RT-qPCR measurements of ribozyme cleavage of gates produced in E. coli. (A) The workflow for expressing RNA in E. coli. Pink lines and gray lines inside cells indicate endogenous RNA and gate RNA, respectively. Gate sequences were cloned into a pET plasmid backbone and then transformed into E. coli BL21* (DE3). Cells cultured with IPTG express T7 RNAP, which transcribes the pET plasmid to produce gate RNA. Total cellular RNA was then extracted for analysis. (B) Fraction uncleaved RNA for gates with a minimal antigenomic HDV ribozyme produced in cells. Reverse transcription was conducted at 37°C. Error bars indicate standard deviation from three technical replicates. Replicate 1 and replicate 2 indicate replicates from two cell cultures and RNA extractions conducted on different days. Amplification curves and primer efficiency plots are presented in Supplemental Sections 2.4, 2.5. (C) Schematic of controls to evaluate whether the cell RNA extraction protocol influenced the ribozyme cleavage measurement. In sample iii, IVT RNA (gray) was taken through the entire cell RNA extraction protocol. In sample iv, cells without the plasmid were grown and lysed to yield endogenous RNA (pink), IVT RNA (gray) was added to the lysed cells, and the cell RNA extraction protocol was followed. The numbers in the box below the RT-qPCR arrow indicate the measured fraction uncleaved. Sample ii through sample iv were all >0.5 fraction uncleaved, as expected. Limitations with plate size prevented technical replicates in this experiment, but the expected standard deviation based on previous G2 technical replicates is approximately ±0.1. These results indicate cell RNA extraction or the presence of cellular RNA cannot account for the lower fraction uncleaved measured in sample i.
