DNA Shape Shifting As a Gene Therapy Tool

DNA supercoiling affects DNA metabolism yet much about how it does so is unknown. Using 336 bp DNA minicircles covering a range of positive to negative supercoiling, we unveiled the first three-dimensional structures of supercoiled DNA using cryo-electron tomography. With supercoiling, DNA can form far more bent and contorted shapes than predicted. We sought to understand how the interplay of DNA sequence and supercoiling drives the formation of these shapes using coarse-grained simulations and biochemical probing. Base pair disruptions indicate regions of high bending, as localized denaturation (from base flipping) creates flexible hinges. At the same time, sharp bending at the apices of highly writhed DNA circles leads to broken base pairs. Probing with nuclease Bal-31 revealed exposed bases as a function of supercoiling. Bal31 cleaved all the negatively supercoiled 336 bp minicircles but the rate increased beyond a distinct negative supercoiling threshold. This threshold shifted to more negative supercoiling for 672 bp minicircles with inherently less curvature, demonstrating the relationship between bending and base accessibility. A sharp positive supercoiling threshold was required for Bal-31 cleavage to even occur. We mapped Bal-31 cleavage sites and, using coarse-grained simulations, determined the DNA register of our cryo-electron micrograph images. Our data reveal two hotspots of Bal-31 cleavage located ∼180° apart along the DNA circumference, suggesting that exposed bases are predominantly located at superhelical apices and that a particular three-dimensional conformation is dominant. The relative probability of Bal-31 cleaving at either site varied as a function of supercoiling. Together these data reveal the interplay among sequence, supercoiling, and shape, resulting in conformational changes that should profoundly influence DNA interactions with proteins. Understanding these changes could facilitate the design of supercoiling-dependent DNA nanostructures for gene therapy.