Nanopore force spectroscopy of aptamer-ligand complexes

Abstract

The stability of aptamer-ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans-cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant Kd ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer-target interactions in this case, the stability of the ligand-free aptamer-containing G-quadruplexes-is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar Kd were identified.

Figure 1

Measurement principle. (I) A potential of 120 mV is applied to capture a DNA molecule with an aptamer structure (red) and a thread sequence (blue) on its 3' end. While the aptamer structure is unfolded on capture, a stable hairpin (green) at the 5' end prevents the DNA from traversing the pore. (II) The DNA is held inside the pore at low bias voltage (50 mV) to allow reforming of the aptamer and target binding on the trans side. (III) A voltage ramp to −200 mV is applied. When the aptamer structure unfolds, the DNA molecule escapes from the pore and a sudden increase in the current signal is observed. The corresponding unfolding voltage is recorded.

Figure 2

ATP aptamer unfolding at varying ATP concentrations. (a) Unfolding voltage distributions at different ATP concentrations, obtained at a loading rate of 10 V/s. With no ATP present, a single population at 27 mV is observed. On addition of ATP, a second, more stable population appears, which increases with increasing ATP concentration. This population is assigned to aptamer structures with bound ATP targets. Histograms are fitted to Eq. 2 (dashed lines) to determine the fraction of bound structures.

Figure 3

Aptamer target specificity. (a) Unfolding voltage distributions for adenosine mono-, di-, and triphosphate at a loading rate of 10 V/s. In all cases, a stable population assigned to target-bound structures is observed. Solid lines correspond to fits using Eq. 2. (b) Fraction of bound aptamer structures versus target concentration for ATP (open squares), ADP (open triangles), and AMP (filled squares). The dashed lines show fits to Eq. 4. The fit yields a dissociation constant of $119\pm 12\mu \text{M}$, $92\pm 10\mu \text{M}$, and $85\pm 14\mu \text{M}$ for ATP, ADP, and AMP, respectively. (c) Voltage-dependent lifetime of aptamer–target complexes determined from measurement data according to Eq. 3. Error bars are standard deviations from 12, 4, and 6 independent measurements for ATP, ADP, and AMP, respectively.

Figure 4

Binding pathways for the ATP aptamers ATPapt and ATPapt-mod. Bases are depicted as circles (A: green, T: red, G: black, C: blue). Black bars indicate free energies of the structures. (a) For ATPapt, to bind ATP, the secondary structure with lowest mean free energy U′ either has to unfold and rearrange to form the structure with the ATP-binding pocket U (conformational selection) or adopt its conformation on ATP binding (induced fit). (b) Binding pathway for ATPapt-mod, for which the binding-competent state is stabilized with two additional A-T basepairs at its end.

Figure 5

Aptamer tailoring. (a) Unfolding voltage distributions for the modified aptamer structure ATPapt-mod with and without addition of ATP at a loading rate of 10 V/s. Note the increase in critical unfolding voltage for the stabilized aptamer compared with the unmodified structure in Fig. 3. (b) Lifetime versus voltage for modified (blue) and unmodified (gray) aptamers (circles) and aptamer–target structures (squares). Error bars are standard deviations from 2 to 12 independent measurements.

Figure 6

Unfolding of the thrombin aptamer. (Top panel) Typical unfolding voltage distributions without (open bars) and with (solid bars) addition of thrombin at different concentrations (gray: 2 μM, red: 3 μM). Experiments were performed at a loading rate of 0.2 V/s. (Bottom panel) Voltage-dependent lifetime of aptamer structures (open triangles) and aptamer–target complexes (solid squares, gray: 2 μM, red: 3 μM thrombin). Error bars are standard deviations from three independent measurements.