Structural transition from antiparallel to parallel G-quadruplex of d(G4T4G4) induced by Ca2+

Abstract

Guanine quadruplex (G-quadruplex) structures are formed by guanine-rich oligonucleotides. Because of their in vivo and in vitro importance, numerous studies have been demonstrated that the structure and stability of the G-quadruplex are dependent on the sequence of oligonucleotide and environmental conditions such as existing cations. Previously, we quantitatively investigated the divalent cation effects on the antiparallel G-quadruplex of d(G4T4G4), and found that Ca2+ induces a structural transition from the antiparallel to parallel G-quadruplex, and finally G-wire formation. In the present study, we report in detail the kinetic and thermodynamic analyses of the structural transition induced by Ca2+ using stopped-flow apparatus, circular dichroism, size-exclusion chromatography (SEC) and atomic force microscopy. The quantitative parameters showed that at least two Ca2+ ions were required for the transition. The kinetic parameters also indicated that d(G4T4G4) underwent the transition through multiple steps involving the Ca2+ binding, isomerization and oligomerization of d(G4T4G4). The parallel-stranded G-wire structure of d(G4T4G4), which is a well controlled alignment of numerous DNA strands with G-quartets, as the final product induced by Ca2+, was observed using SEC and atomic force microscopy. These results provide insight into the mechanism of the structural transition and G-wire formation and are useful for constructing a nanomaterial regulated by Ca2+.

Figure 1

Equilibrium analysis of the transition from the antiparallel to the parallel G-quadruplex. (A) CD spectra of 25 µM d(G₄T₄G₄) (squares), d(G₃T₃G₃) (circles) and d[(G₄T₄)₃G₄] (circles in the inset) in a buffer containing 100 mM NaCl, 50 mM MES (pH 6.1) with (filled symbols) or without 100 mM CaCl₂ (open symbols) at 5°C. (B) Plot of normalized CD intensity d(G₄T₄G₄) at 260 nm versus Ca²⁺ concentration at 5°C. Dotted and stick lines indicate best-fit results for the data with the following equations: (normalized CD intensity at 260 nm) = K_a[Ca²⁺]/(1 + K_a[Ca²⁺]) and (normalized CD intensity at 260 nm) = K_a[Ca²⁺]ⁿ/(1 + K_a[Ca²⁺]ⁿ), respectively. The best fitted value for n is calculated to be 2.11.

Figure 2

SEC profiles for d(G₄T₄G₄) in buffers containing 50 mM MES (thin line), 100 mM NaCl and 50 mM MES (dotted line), and 50 mM CaCl₂, 100 mM NaCl and 50 mM MES (thick line) at 25°C. These buffers were adjusted with LiOH to pH 6.1. The relative retention times in the buffers were calculated from the observed retention times of d(G₄T₄G₄) against those for a 12mer DNA marker, d(T₃C₃T₂CT₂), which forms neither intramolecular nor intermolecular structures under these conditions. In buffer (a), the observed retention times of the marker and d(G₄T₄G₄) were 7.71 and 7.62 min, respectively. In buffer (b), they were 8.71 and 8.32 min, respectively. In buffer (c), they were 9.89 and 9.29 min, respectively.

Figure 3

Kinetic properties of the transition of d(G₄T₄G₄) from the antiparallel to the parallel G-quadruplex. (A) Kinetic traces of the transition of 25 µM d(G₄T₄G₄) traced with CD intensity at 260 nm. Data were collected immediately after 1:1 mixing of 50 µM d(G₄T₄G₄) and various concentrations of CaCl₂. All measurements were performed in a buffer containing 100 mM NaCl and 50 mM MES (pH 6.1) at 45°C. The traces were fitted to a single exponential curve. (B) Plot of k_obs at 400 mM Ca²⁺ at 40°C versus various concentrations of d(G₄T₄G₄) in a buffer containing 100 mM NaCl and 50 mM MES (pH 6.1). (C) Plot of k_obs of 25 µM d(G₄T₄G₄) versus concentration of Ca²⁺ at 45°C in a buffer containing 100 mM NaCl and 50 mM MES (pH 6.1). The solid line indicates the best-fit result for the data using equation 2.

Figure 4

Arrhenius plot of log k₊₂ versus 1/T. The solid line is obtained with the linear least-squares fit to the data.

Figure 5

Schematic mechanism of the structural transition of d(G₄T₄G₄) from antiparallel to parallel G-quadruplex induced by Ca²⁺. Arrows indicate the strand directions. The guanine bases of d(G₄T₄G₄) are shown as rectangles. Thymine bases in the loop have been omitted for clarity. Disks indicate Ca²⁺.