Combinatorial selection for replicable RNA by Qβ replicase while maintaining encoded gene function
To estimate the kinetic parameters, KM and kcat, the rates of complementary strand synthesis were measured using the plus (A) or minus strands (B) of the original and m4 RNA clones. Each template RNA (0, 5, 10, 20, 40, and 100 nM) was incubated with the replicase (10 nM) at 37°C for 5 min, and the synthesized RNA was measured as described in the Materials and Methods section. The plots were fitted to Michaelis-Menten equations (solid lines) for estimating Vmax and KM. To estimate the kcat values, we used the active ratio of the replicase (20%) . The estimated parameters are shown in Table 2 along with the fitting errors. The error bars represent standard errors (n = 3).