Fig. 1. Single-molecule assay for real-time sRNA exchange.

a Biotinylated Hfq (BioHfq) hexamers were complexed with Cy3-labeled resident sRNAs and immobilized on a passivated microscope slide treated with NeutrAvidin. Sparse biotinylation ensures that most hexamers have only one immobilized CTD. The addition of Cy5-labeled competitor sRNA triggers displacement of resident Cy3-sRNA over time (see also Supplementary Fig. 4b). b Percent remaining resident sRNAs 5 min after addition of competitor sRNA, as a function of competitor concentration. Symbols represent the mean and s.d. of 10 different fields of view (FOV) from two independently prepared samples. Solid lines are a visual guide and do not represent a mathematical model. H: Hfq, DA: DsrA, CX: ChiX. c Representative single-molecule trajectory depicting the displacement of a resident sRNA from Hfq following competitor binding. d Representative plot (out of two replicates) showing the percentage of resident remaining on Hfq as a function of time, following the addition of 5 nM competitor sRNA. The number of active Cy3 molecules in a single 100 ms frame was counted every 10 frames (1 s) and divided by the total number of Cy3 molecules at the start of the movie. H-DA (gray) and H-CX (black) represent the dissociation in the absence of a competitor. Source data are provided as a Source Data file.