DNA Nunchucks: Nanoinstrumentation for Single-Molecule Measurement of Stiffness and Bending

Bending of double-stranded DNA (dsDNA) has important applications in biology and engineering, but measurement of DNA bend angles is notoriously difficult and rarely dynamic. Here we introduce a nanoscale instrument that makes dynamic measurement of the bend in short dsDNAs easy enough to be routine. The instrument works by embedding the ends of a dsDNA in stiff, fluorescently labeled DNA nanotubes, thereby mechanically magnifying their orientations. The DNA nanotubes are readily confined to a plane and imaged while freely diffusing. Single-molecule bend angles are rapidly and reliably extracted from the images by a neural network. We find that angular variance across a population increases with dsDNA length, as predicted by the worm-like chain model, although individual distributions can differ significantly from one another. For dsDNAs with phased A₆-tracts, we measure an intrinsic bend of 17 ± 1° per A₆-tract, consistent with other methods, and a length-dependent angular variance that indicates A₆-tracts are (80 ± 30)% stiffer than generic dsDNA.