Detecting Poloidal Bias In DNA Minicircles

Bending of DNA over a few helical periods is anisotropic due to the helical nature of DNA (e.g., roll vs. twist and roll towards major groove vs. minor groove), and the degree of this anisotropy is expected to be DNA-sequence dependent. As a result, when DNA of about ten helical periods in length is covalently closed to form a minicircle, certain minor grooves may prefer facing inward to minimize the total bending energy of the minicircle. This alludes to a bias in the poloidal orientation of the minicircle. Such poloidal bias in the minicircle was seen in a recent molecular dynamics study, but has not been demonstrated experimentally. Here, we introduce a single-molecule fluorescence method to measure the poloidal bias in DNA minicircles. In this method, we exploit a DNA-binding protein, where its binding affinity depends on DNA curvature, and measure its binding kinetics to a series of DNA minicircles that contain a single binding site at different positions. Using the measured protein binding profile, we are able to confirm sequence-dependent poloidal bias of DNA minicircles predicted by a previous computational study.