthogonal alphaviral RNA replication system. It generates a large, continuously diversified library of replicative RNAs through a replicase-limited mode of replication and inducible mutagenesis. We analyzed the variation of RNA mutations induced by two nucleoside analogues over time and at different concentrations. This data was used to guide the construction of RNA mutant libraries. Overall design: EGFP was cloned into the repRNA-v4-derived vector, which was then transcribed in vitro into RNA and delivered into host cells via electroporation. After 24h of electroporation, cells were subjected to continuous puromycin treatment (10 µg/ml) with regular passages. After an additional 5-7 days, a relatively stable and homogeneous population of cells carrying replicative RNA expressing EGFP was established. These cells were then treated with nucleoside analogues at specified concentrations and durations. Total RNA was extracted from the cells and subjected to RNA sequencing to analyze the mutation information.