Multiplex flow magnetic tweezers reveal rare enzymatic events with single molecule precision

Abstract

The application of forces and torques on the single molecule level has transformed our understanding of the dynamic properties of biomolecules, but rare intermediates have remained difficult to characterize due to limited throughput. Here, we describe a method that provides a 100-fold improvement in the throughput of force spectroscopy measurements with topological control, which enables routine imaging of 50,000 single molecules and a 100 million reaction cycles in parallel. This improvement enables detection of rare events in the life cycle of the cell. As a demonstration, we characterize the supercoiling dynamics and drug-induced DNA break intermediates of topoisomerases. To rapidly quantify distinct classes of dynamic behaviors and rare events, we developed a software platform with an automated feature classification pipeline. The method and software can be readily adapted for studies of a broad range of complex, multistep enzymatic pathways in which rare intermediates have escaped classification due to limited throughput.

Topoisomerase VI is a chirally-selective, preferential DNA decatenase

“…Using the magnetic tweezers single-molecule approach, we have not only demonstrated that M. mazei topo VI is a highly distributive and extremely slow supercoil relaxase, confirming findings from Wendorff et al, 2018 (38), but also shown that topo VI activity increases as much ~10-fold on braided DNA, (Figures 1, 2 and 4), approaching rates determined for type IIA topos on their respective optimal substrates (21,43,44). Along with the observation that topo VI has an extremely strict preference for DNA crossing geometries close to 90°, which appear more frequently in catenanes than they do in supercoils (21), these data strongly indicate that M. mazei topo VI is a preferential decatenase, which simultaneously disfavours supercoil relaxation.…”

“…2 nM topo VI), the maximum rate measured for positive and negative supercoil relaxation was only 6.4 ± 0.6 and 3.5 ± 0.5 strand-passage events min -1 , respectively. As detailed in the subsequent section, these rates are 10-50-fold slower than rates measured for the type IIA topos, gyrase and topo IV (21,43,44). Wendorff et al (2018) found the maximal topo VI ATP hydrolysis rate, using the PK/LDH assay, to be ∼3 ATP min -1 during relaxation of negatively supercoiled plasmids (38).…”

“…For instance, E. coli DNA gyrase, measured using a rotor bead tracking technique, showed 1 strand-passage event s -1 (43), or using magnetic tweezers, 1.26 strand-passage events s -1 (44), and E. coli topo IV was demonstrated to relax positively-supercoiled DNA at 2.5 strandpassage events s -1 (21).…”

Highly-parallel microfluidics-based force spectroscopy on single cytoskeletal motors

“…The obtained standard deviations averaged at 32 nm for both x and y . This value corresponds to the experimental value of localization precision, the theoretical limit of the tracking precision in the used configuration was previously calculated to be 6 nm 26 .…”

Nano-particle transport and the prediction of a valid area to be trapped based on a plasmonic antenna array

“…This also may damage the biological cell or specimen due to the heat generated by large incident power. 6,7 As the metal surface plasmon resonance structures with localized strong gradient electric elds can be used to optically trap nanoparticles, [8][9][10][11][12][13] many groups have focused on the optical trapping for a long time. [14][15][16] The optical force on the nanoparticles caused by the evanescent eld can draw the nanoparticles into a 'hot spot'.…”