Figure 3.

Representative single-turnover kinetic data for the minimal ribozyme with high activity (HH_min-AL4:U1.7) shown in Figure 2a, and summarized in the first row (Experiment 1) of Table 1. The fraction of product produced as a function of time, F(t), was quantified in triplicate by polyacrylamide gel electrophoresis separation of fluorescently-labelled substrate and product (gel inset). The data were fit to a single standard exponential function^11,12, where F_o = 0.11 is the initial fraction of cleavage product, and F_sat = 0.9 is the estimated extent of cleavage based upon the final three time points. Non-linear regression analysis was performed to estimate k_obs, as described in the experimental methods. The most conservative estimate for k_obs is 0.77/min at pH 5.6, which is essentially the same as k_obs for an optimized one-stranded full-length hammerhead based on the sTRSV+ natural full-length hammerhead RNA sequence (HH_16-T2)²⁴, as shown in Experiment B in Table 1. The graph inset expands the initial time-points of the data-set. (The data were also fit to a more complex kinetic model with an unrestrained two-phase exponential function, producing an estimated fast-phase k_obs of 3.6 ± 1.5/min corresponding to the initial time-points in the inset, and a slower phase of 0.2 ± 0.1/min, with unrestrained endpoints of F_o = 0.11 ± 0.01 and F_sat = 0.91 ± 0.05.)