A Novel Approach to the Detection of DNA-Enzyme Interaction Processes at a Single-Molecule Level

Abstract

A fundamental problem in both molecular biology and biophysics is how DNA-binding proteins find specific sites among huge amounts of non-specific (chromosomal) DNA. This in turn requires experimental procedures to elucidate the mechanisms of both non-specific and sequence-specific interactions of proteins with DNA. Current techniques are incapable of directly observing these interaction processes. We recently constructed a setup (See Image) for tracking trajectory of single reactant DNA molecules during enzymatic reaction. It uses a microfluidic flow system to pick reacting DNA molecules (previously tagged at one end by a quantum dot) out of the crowd of molecules. Restriction enzymes Not I, Apa I and EcoR I with their target and non-target DNA were analyzed. Quantitative assessments of their interaction time were achieved by sampling surveys of the single-molecule trajectories. With this system, we measured the duration difference of restriction-site-searching by these enzymes. The simplicity and versatility of the method suggest the possibility of more practical process analyses in living organisms.View Large Image | View Hi-Res Image | Download PowerPoint Slide