Andrew Dittmore

A high-speed magnetic tweezer beyond 10,000 frames per second

The magnetic tweezer is a single-molecule instrument that can apply a constant force to a biomolecule over a range of extensions, and is therefore an ideal tool to study biomolecules and their interactions. However, the video-based tracking inherent to most magnetic single-molecule instruments has traditionally limited the instrumental resolution to a few nanometers, above the length scale of single DNA base-pairs. Here we have introduced superluminescent diode illumination and high-speed camera detection to the magnetic tweezer, with graphics processing unit-accelerated particle tracking for high-speed analysis of video files. We have demonstrated the ability of the high-speed magnetic tweezer to resolve particle position to within 1 Å at 100 Hz, and to measure the extension of a 1566 bp DNA with 1 nm precision at 100 Hz in the presence of thermal noise.

Single-molecule methods for ligand counting: linking ion uptake to DNA hairpin folding.

Ligand associations play a significant role in biochemical processes, typically through stabilizing a particular conformation of a folded biomolecule. Here, we demonstrate the ability to measure the changes in the number of ligands associated with a single, stretched biomolecule as it undergoes a conformational change. We do this by combining thermodynamic theory with single-molecule measurements that directly track biomolecular conformation. We utilize this technique to determine the changes in the ionic atmosphere of a DNA hairpin undergoing a force-destabilized folding transition. We find that the number of counterions liberated upon DNA unfolding is a nonmonotonic function of the monovalent salt concentration of the solution, contrary to predictions from common nucleic acid models. This demonstrates that previously unobserved phenomena can be measured with our ligand counting approach.

Evidence for a Solenoid Phase of Supercoiled DNA

In mechanical manipulation experiments, a single DNA molecule overwound at constant force undergoes a discontinuous drop in extension as it buckles and forms a superhelical loop (a plectoneme). Further overwinding the DNA, we observe an unanticipated cascade of highly regular discontinuous extension changes associated with stepwise plectoneme lengthening. This phenomenon is consistent with a model in which the force-extended DNA forms barriers to plectoneme lengthening caused by topological writhe. Furthermore, accounting for writhe in a fluctuating solenoid gives an improved description of the measured force-dependent effective torsional modulus of DNA, providing a reliable formula to estimate DNA torque. Our data and model thus provide context for further measurements and theories that capture the structures and mechanics of supercoiled biopolymers.

Energetic Contributions of Plectoneme Tips and Tails

Global DNA topology is sensed locally by enzymes that act on plectonemes in supercoiled DNA. Here we report that the formation and diffusion of plectonemes are determined by the energetic contributions of their tips and tails. First, to systematically vary the geometry and formation energy of plectoneme end-loops, we introduced base-pair defect regions of variable size (1-16 bp) using a cassette based single-strand nicking template generated by PCR. Direct manipulation measurements with magnetic tweezers revealed that even a single mismatch or abasic site is sufficient to nucleate formation of a plectoneme. Presentation of the defect precisely at an extruded plectoneme tip potentially serves as a damage-sensing mechanism and may facilitate the search process of repair enzymes. Second, our measurements unexpectedly revealed that after twisted DNA abruptly buckles into an initial plectoneme loop, further plectoneme extrusion occurs through a cascade of additional buckling steps in which the torque changes by roughly half of the initial overshoot value. These discrete steps do not match any obvious scale of the system but are consistent with discontinuous feed-in of curving plectoneme tails. In light of these results, theoretical models of plectonemes should include their overall structure, including the often neglected tips and tails.