Association (A) and dissociation (B) kinetics follow a two-step mechanism. Observed rate constants for bimolecular (kobs1) and isomerization (kobs2) steps are a function of microscopic rate constants. In association kinetics (A), kobs1 is linearly dependent on molar concentration of dmDcr-2 (1 µM after twofold dilution upon mixing, see Materials and methods), and therefore, accurate evaluation of the second-order rate constant (k+1, slope) requires kobs1 values to be measured over a fairly wide range of enzyme concentration. Since a non-ideal (aggregative behavior) of dmDcr-2 at higher micromolar concentration precludes determination of kobs1 as a function of enzyme concentration (see Materials and methods), we estimated the second-order rate constant from the kobs1 value measured at a single dmDcr-2 concentration. To enhance accuracy in values of estimated microscopic rate constants as reported in (C), we used the mean value of observed rate constants (BLT, kobs1 = 1.24 s−1, kobs2 = 0.013 s−1; 3’ovr, kobs1 = 0.36 s−1, kobs2 = 0.01 s−1) obtained from three independent experiments; values of kobs measured using different batches of protein and/or dsRNA, in general, varied by less than or equal to twofold. The Kd values for enzyme–substrate interaction were evaluated using all microscopic rate-constants; values of k-1 and k-2 reported in (C) represent an average of three independently determined values from dissociation off-rate measurements. The kinetically determined Kd values reported in (C), Kd = (k-1k-2)/(k+1k+2), are within experimental error of those previously determined with equilibrium binding studies (Donelick et al., 2020).