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Retargeting a Dual-Acting sRNA for Multiple mRNA Transcript Regulation

Wood, D. W., Ohio State University

We are interested in regulating multiple mRNAs within a metabolic pathway for balancing flux of metabolites during fermentation. Bacterial small regulatory sRNAs act by specific "antisense" sRNA:mRNA complementary base pairing interactions with one or more mRNAs. The sRNAs vary the efficiency of translation of mRNA into proteins by perturbing mRNA structures, leading to targeted mRNA turnover or enhanced translation in vivo. Alterations of sRNA antisense sequence frequently diminish sRNA activity or stability. We hypothesized that distinctive stem-loop antisense structural motifs of the E. coli DsrA sRNA were critical to its robust formation of antisense sRNA:mRNA interactions. Two separate DsrA sequences within stem-loops each specify an sRNA:mRNA interaction that targets translation of a native mRNA. We built an E. coli genetic system to quantify perturbation of mRNA translation in vivo by DsrA sRNA and its derivatives. In this system, levels of DsrA and its two mRNA reporters are precisely controlled by orthogonal, small molecule-induced transcription control. Native mRNA target sequences were each fused to distinct (mCherry, GFP) reporter gene fluorescent proteins. The activity of DsrA at these reporter genes was validated during cell growth in a 96-well format for high-throughput screens at different steady-state transcript levels of DsrA and mRNAs. We then rationally designed retargeted DsrA stem-loop antisense regions by editing complementary sequences to make them pair with desired mRNA targets, while conserving the DsrA native-like stem-loop structure. We retargeted each antisense stem-loop of DsrA sRNA to act singly at one of two non-native mRNA reporter gene fusion targets from the n-butanol fermentation pathway of Clostridium acetobutylicum. Most of our singly-retargeted sRNAs functioned as anticipated by tuning down target mRNA translation (~2-9 fold), validating our approach for metabolic engineering by mRNA target regulation. Further, we recombined single-retargeted stem-loops to create dual-retargeted DsrA variants that can act at two non-native mRNA targets in E. coli. This sRNA stem-loop antisense motif is therefore a robust structural element that can be retargeted to govern non-native mRNAs without loss of sRNA function, enabling tailored targeting to desired mRNA sequences. The universality of sRNAs as a genetic control mechanism in bacteria suggests that these sRNA derivatives can be validated in E. coli and then implemented in situ in industrially relevant host organisms such as Clostridium.