Tracking Degradations of Single DNA and Protein Molecules in Fluid

Abstract

Moving images obtained from optical microscopic studies with single biomolecules, including DNA and proteins, provide amazing insights into physico-chemical fundamentals such as dynamics and kinetics in a particular environment. Previously, the observation of large numbers of individual molecules has been used to detect identifiable individual chemical events or components of a chemical synthesis system. These may offer crucial clues towards intricate molecular mechanisms. Despite this importance, analytical applications still have lagged behind the establishment of theoretical principles. Based on the Michaelis-Menten equation, values for the reaction rate constants have traditionally been calculated from the solution phase reaction kinetics. This procedure is predictably effective for discussing a minimal model of the kinetics. However, most biomolecular interactions are thought to involve multiple steps, typically an initial binding followed by a structural rearrangement. Particular attention should be given to the fractionally-sampled molecular steps. Our analysis, described here, uses a technology to determine the detailed molecular information about interactions between DNA and DNA interactive protein. It uses motion capture technology that was originally developed for recording biomechanical movement onto a digital model. We applied it for motion tracking and position sensing of a single DNA molecule undergoing restriction enzyme digestion in a microfluidic device. Quantum dot and total internal reflection fluorescence microscope were used as a marker and a tracker respectively, which allowed motion capture of DNA during interfacial reactions. With our analysis, an enzymatic degradation time was detected at a single molecule level. It was also possible to calculate the observed catalytic rate constant. As an application case of our tracking measurement, protease activity of trypsin was monitored in real time. The geometrical features of the biological process being studied reflect transient molecular actions that are otherwise inaccessible to traditional biochemical methods.