PRJNA666229
Effects of individual base-pairs on in vivo target search and destruction kinetics of small RNA
Base-pairing interactions mediate intermolecular target recognition in many biological systems and applications, including DNA repair, CRISPR, microRNA, small RNA (sRNA) and antisense oligo therapies. Even a single base-pair mismatch can cause a substantial difference in biological activity but presently we do not yet know how the target search kinetics in vivo are influenced by single nucleotide level changes. Here, we used high-throughput sequencing to identify functionally relevant single point mutants of the bacterial sRNA, SgrS, and quantitative super-resolution microscopy to probe the mutational impact on the regulation of its primary target, ptsG mRNA. Our super-resolution imaging and analysis platform allowed us to further dissect mutational effects on SgrS lifetimes, and even subtle changes in the in vivo rates of target association, k on , and dissociation, k off . Mutations that disrupt Hfq binding and are distal to the mRNA annealing region still decreased k on and increased k off , providing an in vivo demonstration 37 that Hfq directly facilitates sRNA-mRNA annealing. Single base-pair mismatches in the annealing region reduced k on by 24-31% and increased k off by 14-25%, extending the time it takes to find and destroy the target mRNA by about a third, depending on whether an AU or GC base-pair is disrupted. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq also buffers base-pair disruptions.