Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

Abstract

Human telomeric DNA consists of tandem repeats of the sequence 5'-TTAGGG-3' that can fold into various G-quadruplexes, including the hybrid, basket, and propeller folds. In this report, we demonstrate use of the α-hemolysin ion channel to analyze these subtle topological changes at a nanometer scale by providing structure-dependent electrical signatures through DNA-protein interactions. Whereas the dimensions of hybrid and basket folds allowed them to enter the protein vestibule, the propeller fold exceeds the size of the latch region, producing only brief collisions. After attaching a 25-mer poly-2'-deoxyadenosine extension to these structures, unraveling kinetics also were evaluated. Both the locations where the unfolding processes occur and the molecular shapes of the G-quadruplexes play important roles in determining their unfolding profiles. These results provide insights into the application of α-hemolysin as a molecular sieve to differentiate nanostructures as well as the potential technical hurdles DNA secondary structures may present to nanopore technology.

Keywords: single-molecule detection; α-hemolysin nanopore.

Fig. 1.

Structures and characterization of the G-quadruplexes formed by the human telomere sequence. (A) Schematic structures and dimensions of hybrid-1, hybrid-2, basket, and propeller folds. (B) Structure and dimensions of the α-HL ion channel (3).

Fig. 2.

Interactions between G-quadruplexes [5′-TAGGG(TTAGGG)₃TT-3′] and the α-HL ion channel reveal the size-selective property of the protein nanopore. (A) Space-filling models of the G-quadruplexes interacting with α-HL constructed using PBD structures 7AHL (3), 2JSQ (6), 143D (4), and 1K8P (1), respectively. (B–D) Stick models of the proposed interaction mechanisms and i-t traces yielded by (B) the hybrid folds (50 mM KCl, 950 mM LiCl, 25 mM Tris, pH 7.9) (33), (C) the basket fold (1 M NaCl, 25 mM Tris, pH 7.9), and (D) the propeller fold (20 mM KCl, 5 M LiCl, 25 mM Tris, pH 7.9). The hybrid fold studies were reported previously and are illustrated here for comparison (33). All measurements were done under 120 mV (trans vs. cis). I and I_M values are indicated as a percentage of the open-channel current I_o.

Fig. 3.

Translocation study of the G-quadruplexes after attachment of a 5′-dA₂₅ tail [5′-A₂₅-TAGGG(TTAGGG)₃TT-3′]. (A–C) Stick models of G-quadruplexes unraveling within the α-HL ion channel and i-t traces yielded by interactions between the α-HL and (A) hybrid folds (50 mM KCl, 950 mM LiCl, 25 mM Tris, pH 7.9), (B) basket fold (1 M NaCl, 25 mM Tris, pH 7.9), and (C) propeller fold (20 mM KCl, 5 M LiCl, 25 mM Tris, pH 7.9). The sample traces presented here were collected under 120 mV (trans vs. cis). I and I_M values are indicated as a percentage of the open-channel current I_o.

Fig. 4.

Unfolding and translocation rates for the G-quadruplexes under various potentials. Plots of the decay constants of translocation events as a function of applied voltage (trans vs. cis) for (A) the control oligonucleotide and (B) G-quadruplexes, where red indicates basket folded and green propeller folded.