Single-Molecule Study of the Enzymatic Mechanism and Kinetics of 10-23 Deoxyribozyme using Total Internal Reflection Fluorescence Microscopy

Abstract

Total internal reflection fluorescence (TIRF) microscopy, with the aid of the fluorescence resonance energy transfer (FRET) phenomenon, enables the real-time observation of nanometer-scale structural changes, which are often hidden in ensemble measurements, from a fluorescently labeled single biomolecule. Using TIRF microscopy, we investigated the stepwise enzymatic reaction mechanism of 10-23 deoxyribozyme, an RNA-cleaving DNA enzyme which has been widely studied in various fields of applied science. First, we analyzed the kinetics of the four normal reaction sub-steps of 10-23 deoxyribozyme of enzyme-substrate binding, substrate cleavage, and dissociations of each of the two products by varying the molecular environments specifically the temperature, the pH, and the residue of the cleavage site. Kinetic parameters and rates depending on the environmental changes revealed significant features with regard to the molecular mechanisms of each reaction. Secondly, we identified two additional individual reaction sub-steps which involve the half-binding of a new substrate before the second product dissociation that we termed shortcut binding and the subsequent strand displacement of the remaining product by the half-bound substrate. We determined that shortcut binding and strand displacement can occur and conducted a kinetic analysis of each reaction sub-step while varying the substrate concentration. Shortcut binding occurs more frequently at a higher substrate concentration and the rate of shortcut binding and the second dissociation are dependent on the substrate concentration, as the two reactions compete with each other. We calculated the rate equation, noting that shortcut binding accelerates the enzymatic turnover rate. To sum up, by adapting TIRF, we revealed the characteristics of 10-23 deoxyribozyme. These may be essential in the study of the chemical mechanisms and for explorations of the wider use of catalytic nucleic acids.