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

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 high-throughput productivity. This sRNA design platform consists of three modular plasmids: one plasmid contains an inducible sRNA and the RNA chaperone Hfq; the second contains an inducible fluorescent reporter protein and a LacY mutant transporter protein for inducer molecules; and the third plasmid contains a second inducible fluorescent reporter protein. The second reporter gene makes it possible to screen for sRNA regulators that have activity against multiple mRNAs. We describe the protocol for engineering sRNAs with novel regulatory activity using this system. This sRNA prototyping regimen could also be employed for validating predicted mRNA targets of uncharacterized, naturally occurring sRNAs or for testing hypotheses about the predicted roles of genes, including essential genes, in cellular metabolism and other processes, by using customized antisense sRNAs to knock down or tune down gene expression.

Retargeting a Dual-Acting sRNA for Multiple mRNA Transcript Regulation.

Multitargeting small regulatory RNAs (sRNAs) represent a potentially useful tool for metabolic engineering applications. Natural multitargeting sRNAs govern bacterial gene expression by binding to the translation initiation regions of protein-coding mRNAs through base pairing. We designed an Escherichia coli based genetic system to create and assay dual-acting retargeted-sRNA variants. The variants can be assayed for coordinate translational regulation of two alternate mRNA leaders fused to independent reporter genes. Accordingly, we began with the well-characterized E. coli native DsrA sRNA. The merits of using DsrA include its well-characterized separation of function into two independently folded stem-loop domains, wherein alterations at one stem do not necessarily abolish activity at the other stem. Expression of the sRNA and each reporter mRNA was independently controlled by small inducer molecules, allowing precise quantification of the regulatory effects of each sRNA:mRNA interaction in vivo with a microtiter plate assay. Using this system, we semirationally designed DsrA variants screened in E. coli for their ability to regulate key mRNA leader sequences from the Clostridium acetobutylicum n-butanol synthesis pathway. To coordinate intervention at two points in a metabolic pathway, we created bifunctional sRNA prototypes by combining sequences from two singly retargeted DsrA variants. This approach constitutes a platform for designing sRNAs to specifically target arbitrary mRNA transcript sequences, and thus provides a generalizable tool for retargeting and characterizing multitarget sRNAs for metabolic engineering.