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Real-time Dynamics and Kinetic Study of 10-23 Deoxyribozyme Using Single-molecule Microscopy Technique
단분자 현미경법을 이용한 10-23 디옥시라이보자임의 실시간 운동 및 동역학적 연구

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Authors
정지원
Advisor
김성근
Major
자연과학대학 화학부
Issue Date
2015-02
Publisher
서울대학교 대학원
Keywords
단분자분광학
Description
학위논문 (박사)-- 서울대학교 대학원 : 화학부, 2015. 2. 김성근.
Abstract
We studied the dynamic motion and kinetics of 10-23 deoxyribozyme which has RNA-cleaving enzymatic activity. Observing dynamic motion of the enzyme is essential to elucidate the mechanism of the enzymatic reaction. Also, kinetic information of the reaction is To observe every reaction sub-steps, we developed temperature-controlled single-molecule total internal reflection fluorescence (smTIRF) microscopy. For a good signal-to-noise level, we applied prism-type smTIRF microscopy rather than objective-type smTIRF microscopy. Also, we used fluorescence resonance energy transfer (FRET) to distinguish the structural changes of 10-23 deoxyribozyme in nano-meter scale.

i) Real-time dynamic trace shows four reaction steps, binding, cleavage, dissociation1, and dissociation2 steps, for a single-turnover reaction. At the binding step, tightly coiled 10-23 deoxyribozyme waits for the binding of the substrate with a high FRET value, E~0.82. Because of the negative charges in the phosphate backbone of the DNA, the coiled structure of the enzyme is critically affected by the divalent cations in the environment. In our experiments, Mg2+ divalent cations, which are injected in the solution to turn on the enzymatic reaction, neutralized negative charges of the DNA strands, so that the repulsion forces between negative charges are sufficiently reduced. After the binding of the substrate, double-stranded enzyme-substrate complex forms the double-strand moiety at the
two binding sites that the structure changes from the single strand to the nearly linear double strand, E~0.35. After the cleavage of the substrate, FRET value is further decreased to E~0.30 which means the distance of
the each ends of the enzyme-products complex is more far than that of the enzyme-substrate state, E~0.35. Unfortunately, we could not observe the transient structure of the enzymatically active state. Finally, FRET value was sequentially restored its initial value, E~0.30 → 0.49 → 0.82, by the dissociation of the two products made the enzyme to be a freely coiled structure of the initial state. In conclusion, we directly observed four enzymatic reaction steps which are impossible to distinguish in conventional ensemble experiments.

ii) In ensemble tests, no one can concisely measure the kinetic rate constants because of the error from the ensemble averaging problem. However, exact kinetic rate constants can be obtained by single-molecule experiments even in single-reaction-step level. Here, we exactly measured the kinetic rate constants for the previously observed enzymatic reaction distinguishing each reaction steps. Four reaction steps differently responded to changes of the environmental conditions, temperature, pH,
RNA residue at the cleavage site, and viscosity. When we varied temperature of the reaction chamber, the reaction rates of entire reaction steps are quickened in the range of 26~34℃ at pH 7.52. For more kinetic information, Eyring-Polanyi equation (derived from transition state theory) was applied to the temperature dependence data. Transition state theory (TST) shows the binding and the cleavage steps are associative reactions in contrast to that the dissociation steps are dissociative reactions. And, we concluded that the melting of the strands, the catalytic effect for the transesterification reaction, and the dissociation of the Watson-Crick base pairing and base-base stacking interaction is important for the binding, the cleavage, and the dissociation steps, respectively. In pH test, we observed that the only cleavage step is critically affected by pH change. The pH
dependency for the cleavage reaction shows a log-linear plot that means the single deprotonation reaction is the rate-determining step for the cleavage reaction. The binding and the dissociation steps show no response to the change of pH in the range of 7.33~7.81 at 30℃. Changing an RNA residue at the middle of the substrate shows similar tendency to pH test. The only cleavage reaction was critically affected by the change of an RNA residue of the substrate. In last, we tested the effect of the viscosity on the enzymatic reaction rates. We changed the concentration of the glycerol up to 25% to control the viscosity coefficient of the solution. Because of the viscosity and the chemical effect of the addition of the glycerol, the cleavage step and the dissociation steps are affected in the different tendencies. The cleavage reaction was slowed down as half as the rate for the water-only environment. In contrast to the cleavage step, sequential dissociation steps were quickened as twice as the rate for the water-only environment. We concluded that the effect of the glycerol on the kinetic rate of enzymatic reaction is come from the viscosity effect that causes the slowdown effect on the folding of the enzymatic structure and the chemical effect that leads to the weakness of the H-bonds. We expect that the single-molecule kinetics for 10-23 deoxyribozyme could be expanded to the other RNA/protein enzyme kinetics.

iii) To complete the enzymatic reaction, the enzyme should bind to the substrate at first. Because of that, the concentration of the substrate is critical to the entire kinetic rate of the enzyme-substrate system. The
enzyme typically shows multi-turnover behavior when the substrate concentration is sufficiently high relative to the concentration of the enzyme. Also, Michaelis-Menten mechanism tells us that the rate maximum is achieved when all enzymes is bound to the substrates. To measure rate
maximum of 10-23 deoxyribozyme at the single-molecule level, we increased the concentration of the substrate from 50 nM up to 800 nM. Surprisingly, the dissociation step was not found in most dynamic traces at 800 nM substrate concentration. In contrast to single-molecule experiment, gel electrophoresis results show that the enzyme reaction at high substrate concentration have sufficiently fast as reported in the other reports. With
several experiments in control, we concluded that the final dissociation of the product is skipped or accelerated by the invasion of the new substrate. To finish the reaction, the products should dissociate from the enzyme to empty the binding site for the next binding of the new substrates. However, we found out that the new substrate binds to the half binding site before the complete of the second dissociation step. Also, single-molecule kinetic analysis shows that the dissociation of the second product is accelerated by the half-bound new substrate. Here, we propose the new mechanism of 10-23 deoxyribozyme, named shortcut binding and strand displacement, which shows a different behavior with the kinetics expected by Michaelis-Menten mechanism. In addition, we expect that the shortcut binding and
the strand displacement is important for the enzymatic reaction of the RNA-cleaving ribozyme for better activity in vivo. We observed the real-time analysis of the 10-23 deoxyribozyme reaction and found the new mechanism that accelerates the enzymatic reaction. Also, we assigned all kinetic rate constants in the single-reaction-step level,
and the results show that there is the different behavior of each reaction steps for the change of the environment.
Language
English
URI
http://hdl.handle.net/10371/125279
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College of Natural Sciences (자연과학대학)Dept. of Chemistry (화학부)Theses (Ph.D. / Sc.D._화학부)
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