Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds

Abstract

Single-molecule data are of great significance in biology, chemistry, and medicine. However, new experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are still needed. Acoustic force spectroscopy is an emerging manipulation technique which generates acoustic waves to apply force in parallel on multiple microbeads tethered to a surface. We here exploit this configuration in combination with the recently developed modular junctured-DNA scaffold that has been designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measure its unbinding kinetics under force at the single-bond level. Special efforts are made in analyzing the data to identify potential pitfalls. We propose a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well-established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy to study the force-dependent rupture of a single-domain antibody with its antigen. Overall, we get a good agreement with the published parameters that have been obtained at zero force and population level. Thus, our technique offers single-molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.

Figure 1

Principle of the method and sample trace. (A) Beads are tethered in a microfluidic chamber equipped with a piezo generating the acoustic waves and a temperature control system (not to scale). (B) Part of the field of view showing beads that are selected (red squares) to be tracked in real time and in 3D thanks to their diffraction fringes. (C) Each bead is tethered to the chamber surface through a J-DNA scaffold (red) maintaining the protein partners (blue) in close proximity via a leash. Bond formation occurs in absence of significant acoustic force. Upon application of an acoustic force, the difference in extension of the scaffold in the closed and in the open state allows one to distinguish the unbinding of the protein complex. (D) Corresponding power and height profile of the bead. To see this figure in color, go online.

Figure 2

Tracking of a single bead under force cycling, bond lifetime determination, and force calibration. (A) Height trace Z obtained during 100 low/test power cycles. (B) Zoom of the trace in (A), indicated by the dashed blue rectangle. Based on power value and jump detection at high power, the trace is divided in three subsets: low power (gray), closed state at test power (orange), and open state at test power (red). $\Delta t$, which corresponds to the duration of the orange step, represents the dwell time before scaffold opening. $\Delta Z$ represents the height jump between the closed and the open state. The applied force obtained after calibration is specified on the right axis. (C) Survival curve calculated from the dwell time $\Delta t$ distribution for one representative bead at different powers in percent (calibrated force in pN is shown in the inset). Superimposed monoexponential fits: S($\Delta t$) = exp(−$\Delta$t.k_off), with $k_{\mathrm{o}\mathrm{f}\mathrm{f}}$ the rate constant of the opening reaction. The value of the calibrated force (ID13) for each applied acoustic power is specified in the legend. (D) Density kernel of jump distance $\Delta$Z for the same bead as in (C) at different powers. (E) Pulling angle versus power for three representative beads. (F) x-power spectrum density for the same bead as in (D), at various test powers $P_i$. Curve fits used to determine the bead radius $R^x$ and the stiffnesses $k_i^x$ are shown as black lines. (G) Force versus power for three representative beads, the same as in (E). Full symbols correspond to the results obtained from analyzing the bead Brownian motion along the x-direction and open symbols from the y-direction. To see this figure in color, go online.

Figure 3

Bell plots for the FKBP12-rapamycin-FRB complex and corresponding fit. (A) The off-rate at different forces of individual molecules is shown in different colors. The error bars on individual off-rate values are calculated as indicated in the materials and methods and, for the sake of clarity, they are omitted here and displayed in Fig. S18. The average values on force bins of equal size are shown as black circles, the vertical error bars showing the standard deviation of off-rate values in each force bin. The black line is the fit with Bell equation (7), taking into account the error bars. See Tables S3 and S6 for detailed statistics. (B) Off-rate in absence of applied force $k_{\mathrm{o}\mathrm{f}\mathrm{f}}^0$ as a function of $x_b$, the distance from the potential well to the barrier on the energy landscape of the interaction, as obtained from the individual Bell fits shown in Fig. S18. The same color code is used as in (A) to specify the different molecules measured. Each color dot relies on the observation of 250–1020 rupture events. The error bars are the fitting errors when applying the Bell model. The red dashed lines represent the values of off-rate measured by surface plasmon resonance (50,51). The green dashed-and-dotted lines were obtained using SMFS (25,20). The black circle represents the parameter obtained from the global Bell fit represented as a black line in (A) (error bar = error on the fit). The black square represents the average of the colored points (error bar = SD). To see this figure in color, go online.

Figure 4

Bell plot for the Nef-Nef19 complex and corresponding fit. The off-rate at different forces of individual molecules is shown in different colors. The black circles correspond to the $k_{\mathrm{o}\mathrm{f}\mathrm{f}}$ averaged by equal force bins. The black line is the fit with Bell equation (7). The vertical error bars on the black points are calculated as the standard deviation of off-rate values in each force bin. The red horizontal dashed line represents the value of the off-rate measured by SPR (61). To see this figure in color, go online.