DNA Y Structure: A Multidimensional Single Molecule Assay

Abstract

Single molecule techniques have enabled significant advancement in the understanding of biological systems. However, current optical trapping techniques allow for force, extension and torque to be measured only along the axis of applied tension. This one dimensional aspect limits studies of complex biological systems due to the difficulty of data interpretation. Next generation optical trapping assays need to expand the number of measurement dimensions to capture the behaviors of multi-component biological systems. In addition, it would be advantageous for these new assays to combine optical trapping with fluorescence. Here, we present a novel assay which utilizes a dual beam optical trap to hold a three-arm DNA construct, which we call a Y structure. This design combines all manipulation capabilities of existing optical trapping techniques and enables simultaneous stretching, unzipping, and twisting of the same piece of DNA. We have characterized the mechanical properties of the Y structure by unzipping under force and torque, and have theoretically modeled the resulting data. We have also demonstrated that this assay is compatible with fluorescence by unzipping through a fluorescently labeled DNA-bound protein and observing its subsequent fate. These features, taken together, should enable the study of more complex biological systems.