Fig. 4: Seed-independent binding of non-seed target mimic probes to miR-21 RISC with heterogeneous kinetics.

A Schematic showing base complementarity between RISC-loaded miR-21 and target mimic probes of different lengths. B Scatter plots of the median τ_bound and τ_unbound dwell times in the presence of Cy5-labeled 8-nucleotide (t8_NS, upper scatter plot) and 10-nucleotide (t10_NS, lower scatter plot) target mimics interacting with miR-21 RISC. Two distinct clusters of traces characterized by different binding kinetics (blue squares and orange filled circles) are seen in each scatter plot with respective total accepted molecules (N) indicated. C Two representative intensity-versus-time trajectories showing distinct kinetics of interaction of the same non-seed probe (t8_NS) with different copies of miR-21 RISC. The upper trace (orange) shows transient binding events, while the lower trace (blue) shows long-lived binding interactions. D Normalized relative populations of the long-lived and transient binding components for the respective probes employed; mean of independent triplicates ±SEM. E, F Association (k_a) and dissociation (k_dis) rate constants for the transiently binding component (E) and long -lived binding component (F) were calculated from exponential fitting to the cumulative distributions of τ_unbound and τ_bound dwell times, respectively (error bars represent the SEM from three independent replicates; Statistical significance of differences in the rate constants was determined using two-tailed Student’s test (t test). P values < 0.1 were considered significant ***p < 0.01, **p < 0.05, *p < 0.1). G Binding affinity (dissociation equilibrium constant, K_D) of target mimics having varying degrees of guide complementarity to the supplementary and 3’NS region of miR-21 RISC. Mean of independent triplicates ± SEM. H, I Calculated Fano factor for transiently binding component (H) and long-lived binding component (I) indicates non-Poissonian statistics. The black dots represent the Fano factor of the simulated data. The dashed lines indicate the 95% confidence interval for Poisson behavior. J Cumulative histogram displaying the distribution of inter spike intervals (ISIs) during burst period for transient (orange shade) and long-lived component (blue shade). K Cumulative histogram displaying the distribution of inter spike intervals (ISIs) during non-burst period for transient (orange shade) and long-lived component (blue shade). Created in BioRender. Nils, W. (2025) https://BioRender.com/k6iju48.