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. 2022 May 18;12(23):14883-14887.
doi: 10.1039/d2ra02131e. eCollection 2022 May 12.

Single-molecule study of the effects of temperature, pH, and RNA base on the stepwise enzyme kinetics of 10-23 deoxyribozyme

Jiwon Jung  1 , Seon Yong Kim  1 , Seong Keun Kim  1
Affiliations

Single-molecule study of the effects of temperature, pH, and RNA base on the stepwise enzyme kinetics of 10-23 deoxyribozyme

Jiwon Jung et al. RSC Adv. .

Abstract

We investigated how the stepwise enzyme kinetics of 10-23 deoxyribozyme was affected by temperature, pH, and RNA residue of the substrate at the single-molecule level. A deoxyribozyme-substrate system was employed to temporally categorize a single-turnover reaction into four distinct steps: binding, cleavage, dissociation of one of the cleaved fragments, and dissociation of the other fragment. The dwell time of each step was measured as the temperature was varied from 26 to 34 °C, to which the transition state theory was applied to obtain the enthalpy and entropy of activation for individual steps. In addition, we found that only the cleavage step was significantly affected by pH, indicating that it involves deprotonation of a single proton. We also found that different RNA residues specifically affect the cleavage step and cause the dwell time to change by as much as 5 times.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. (a) Experimental scheme for 10–23 deoxyribozyme immobilized on PEG-coated quartz slide via biotin-avidin interaction. The Cy3 (green)–Cy5 (red) FRET pair is labeled at each end of the 10–23 enzyme sequence, while the DNA substrate is designed with an RNA-based cleavage site in the middle of its own sequence shown by the dotted green circle. A 532 nm laser diode is used to generate an evanescent wave. (b) Schematic representation for cleavage reaction between 10–23 deoxyribozyme and DNA substrate. Upon injection, the substrate first binds to the immobilized 10–23 deoxyribozyme, becomes cleaved in half, and each piece dissociates from the enzyme successively under thermal energy. (c) Typical single-molecule time trace of FRET signals for the above reaction. A freely coiled enzyme (E ∼ 0.8) becomes stretched upon substrate binding to form an enzyme–substrate dsDNA (E ∼ 0.8→0.4). Substrate cleavage gives more structural freedom and allows further stretch of the enzyme (E ∼ 0.4→0.3), but sequential dissociation of the cleaved DNA pieces leads back to the original coiled form for the enzyme (E ∼ 0.3→0.4→0.8).
Fig. 2
Fig. 2. Arrhenius plot for each step of the overall reaction in the temperature range from 26 to 34 °C at pH 7.52. Optimized linear fitting parameters yield the values of enthalpy, entropy, and Gibbs free energy of activation given in Table 1.
Fig. 3
Fig. 3. pH-dependence of the kinetic rate of the individual reaction steps. (a) Only the rate of cleavage (red) is significantly affected by pH. The cleavage step plays the role of the rate-determining step, which becomes less distinct as pH increases. (b) A log–linear plot of the kinetic rate vs. pH for the cleavage step. A slope of 0.86 indicates that the cleavage step involves deprotonation of a single proton.
Fig. 4
Fig. 4. Effect of a mismatched residue in the substrate for each reaction step. Only the cleavage step is affected drastically while other steps are little affected.
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