Calibrating Mechanical Amplifiers of DNA Bend Dynamics
We are developing nanostructures that can be used to amplify the dynamic bending of DNA to better understand how the structure and movement of DNA affects gene expression. Our nanostructures, called nunchucks, consist of a double stranded piece DNA with a DNA nanotube on each end that magnify the strand’s bending. However, the tile-based nanotubes nucleate off of DNA origami seeds that are difficult to determine the concentration of, and produce highly variable yields each time. In order to consistently increase the yield of nunchucks, we are calibrating a fluorescence-based assay (Qubit® Fluorometer 3.0) against a microscopy assay to quantify the seed concentration. Varying volumes of seeds were added to a solution of supersaturated tiles and nunchucks were counted by microscopy. We determined the optimal amount of seeds to add based on Qubit reading to ensure an adequate amount of sufficient length nunchucks. Higher Qubit readings correlated with greater seeds concentrations that could be added in smaller amounts to maintain tube length, whereas lower Qubit readings indicated lower seed concentration that required being added in greater amounts to boost yield. Qubit measurements provide a quick and easy way to achieve consistent nunchuck yields given the variability of origami seed concentration.