Encapsulating a single G-quadruplex aptamer in a protein nanocavity

Abstract

The alpha-hemolysin (alphaHL) protein pore has many applications in biotechnology. This article describes a single-molecule manipulation system that utilizes the nanocavity enclosed by this pore to noncovalently encapsulate a guest molecule. The guest is the thrombin-binding aptamer (TBA) that folds into the G-quadruplex in the presence of cations. Trapping the G-quadruplex in the nanocavity resulted in characteristic changes to the pore conductance that revealed important molecular processes, including spontaneous unfolding of the quartet structure and translocation of unfolded DNA in the pore. Through detection with Tag-TBA, we localized the G-quadruplex near the entry of the beta-barrel inside the nanocavity, where the molecule vibrates and rotates to different orientations. This guest-nanocavity supramolecular system has potential for helping to understand single-molecule folding and unfolding kinetics.

Figure 1

Molecular graphic representations of α-hemolysin (αHL) transmembrane protein pore, thrombin-binding aptamer (TBA) and their interaction. a. Sagittal plane through the αHL pore and a G-quartet TBA encapsulated in the nanocavity of αHL. b. The NMR structure of TBA (DOI 10.2210/pdb1c38/pdb, RCSB Protein Data Bank) in the presence of the potassium ion. The two G-tetrad planes (orange) formed by eight guanines (green) are illustrated. c. Top view of the cis vestibule of the αHL pore superimposed on an identically-scaled TBA molecule in the center, showing the pore entry that is wide enough to allow a G-quadruplex to enter.

Figure 2

Single channel current traces and histograms showing blocks to the αHL pore with the control oligonucleotides in different metal ions. Both concentrations of Ctrl-1 and Ctrl-2 were 2.5 μM. All traces were recorded at +100 mV in either a 1 M NaCl or a 1 M KCl solution buffered with 10 mM Tris HCl (pH 7.2). The left panels were current traces recorded at a 5 kHz filtering bandwidth and a 20 ks^-1 sampling rate; middle panels were histograms constructed from traces in left; and right panels were histograms constructed from recordings at a 50 kHz filtering bandwidth and a 200 ks^-1 sampling rate. Calculated t_P and ${\tau }_b^S$ values are given in Table 1. a. Ctrl-1 in NaCl. b. Ctrl-1 in KCl. c. Ctrl-2 in NaCl. d. Ctrl-2 in KCl. e. A model showing a linear DNA transporting in a αHL pore.

Figure 3

Current traces from a single αHL pore showing blocks with TBA in different metal ion and ligand conditions. The DNA concentration and the solution condition were the same as in Fig.2. a. TBA in NaCl. b. TBA in KCl. Left inset was the expansion of a Level-2 block at the long event terminal, and the right inset shows a long-lived event without terminal block. c. A model showing the encapsulation of a G-quartet TBA in the nanocavity of αHL followed by the spontaneous unfolding process. d. TBA in a KCl solution in the presence of 2.5 μM human α-thrombin.

Figure 4

Influence of voltage on the Level-2 block and the long block duration. a. Nonlinear decrease of the Level-2 block duration with the voltage. b. voltage-independence of the long-lived block duration.

Figure 5

Current traces from single αHL pores showing blocks with Tag-TBA. The DNA concentration and the solution condition were the same as in Fig.2. a. Characteristic blocks produced by tag-TBA (top) and the model showing the molecular location and position in the cavity (bottom). b. Another type of block by tag-TBA (top) and the corresponding model showing the change in position of the molecule (bottom). c. Unfolding of tag-TBA in nanocavity (top) and the model of this process (bottom).