A Modular Genetic System for High-Throughput Profiling and Engineering of Multi-Target Small RNAs

Methods in Molecular Biology
Samuel D StimpleRichard A Lease

Abstract

RNA biology and RNA engineering are subjects of growing interest due to recent advances in our understanding of the diverse cellular functions of RNAs, including their roles as genetic regulators. The noncoding small RNAs (sRNAs) of bacteria are a fundamental basis of regulatory control that can regulate gene expression via antisense base-pairing to one or more target mRNAs. The sRNAs can be customized to generate a range of mRNA translation rates and stabilities. The sRNAs can be applied as a platform for metabolic engineering, to control expression of genes of interest by following relatively straightforward design rules (Kushwaha et al., ACS Synth Biol 5:795-809, 2016). However, the ab initio design of functional sRNAs to precise specifications of gene control is not yet possible. Consequently, there is a need for tools to rapidly profile uncharacterized sRNAs in vivo, to screen sRNAs against "new/novel" targets, and (in the case of metabolic engineering) to develop engineered sRNAs for regulatory function against multiple desired mRNA targets. To address this unmet need, we previously constructed a modular genetic system for assaying sRNA activity in vivo against specifiable mRNA sequences, using microtiter plate assays for...Continue Reading

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