Effect of Seed Length and Binding Motifs on Hfq-Mediated sRNA-mRNA Annealing Analyzed Using Single-Molecule FRET

Defining the rules that govern annealing between bacterial small RNAs (sRNAs) and their mRNA targets is challenging because these RNAs are highly heterogeneous in sequence and structure. To disentangle the contribution of individual features, we systematically varied key determinants, including seed strength, Hfq-binding motifs, and the spacing between them. The effects of these features on sRNA-mRNA duplex stability were tested using single-molecule Förster resonance energy transfer (smFRET), which enables monitoring of individual RNA molecules on the millisecond timescale and provides access to dynamic events often obscured in ensemble assays. We demonstrate that short seed regions (4-5 bp) form unstable, short-lived complexes, whereas longer seeds (8-10 bp) produce stable duplexes. Extending Hfq-binding motifs or introducing a spacer between the binding sites on mRNA further increased interaction lifetimes, highlighting the importance of motif strength and spatial arrangement in Hfq-mediated annealing efficiency. Moreover, this approach provides a robust and reproducible framework for dissecting RNA-RNA interaction dynamics at single-molecule resolution and for probing RNA chaperone mechanisms.