A novel synthetic sRNA promoting protein overexpression in cell-free systems.

Bacterial small RNAs (sRNAs) that regulate gene expression have been engineered for uses in synthetic biology and metabolic engineering. Here, we designed a novel non-Hfq-dependent sRNA scaffold that makes use of a 20 nt antisense binding region to selectively target mRNAs and influence expression. The system was developed for regulation of a fluorescent reporter in vivo using E. coli, but the system was found to be more responsive and produce statistically significant results when applied to protein synthesis in in vitro cell-free systems (CFS). Antisense binding sequences were designed to target not only translation initiation regions but various secondary structures in the reporter mRNA. Targeting a high energy stem loop structure and the 3' end of the mRNA produced expression knock-downs that approached 70%. Notably, targeting a low energy stem structure near a potential RNase E binding site led to a statistically significant 65% increase in gene expression. These results were not obtainable in vivo, and the underlying mechanism was translated from the reporter system to achieve better than 75% increase in recombinant diaphorase expression in an in vitro CFS. It is possible the designs developed here can be applied to improve expression of other proteins in a CFS. This article is protected by copyright. All rights reserved.