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, kon, and dissociation, koff. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decreased kon and increased koff, providing an in vivo demonstration that Hfq directly facilitates sRNA-mRNA annealing. Single base-pair mismatches in the annealing region reduced kon by 24-31% and increased koff 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.