Fig. 5: Seed and non-seed binding within single miRISC complexes.

A Schematic showing the design of Cy3-labeled seed (t10_7S) and Cy5 labeled non-seed probes (t8_NS) employed to simultaneously investigate seed and non-seed accessibility of miR-21 RISC. B Scatter plot of the median τ_bound and τ_unbound dwell times when both seed (t10_NS) and non-seed probe (t8_NS) are employed simultaneously. The scatter plot shows three distinct clusters whose kinetics are consistent with miRISC populations accessible to either the seed (green cluster) or non-seed (orange and blue clusters) probe. C Representative intensity versus time traces of miR-21 RISCs whose chromato-kinetic properties are suggestive of interaction with t8_NS (upper and middle traces), and t10_7S (lower trace). The relative percentage of each kind of trace accepted in the collected movie are mentioned. D Venn diagram showing that a 20% of long-lived non-seed binding component has also seed accessibility and vice versa. However, the transient non-seed component and a fraction of long-lived non-seed binding components have no seed accessibility. E Schematic showing the design of FRET probes for probing simultaneous seed and non-seed accessibility. Cy3 labeled 10-nt seed probe serve as a FRET donor, and a Cy5-labeled 8-nt non-seed probe acts as a FRET acceptor. F Representative FRET trajectories showing simultaneous seed and long-lived supplementary plus non-seed binding, evident from anti-correlated changes in Cy3 and Cy5 signal. Total number of traces showing FRET is N_FRET = 135 and Number of traces that show only seed binding is N_{5’S only} = 180. G Plot showing the fraction of seed-accessible miR-21 RISCs that also show binding to the supplementary and non-seed region by the t8_NS or t10_NS probe. Error bars represent the SEM from two independent replicates. The fraction of molecules showing 3’NS interaction is \({f}_{t10{ns}}^{3^{\prime} {NS}/({3}^{{\prime} }{NS}+{5}^{{\prime} }S)}=0.7\pm 0.05\) and \({f}_{t8{ns}}^{3^{\prime} {NS}/({3}^{{\prime} }{NS}+{5}^{{\prime} }S)}=0.4\pm 0.05.\) H The percentage of different miRISC conformations present in cell extract. The purple bar represents the percentage of miRISC complexes (~70%) showing stable 5’S binding and the yellow bar represents the percentage of supplementary and 3’NS binding miRISC (~30%, sum of transient and long-lived binding component) that do not show 5’S binding. Mean of independent triplicates ±SEM has been shown. Created in BioRender. Walterlab, B. (2025) https://BioRender.com/uucpy2t.