Multiplexed single-molecule force spectroscopy using a centrifuge

We present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. Incorporating DNA nanoswitches to enable repeated interrogation by force of single molecular pairs, we demonstrate increased throughput, reliability and the ability to characterize population heterogeneity. We perform spatiotemporally multiplexed experiments to collect 1,863 bond rupture statistics from 538 traceable molecular pairs in a single experiment, and show that 2 populations of DNA zippers can be distinguished using per-molecule statistics to reduce noise.

The top panel shows a scatter plot of the persistence length and contour length obtained from fits to the worm-like chain model performed for each tether (n=113)., Histograms projecting the persistence length and contour length of the model onto the x-and yaxis, respectively, are shown with red lines indicating expected values. The bottom panel shows the force-extension curves of single-tethered DNA data filtered by persistence length and contour length (n=30). Ranges were selected from the peaks of the histograms (one bin-width on either side), yielding filtering ranges of 43-48 nm and 7.8-8.6 µm for the persistence length and contour length, respectively. The overlayed red curve represents the expected force-extension curve. The centrifuge bucket is at angle, θ, therefore the centrifugal force has components both perpendicular and parallel to the sample surface (x-y plane). When the bond is ruptured, the DNA Nanoswitch goes from looped (blue) to unlooped (red) experiencing a change in length ΔL. This is identified by measuring the projected change in length in the xy plane ΔL obs = ΔL cos θ. The orientation of the miniCFM in the bucket sets the direction of ΔL obs . (b) Images of a bead before (above), and after (below) a loop opening transition. A bead which is tethered to the surface with a single DNA nanoswitch will undergo a discontinuous change in position with a well-defined length ΔL obs, and direction φ. (c) A scatter plot of all contour length changes detected for all directions. Color represents data density. Only transitions within the boxed region are accepted as nanoswitch transitions. Inset, a histogram of transition forces for three different types of transitions: beads which leave the surface (yellow), beads which display discontinuous transition with (green) and without (light-blue) correct direction and magnitude. (d) A scatter plot of the symmetry ratio and root mean square displacement based on the lateral fluctuations of all tracked beads, with color representing data density. The overlayed black data points are for tethered beads that undergo validated nanoswitch transitions.

Supplementary Figure 6 | Force and loading rate range of the benchtop CFM. (a)
Force as a function of rotational speed calculated using four different types of beads (1 and 2.8 um Dynabeads, and 5 and 10 um silica beads) for the benchtop CFM. Using this set of beads, the CFM is capable of applying a force range that spans eight orders of magnitude (10 -4 to 10 4 pN). (b) The fastest and slowest force-loading rates of the benchtop CFM measured using the Thermo Scientific Heraeus X1R Centrifuge. The fastest ramping rate of 317 g/s, shown in blue, can correspond to a 1,300 pN/s loading rate using a large 10 um silica bead. The slowest ramping rate of 0.373 g/s, show in red, can corresponds to a 1.15 fN/s loading rate using a small 1.0 um Dynabead (Invitrogen). Based on the geometry as illustrated in (Supplementary Fig. 2a), extension of the molecular tether can be measured by tracking the tethered microsphere's motion parallel to the coverslip. Specifically, changes in tether extension will appear as a lateral displacement of the bead (∆L obs ). The actual changes in extension (∆L) in the direction parallel to the force can be calculated based on the angle of the bucket (θ) as follows:

Supplementary
The minimum tether length that can be measured in this way depends on both the bead size used in the experiment and the angle of the centrifuge bucket. The bead can make direct contact with the cover glass surface if the tethered length is not long enough for the bead to be pulled away from the surface. The minimum tether length (L min ) as a function of bead radius (R bead ) and bucket angle (θ) is given by: The tracking resolution of tether extension (∆L) and values of L min in units of bead radius ( !"#$ ) are calculated for bucket angles between 20 o to 85 o as shown in Supplementary Fig. 2c.

Supplementary Note 2.
The unzipping force of the 29 bp DNA duplex measured here increases as the temperature decreases in reasonable quantitative agreement with simple thermodynamic theoretical predictions. Interestingly, the theoretical prediction slightly underestimates the unzipping force at low temperature (Fig. 2c)

Supplementary Note 3.
The benchtop CFM bill of materials is summarized in Supplementary Table 1 and the exploded view drawing of the assembly is shown in Supplementary Fig. 1e.
The majority of the opto-mechanical components can be purchased from Thorlabs. Ethernet connection to an optical signal could be omitted if a camera with a 10 GigE optical fiber output is used. Data output using a compact wireless router is an alternative approach, but may result in a slower acquisition rate. Additional customized parts such as the turning mirror housing can be replaced with a Thorlabs compact cage cube system, but for added stability of the imaging path, we recommend a solid aluminum construction. Moreover, larger floor model centrifuges with 1 Liter buckets can provide sufficient bucket depth that the turning mirror is no longer needed. The CAD drawing of the solid aluminum turning mirror housing and the full CAD assembly of the CFM show in Supplementary Fig. 1 is available in the Supplemental Files.