Interactions of the human telomere sequence with the nanocavity of the α-hemolysin ion channel reveal structure-dependent electrical signatures for hybrid folds

Abstract

Human telomeric DNA consists of tandem repeats of the sequence 5'-TTAGGG-3', including a 3' terminal single-stranded overhang of 100-200 nucleotides that can fold into quadruplex structures in the presence of suitable metal ions. In the presence of an applied voltage, the α-hemolysin (α-HL) protein ion channel can produce unique current patterns that are found to be characteristic for various interactions between G-quadruplexes and the protein nanocavity. In this study, the human telomere in a complete sequence context, 5'-TAGGG(TTAGGG)3TT-3', was evaluated with respect to its multiple folding topologies. Notably, the coexistence of two interchangeable conformations of the K(+)-induced folds, hybrid-1 and hybrid-2, were readily resolved at a single-molecule level along with triplex folding intermediates, whose characterization has been challenging in experiments that measure the bulk solution. These results enabled us to profile the thermal denaturation process of these structures to elucidate the relative distributions of hybrid-1, hybrid-2, and folding intermediates such as triplexes. For example, at 37 °C, pH 7.9, in 50 mM aqueous KCl, the ratio of hybrid-1:hybrid-2:triplex is approximately 11:5:1 in dilute solution. The results obtained lay the foundation for utilizing the α-HL ion channel as a simple tool for monitoring how small molecules and physical context shift the equilibrium between the many G-quadruplex folds of the human telomere sequence.

Figure 1. Structures of the G-quadruplexes formed by the human telomere sequence

a, The sequence, structures and dimensions of hybrid-1 and hybrid-2 folds. b, Structure and dimensions of the α-HL ion channel.^,

Figure 2. The various interactions between human telomeric sequence and the α-HL

a, Three types of current-time traces observed. b, The percentage distributions of these event types. These results were obtained in 50 mM KCl, 950 mM LiCl in 25 mM Tris buffer (pH 7.9) at 25 °C.

Figure 3. Resolving the coexistent hybrid-1 and -2 folds at a single-molecule level

Incorporation of two 8-Br-dGs at different positions into the natural sequence can induce the G-quadruplexes to adopt exclusively hybrid-1 or -2 folds. %I_M/I_o histograms of the type 1 events from the natural sequence without any substitutes, N: 5′-TAGGG(TTAGGG)₃TT-3′ (top) were compared to those of the substituted strands, H1: 5′-TAGGGTTAGGGTTAGXGTTAXGGTT-3′ (middle) and H2: 5′-TAGGGTTAGXGTTAXGGTTAGGGTT-3′ (bottom), where X = 8-Br-dG. All three histograms represent analysis of ~500 molecules for each experiment.

Figure 4. Studies of tail effects on the current signatures

The representative i-t traces for three sequences: a, natural human telomere sequence N: 5′-TAGGG(TTAGGG)₃TT-3′ b, 5′ tail: 5′-TAGGGTTAGXGTTAXGGTTAGGG-3′, where X = 8-Br-dG c, No tails: 5′-GGG(TTAGGG)₃-3′.

Figure 5. Illustration of the proposed mechanism for the current signatures

a, Space-filling model of the hybrid fold entering the α-HL vestibule using PBD structures 7AHL and 2JSQ. b, Stick model of the proposed interaction mechanism. Intermediate current levels (I_M) at the beginning and end of an event correlated to the interaction between the DNA and the opening of the protein vestibule. In the presence of an electrical potential (−120 mV, cis vs. trans), the G-quadruplexes can be driven closer to the β-barrel, producing deeper blockages I with one tail protruding into the constriction, or diffuse back to the opening of the vestibule, leading again to I_M current levels.

Figure 6. Proposed triplex folding intermediates of the hybrid folds

Replacement of 5′ vs. 3′ GGG with TTT leads to 5′ tail (5′T) and 3′ tail (3′T) triplexes, proposed to be the folding intermediates of hybrid-1 (a) and hybrid-2 folds (b).

Figure 7. Nanopore measurements of the triplexes (5′T and 3′T)

a. Examples of i-t traces of the triplexes and the durations of the events (t_D) that were fit to a single exponential decay model, shown in b with a time constant τ. c. Plot of the decay constants of translocation events (blue: type 3 events from the natural sequence, red: 5′-T triplex, green: 3′-T triplex) as a function of applied voltage (cis vs. trans).

Figure 8. Entry rate corrections between the hybrid species and the triplex

Plots of event frequency ratio vs. concentration ratio for a, Hybrid-2 (H2) and Hybrid-1 (H1) model sequences and b, Triplex and H1. The ODNs sequences used are: H1: 5′-TAGGGTTAGGGTTAGXGTTAXGGTT-3′, H2: 5′-TAGGGTTAGXGTTAXGGTTAGGGTT-3′ (X = 8-Br-dG) and Triplex: 5′-TATTT(TTAGGG)₃TT-3′.

Figure 9. Thermal denaturation profile of the human telomeric sequence

Plot of percentage abundance vs. temperature after entry rate corrections for hybrid-1, hybrid-2, and triplex plus other non-hybrid folds, respectively. The system temperature limit is ~55 °C.