Dissecting the multistep reaction pathway of an RNA enzyme by single-molecule kinetic “fingerprinting”

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   Fig. 1.

   Multistep catalytic reaction by the hairpin ribozyme. (A) The 2WJ hairpin ribozyme used in this study. An AC₅ linker was added to the ribozyme to keep substrate or the 5′ product linked to the ribozyme. It has been shown that the ribozyme with AC₅ linker behaves similarly to the nicked 2WJ ribozyme (11, 17). Substrate is colored in orange. The cleavage site is indicated by a red arrow. Biotin and fluorophores Cy3 and Cy5 were attached as indicated. (B) Multistep cleavage reaction scheme. (C) Representative time trace of Cy3 and Cy5 fluorescence and the corresponding FRET values of a ribozyme molecule undergoing cleavage. Mg²⁺ (12 mM) was added at 100 s. The trace was recorded at 1 frame per second (fps), and the HMM analysis was applied to the FRET trace (pink line). (D) Histogram of FRET values obtained from many time traces as shown in C, but counting only the part after Mg²⁺ addition. The three peaks at FRET = 0.18, 0.30, and 0.76 represent product-released (P_R), undocked (U_L and U_C), and docked (D_L and D_C) states, respectively. (E) FRET histogram for ribozymes complexed with noncleavable substrate (with the native 2′-OH of A-1 substituted with 2′-OMe) in the presence of 12 mM Mg²⁺.

   
   
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   Fig. 2.

   Binding and dissociation of the cleavage product 3′P. (A) Schematic of experimental principle. 3′P with fluorescence quencher dabcyl (3′P-D) was added to the immobilized and cleaved ribozyme in the presence of Mg²⁺. The green, red, and black dots indicate Cy3, Cy5, and dabcyl, respectively. (B) Representative time traces of Cy3 and Cy5 fluorescence of a single ribozyme molecule in the presence of 500 nM 3′P-D. The trace was recorded at the frame rate of 33 fps. In the undocked state, 3′P-D stochastically bound to and dissociated from the ribozyme, leading to repetitive quenching and dequenching of the Cy5 signal, whereas the signal from the more distant Cy3 stayed large. In the docked state, 3′P-D remained bound, quenching signals from Cy3 and Cy5, now both in proximity to the quencher. (C) Cumulative dwell-time histogram of the 3′P unbound state in the presence of 500 nM 3′P-D. The histogram (circles) is fit to a single exponential (red line), yielding an observed binding rate constant of 2.8 s⁻¹. (D) Cumulative dwell-time histogram of the 3′P bound state (circles), which is fit to a single exponential (red line), yielding a dissociation rate constant of 2.6 s⁻¹.

   
   
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   Fig. 3.

   Docking kinetics of the ligated and cleaved forms of the ribozyme. (A) Reaction scheme to determine k _dock ^L with Mg²⁺ added at 100 s to induce docking, shown together with a representative single-molecule FRET time trace (1 fps) and HHM analysis (pink line). k _dock ^L is determined from the time (T _L) between Mg²⁺ addition and docking. (B) Reaction scheme to determine k _dock ^C with 3′P added at 100 s to induce docking, shown with a representative FRET time trace (1 fps) and HHM analysis (pink line). k _dock ^C is determined from the time (T _C) between 3′P binding and the first docking event. (C) The cumulative histogram of T _L (circles), which is fit to a single exponential (red line), yielding a docking rate constant k _dock ^L = 0.013 s⁻¹. (D) The cumulative histogram of T _C (circles), which is fit to a single exponential (red line), yielding a docking rate constant k _dock ^C = 0.012 s⁻¹.

   
   
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   Fig. 4.

   Equilibrium constants for docking and the internal chemistry. (A) Schematic of the sequential buffer exchange experiment. Equilibrium among state U_L, D_L, D_C, and U_C was first reached by placing molecules in a solution containing 12 mM Mg²⁺ and 30 μM 3′P for >1 h. At time t ₀, an unfolding buffer containing no Mg²⁺ or 3′P but 10 mM EDTA was added to force undocking and product release. Then a folding buffer containing 12 mM Mg²⁺ but no 3′P was added at t ₁ to trigger docking. Molecules in state U_L, D_L, D_C, and U_C at t ₀ showed distinct kinetic fingerprints, allowing them to be clearly distinguished. (B) Representative FRET time traces (1 fps) for each of the four scenarios. The first and second buffer exchanges were at 0 and 100 s, respectively. Scenarios 1, 2, 3, or 4 correspond to a molecule at state U_L, D_L, D_C, or U_C at t ₀, respectively.

   
   
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   Fig. 5.

   Dwell-time analysis of the last docking event before 3′P release. (A) Reaction scheme with Mg²⁺ added at 100 s to induce docking and cleavage, shown with a representative FRET time trace (1 fps). The HMM analysis is shown in a pink line. The dwell time T of the last FRET = 0.8 event before releasing 3′P was analyzed. (B) The cumulative histogram of T (circles) is fit to a single exponential (red line) with a decay constant of m = 0.0032 s⁻¹, where m is a function of k _undock ^L, k _undock ^C, k _lig, and k _cleav.

   
   
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   Fig. 6.

   The overall cleavage kinetics catalyzed by the 2WJ hairpin ribozyme. (A) Summary of the rate constants along the reaction pathway for the major (stably docked) population of the enzyme determined in this work. (B) (Upper) Number of molecules transitioned to the P_R state per 30-s interval (N) as a function of time with Mg²⁺ being added at t = 0. Red curve indicates data simulated (N _sim,i) using the kinetic rate constants determined above without any adjustable parameter and black curve indicates data obtained experimentally (N _exp,i). (Inset) Integrated number of molecules accumulated in the P_R state as a function of time (simulated curve: red; experimental curve: black). (Lower) The residual plot showing the difference between the simulated and experimental data. A standardized residual is calculated as ( .