UC Santa Barbara

We present the design and application of a novel nanoscale instrument, the “DNA nunchuck”, which enables the dynamic measurement of bend angles in short double- stranded DNA (dsDNA) molecules. The DNA nunchuck, leveraging stiff, fluorescently- labeled DNA nanotubes, mechanically magnifies the orientations of an embedded dsDNA strand, allowing for precise angle determination through fluorescence imaging techniques combined with neural network image analysis. The validation of the DNA nunchuck tech- nique is demonstrated by reproducing expected relationships between nunchuck angular variance and dsDNA length, corroborating its effectiveness in accurately measuring DNA bending. Further application of the DNA nunchuck uncovers the dynamic mechanical behavior of short dsDNA sequences, particularly the existence of meta-stable bent states that have eluded detection with previous methodologies. Detailed analysis of nunchucks with a 37 bp embedded strand identifies a significant bent state, or “kink”, with a mean angle of 54 ± 2◦, providing insight into the anomalous high cyclization frequency ob- served in short dsDNA. This kinked state accounts for a substantial proportion of the observed bending dynamics at room temperature. The combined findings from the DNA nunchuck’s applications significantly advance our understanding of mechanical properties of dsDNA, contributing to a deeper understanding of its role in biological processes and its potential in nanoscale engineering. The work sets a precedent for future quantitativestudies of DNA mechanics and dynamics at the single-molecule level.