RNA-based fluorescent biosensors for live cell detection of bacterialsRNA

Bacteria contain a diverse set of RNAs to provide tight regulation of gene expression in response to environmental stimuli. Bacterial small RNAs (sRNAs) work in conjunction with protein cofactors to bind complementary mRNA sequences in the cell, leading to up- or downregulation of protein synthesis.In vivoimaging of sRNAs can aid in understanding their spatiotemporal dynamics in real time, which inspires new ways to manipulate these systems for a variety of applications including synthetic biology and therapeutics. Current methods for sRNA imaging are quite limitedin vivoand do not provide real-time information about fluctuations in sRNA levels. Herein, we describe our efforts toward the development of an RNA-based fluorescent biosensor for bacterial sRNA bothin vitroandin vivo. We validated these sensors for three different bacterial sRNAs inEscherichia coliand demonstrated that the designs provide a bright, sequence-specific signal output in response to exogenous and endogenous RNA targets.