Abstract
Gene expression is achieved by enzymes as RNA polymerases that translocate along nucleic acids with steps as small as a single base pair, i.e., 0.34 nm for DNA. Deciphering the complex biochemical pathway that describes the activity of such enzymes requires an exquisite spatiotemporal resolution. Magnetic tweezers are a powerful single molecule force spectroscopy technique that uses a camera-based detection to enable the simultaneous observation of hundreds of nucleic acid tethered magnetic beads at a constant force with subnanometer resolution [1,2]. High spatiotemporal resolution magnetic tweezers have recently been reported [3–5]. We present data acquired using a bespoke magnetic tweezers instrument that is able to perform either in high throughput or at high resolution. The data reports on the best achievable resolution for surface-attached polystyrene beads and DNA tethered magnetic beads, and highlights the influence of mechanical stability for such assay. We also present data where we are able to detect 0.3 nm steps along the z-axis using DNA tethered magnetic beads. Because the data presented here are in agreement with the best resolution obtained with magnetic tweezers, they provide a useful benchmark comparison for setup adjustment and optimization.
Highlights
Subject Specific subject area Type of data How data were acquired Data format Parameters for data collection
Single-molecule force spectroscopy using magnetic tweezers Figure, image, graph, table Custom built magnetic tweezers setup interfaced using a custom software written in Labview 2016 for three-dimensional bead position tracking
We tracked the position of: (1) a surface-attached polystyrene reference bead at 50x and 100x magnifications and several acquisition frequencies, subjected to various drift in order to characterize the limiting factors for the best achievable resolution in our assay (Figs. 2 and 3); (2) a magnetic bead tethered by dsDNA under constant force in order to quantify the thermal noise to which the bead is subjected and the relationship between thermal noise and applied force)(Fig. 4(a) and (b)); (4) both immobilized and tethered bead while performing steps with the piezo stage along the z-axis(Fig. 4(c) and (d))
Summary
Single-molecule force spectroscopy using magnetic tweezers Figure, image, graph, table Custom built magnetic tweezers setup interfaced using a custom software written in Labview 2016 for three-dimensional bead position tracking. The data reported here provide a benchmark of the best achievable spatiotemporal resolution of magnetic tweezers for a surface-attached reference bead and a DNA tethered magnetic bead. High frequency noise appears along the x-axis and y-axis, which is corrected by reference bead subtraction (Fig. 3(c) and (d), respectively) This high frequency noise likely originates from the piezo stage (P-726, Physik Instrumente) on which is mounted the microscope objective, as characterized for optical tweezers [7] and observed in magnetic tweezers using a similar piezo stage [4]. We tracked the position of: (1) a surface-attached polystyrene reference bead at 50x and 100x magnifications and several acquisition frequencies, subjected to various drift in order to characterize the limiting factors for the best achievable resolution in our assay We tracked the position of: (1) a surface-attached polystyrene reference bead at 50x and 100x magnifications and several acquisition frequencies, subjected to various drift in order to characterize the limiting factors for the best achievable resolution in our assay (Figs. 2 and 3); (2) a magnetic bead tethered by dsDNA under constant force in order to quantify the thermal noise to which the bead is subjected and the relationship between thermal noise and applied force)(Fig. 4(a) and (b)); (4) both immobilized and tethered bead while performing steps with the piezo stage along the z-axis (of 1, 0.7, 0.5, 0.3 nm)(Fig. 4(c) and (d))
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