EQuAL Student Seminar: Ruiyao Liu

Metastable Bent States of Short, Double-Stranded DNA with and without Defects Revealed by DNA Nunchucks

It is now well-established that short dsDNA can cyclize much more easily than implied by the bending stiffness deduced from the worm-like chain model that fits well to measurements of long dsDNA. Indeed, high-throughput DNA cyclization measurements are now used on the genome-scale to characterize sequence encoded DNA mechanics. However, such measurements reveal little about the nature of short dsDNA bending. Do distinct bent states exist? If so, what are their angles and angular variances? How frequently do they arise? How are they altered, if at all, by the presence of insertions/deletions (ie, “bulges”) and mismatches (ie, “bubbles”)? To address these fundamental questions, we have developed modular DNA nunchucks that enable single-molecule measurement of DNA bending in aqueous solution and in real time via conventional fluorescence microscopy. We find that the aggregate bend angle distribution of nunchucks with perfectly complementary 37-bp dsDNA linkers is not consistent with a normal distribution centered at (and folded about) μ = 0°. By aggregating the bend angles from only those nunchucks whose distributions fit well to a folded normal with μ = 0°, we find an angular variance consistent with the measured persistence length of long dsDNA. Assuming this unbent state is the ground state of the linker, we characterize its bent states by fitting the aggregated bend angles of the remaining nunchucks to the sum of two folded normal distributions. The predominant bent state consistently accounts for ~7% of the observed bend angles. When a defect is placed in the center of a 37-bp linker, the aggregate nunchuck bend angle distribution is measurably different. A pair of tandem thymine mismatches (a 2-T bubble) stiffens the linker, whereas a string of six tandem thymine mismatches (a 6-T bubble) makes it much more flexible. Linkers with a 3-base bulge explore one predominant 73o-bent state, while the predominant bent state of linkers with a 6-base bulge is bent 91°. These results may help guide the design of dynamic, actuatable DNA nanostructures.