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. 2004 Jun 24;101(27):10066–10071. doi: 10.1073/pnas.0403575101

Table 1. Single-molecule docking and undocking rate constants of modified hairpin ribozyme–substrate and –product analog complexes compared to WT.

Variant (number of trajectories) Subpopulation Fraction kdock (s-1) kundock (s-1) k′undock (s-1) k′dock (s-1)
WT (454) I 0.62 0.0086 ± 0.0010 0.004 ± 0.001 0.011 ± 0.001 0.020 ± 0.001
II 0.08 0.079 ± 0.020 0.076 ± 0.002
III 0.13 0.59 ± 0.10 0.4 ± 0.2
IV 0.17 4.5 ± 1.0 5 ± 3
dC12 (201) I 0.62 0.0049 ± 0.0006 0.0223 ± 0.0004 0.142 ± 0.001 0.016 ± 0.001
II 0.11 0.83 ± 0.07 5 ± 5
III (u.a.) 0.27 n.d. n.d.
dA38 (252) I 0.64 0.020 ± 0.002 0.26 ± 0.01 0.63 ± 0.10 0.034 ± 0.003
II 0.10 1.7 ± 0.1 5 ± 3
III (u.a.) 0.26 n.d. n.d.
C39S3 (370) I 0.64 0.033 ± 0.002 0.034 ± 0.001 0.095 ± 0.003 0.071 ± 0.006
II 0.09 0.3 ± 0.1 0.23 ± 0.06
III 0.09 3 ± 1 3.4 ± 0.6
IV (u.a.) 0.18 n.d. n.d.
RzAS3 (835) I 0.48 0.30 ± 0.04 0.0061 ± 0.0001 0.020 ± 0.003 0.23 ± 0.01
II 0.06 0.05 ± 0.02 0.19 ± 0.01
III 0.06 0.68 ± 0.40 0.91 ± 0.001
IV 0.04 4.5 ± 0.3 6 ± 2
V (u.a.) 0.07 n.d. n.d.
I′ 0.09 0.038 ± 0.03 0.0061 ± 0.0004 0.02 ± 0.001 0.029 ± 0.005
II′ 0.07 0.05 ± 0.02 0.19 ± 0.01
III′ 0.05 0.68 ± 0.40 0.91 ± 0.04
IV′ 0.05 4.5 (3) 6 ± 2
V′ (u.a.) 0.03 n.d. n.d.
RzAS3/C39S3 (560) I 0.53 1.0 ± 0.1 0.022 ± 0.001 0.17 ± 0.01 0.51 ± 0.03
II 0.05 0.55 ± 0.04 0.83 ± 0.08
III 0.02 5 ± 1 5 ± 5
IV (u.a.) 0.08 n.d. n.d.
I′ 0.13 0.13 ± 0.03 0.022 ± 0.001 0.17 ± 0.01 0.10 ± 0.03
II′ 0.08 0.55 ± 0.04 0.83 ± 0.08
III′ 0.07 5 ± 1 5 ± 5
IV′ (u.a.) 0.04 n.d. n.d.

Docking and undocking rate constants were determined under standard conditions (50 mM Tris-HCl, pH 7.5/12 mM Mg2+/25°C) by statistical analysis of single-molecule FRET time trajectories, as described in Materials and Methods and Supporting Text. The estimated confidence intervals have the same accuracy as the last digit. In case of the dC12, dA38, C39S3 and RzAS3/C39S3 variants, undocking is significantly faster than that of the WT. It is thus likely that the fastest undocking rate constants become faster than our experimental time resolution of 100 ms. This is consistent with larger unassigned subpopulations in these variants compared to the WT. u.a., unassigned subpopulation; n.d., undetermined rate constant, in kinetic simulations assumed to be equivalent to our lower-limit estimate of 10 s-1.