Bacterial small RNAs (sRNAs) have been established as powerful parts for controlling gene expression. However， development and application of engineered sRNAs has primarily focused on regulating novel synthetic targets. In this work， we demonstrate a rational modular RNA engineering approach that uses in vivo structural accessibility measurements to tune the regulatory activity of a multisubstrate sRNA for differential control of its native target network. Employing the CsrB global sRNA regulator as a model system， we use published in vivo structural accessibility data to infer the contribution of its local structures (substructures) to function and select a subset for engineering. We then modularly recom... More
Bacterial small RNAs (sRNAs) have been established as powerful parts for controlling gene expression. However， development and application of engineered sRNAs has primarily focused on regulating novel synthetic targets. In this work， we demonstrate a rational modular RNA engineering approach that uses in vivo structural accessibility measurements to tune the regulatory activity of a multisubstrate sRNA for differential control of its native target network. Employing the CsrB global sRNA regulator as a model system， we use published in vivo structural accessibility data to infer the contribution of its local structures (substructures) to function and select a subset for engineering. We then modularly recombine the selected substructures， differentially representing those of presumed high or low functional contribution， to build a library of 21 CsrB variants. Using fluorescent translational reporter assays， we demonstrate that the CsrB variants achieve a 5-fold gradient of control of well-characterized Csr network targets. Interestingly， results suggest that less conserved local structures within long， multisubstrate sRNAs may represent better targets for rational engineering than their well-conserved counterparts. Lastly， mapping the impact of sRNA variants on a signature Csr network phenotype indicates the potential of this approach for tuning the activity of global sRNA regulators in the context of metabolic engineering applications.
