Cooperative control of a DNA origami force sensor

Abstract

Most biomolecular systems are dependent on a complex interplay of forces. Modern force spectroscopy techniques provide means of interrogating these forces. These techniques, however, are not optimized for studies in constrained or crowded environments as they typically require micron-scale beads in the case of magnetic or optical tweezers, or direct attachment to a cantilever in the case of atomic force microscopy. We implement a nanoscale force-sensing device using a DNA origami which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, functions as a binary (open or closed) force sensor that undergoes a structural transition under an external force. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). This actuation of the NanoDyn is reversible and the design parameters strongly influence the efficiency of resetting the initial state, with higher stability devices (≳10 pN) resetting more reliably during repeated force-loading cycles. Finally, we show that the opening force can be adjusted in real time by the addition of a single DNA oligonucleotide. These results establish the NanoDyn as a versatile force sensor and provide fundamental insights into how design parameters modulate mechanical and dynamic properties.

Figure 1:. DNA origami NanoDyn schematic and experimental design.

(a) Schematic drawing of a DNA origami NanoDyn (ND) which consists of 2 honeycomb lattice barrels held together by 6, 116 nt ssDNA crossover strands called loops. Each loop can be folded into a “force-sensing” loop or a “hinged” loop through annealing of a zipper strand or blocking strands, respectively. The force-sensing loop can be in an open or closed state. TEM images provide visualization of these two states. (b) The ND is attached to a 2.8 µm superparamagnetic bead through 4 biotin-streptavidin linkages. The opposite end of the ND is annealed to a dsDNA tether, which itself is annealed to an oligonucleotide covalently bonded to a microscope slide via click chemistry. Repeated actuation of the ND is achieved by repeatedly increasing and then decreasing the force using a magnetic tweezers system. A sudden increase in length during the force loading step is indicative of an opening event.

Figure 2:. Zipper region length within a single force-sensing loop modulates the opening force.

(a) Schematic design for ND with one force-sensing loop at L6 and five hinged loops at L1-L5. (b) Representative force-extension curves for L6-13nt (blue) and L6-21nt (red). Data was fit to a Torsional spring + worm-like chain model (TorWLC) for both the closed (TorWLC_closed) and open (TorWLC_open) states. (c) Cumulative probability distribution of the opening forces for multiple pulls across multiple devices. (d) Violin plots of the opening force distributions. The white dot is the median opening force value, which is indicated above or below each respective distribution and the black bar indicates the first quartile above and below the median. The median, mean, SD, SEM, and interquartile values for each ND version can be found in Supplementary Table S1.

Figure 3:. Multiple force-sensing loops increases the ND opening force.

(a) Schematic design for force-sensing loops with (i) one force-sensing loop at loop 6 (blue), 3 (purple), and 1 (grey); (ii) two force-sensing loops at loops 6 and 3 (pink); and (iii) three force-sensing loops at loops 6, 3, and 1 (dark red). (b) Representative force-extension curves for L3-11nt (purple) and L6-13nt + L3-11nt + L1-13nt (dark red - abbreviated L6+L3+L1). Data was fit to the TorWLC model. (c) Cumulative probability distributions of the opening forces for the ND devices. (d) Violin plots of the opening force distribution for each ND device. The white dot indicates the median opening force, which is shown above or below each respective distribution and the black bar indicates the first quartile above and below the median. The median, mean, SD, SEM, and interquartile values for each ND version can be found in Supplementary Table S1.

Figure 4:. Effect of zipper region length and multiple force-sensing loops on ND closure and subsequent re-opening.

(a) Violin plots of the fraction of extensions with an opening event for each individual ND. The triangles indicate the position in the distribution of the devices shown in (b-d). The white dot indicates the median opening fraction, which is indicated above or below each respective distribution. The black bar indicates the first quartile above and below the median. The median, mean, SD, SEM, and interquartile values for each ND version can be found in Supplementary Table S4. (b-d) Examples of repeated force-extension curves of single (b) L1-13nt, (c) L6-13nt, and (d) L6+L3+L1 ND devices. Each plot contains 10 consecutive extensions with arrows indicating opening events. The corresponding retraction cycles can be found in Supplementary Fig. S11.

Figure 5:. Iterative introduction of multiple force-sensing loops into a single ND.

(a) Diagram of the iterative introduction of one and then two force-sensing loops into the ND. (b) Force-extension of the ND prior to incorporation of a force-sensing loop never results in an opening event. (c) Following incubation with a single zipper strand that forms the L6-13nt force-sensing loop, a well-defined step is observed upon force-extension. (d) Following incubation with a second zipper strand that forms the L3-11nt force-sensing loop, an additional increase in the opening force is observed.