Abstract
Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the “Broccoli” RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, covariation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro.
Highlights
Methods to monitor transcription in vitro largely relied on the incorporation of radioactive nucleotides or detection of transcripts by hybridization-based methods [1,2,3,4]
The results of this study provide a resource to researchers interested in the properties of T7 RNA polymerase (RNAP) or that use T7 RNAP for routine synthesis of RNA in the laboratory
Fluorescence was typically measured every minute in a standard quantitative PCR instrument in small 10-μl reactions in a 96-well format
Summary
Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, covariation of magnesium and nucleoside triphosphates, and the effects of several typical additives When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. Screening approach that uses common laboratory equipment would enable cost-effective screening of focused small chemical libraries Inspired by this need, we designed a simple, rapid throughput assay for monitoring transcriptional output in realtime with a fluorescent RNA aptamer reporter transcript.
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