DNA G-quadruplexes are uniquely stable in the presence of denaturants and monovalent cations

Abstract

Ions in the Hofmeister series exhibit varied effects on biopolymers. Those classed as kosmotropes generally stabilize secondary structure, and those classed as chaotropes generally destabilize secondary structure. Here, we report that several anionic chaotropes exhibit unique effects on one DNA secondary structure - a G quadruplex. These chaotropes exhibit the expected behaviour (destabilization of secondary structure) in two other structural contexts: a DNA duplex and i-Motifs. Uniquely among secondary structures, we observe that G quadruplexes are comparatively insensitive to the presence of anionic chaotropes, but not other denaturants. Further, the presence of equimolar NaCl provided greater mitigation of the destabilization caused by other non-anionic denaturants. These results are consistent with the presence of monovalent cations providing an especially pronounced stabilizing effect to G quadruplexes when studied in denaturing solution conditions.

Keywords: DNA; Denaturant; G quadruplex; Hofmeister; Nucleic acids.

Fig. 1

Melting temperatures of three representative DNA structures with varying [NaClO₄]. a) The Watson-Crick duplex Duplex40 exhibits an increase in T_M between 0.1 and 1 M, presumably due to relief of electrostatic repulsion, whereas at higher concentrations of NaClO₄, chaotropic effects predominate, with an average ΔT_M/d[NaClO₄] of −6.6 °C/M NaClO₄ from 1 to 9.5 M NaClO₄. b) The intramolecularly folded i-Motif iMotif19 does not exhibit an initial “electrostatic regime” of increasing T_M, but it instead exhibits ca. linear dependence for T_M vs. NaClO₄ intermediate to Duplex40 and HumTel, with an average ΔT_M/d[NaClO₄] of −3.9 °C/M NaClO₄. c) The intramolecularly folded G quadruplex HumTel exhibits an initial increase in T_M as well (up to 2.5 M NaClO₄), with a significantly smaller change in thermal stability at higher concentrations of NaClO₄ and an average ΔT_M/d[NaClO₄] of ca. −2.1 °C/M NaClO₄ from 2.5 M to 9.5 M NaClO₄. d-f) CD spectra of Duplex40, iMotif19, and Humtel in varying [NaClO₄].

Fig. 2

The inclusion of stoichiometric sodium chloride ameliorates the denaturing effect of urea and GuCl to a greater extent in G quadruplex versus Duplex DNA. a) The thermal stability of the duplex formed by Duplex40 (circles) and the G quadruplex formed by HumTel (squares) was measured in varying concentrations of urea with 100 mM KCl (open symbols) and equimolar urea/sodium chloride (closed symbols). Urea destabilized both structures (average dT_M/d[Urea] of −6.6 °C/M from 1 to 4 M urea, Duplex40 and -4.7 °C/M, HumTel). The inclusion of equimolar sodium chloride at high denaturant concentrations decreased urea-induced destabilization to a greater extent in HumTel (dT_M/d[Urea] = [NaCl] −1.7 °C/M from 1 to 4 M Urea/NaCl) than in Duplex40 (−5.3 °C/M from 1 to 4 M Urea/NaCl). b) A cationic chaotrope exerts similar destabilizing effects on a DNA duplex and G quadruplex. Duplex40 and HumTel both exhibit destabilization in the presence of increasing concentrations of 1–3 M guanidinium chloride (GuCl), with a greater destabilization for HumTel (dT_M/d[GuCl]: HumTel, −7.5 °C/M; Duplex40, -4.2 °C/M). When equimolar sodium is added, HumTel is greatly stabilized relative to Duplex40 as measured from 1 to 3 M GuCl (dT_M/d[GuCl] = [NaCl]: HumTel, −6.1 °C/M; Duplex40, -8.7 °C/M). c-f) CD spectra of HumTel in (c) Urea, (d) GuCl, (e) Equimolar Urea/NaCl, and (f) Equimolar GuCl/NaCl-containing solution.

Fig. 3

G quadruplexes of varied tetrad number exhibit the same destabilization trend in perchlorate solutions. a) A human telomere-derived G quadruplex (HumTel, 3 tetrads) as well as a more stable expanded analogue (HumTel + Tetrad, 4 tetrads) and less stable reduced analogue (HumTel-Tetrad, 2 tetrads) exhibit the same relative low sensitivity to perchlorate-induced destabilization. b) This is also observed for a second G quadruplex sequence (thrombin-binding aptamer, TBA, 2 tetrads) and two expanded (TBA + Tetrad, TBA+2Tetrad, 3 and 4 tetrads) analogues. c-d) In contrast to HumTel, which exhibits a conformational change with increasing sodium perchlorate (Fig. 1f) as observed by CD spectroscopy, (c) HumTel-Tetrad does not exhibit a large spectral change, (d) HumTel + Tetrad exhibit a structural change only at high sodium perchlorate concentrations, and (e–g) TBA and its derivatives do not exhibit large-scale CD spectral changes regardless of tetrad number.

Fig. 4

Sodium salts of multiple anionic chaotropes exhibit similar effects on a G quadruplex. HumTel exhibits similar behaviour with increasing chaotrope in the presence of NaSCN, NaI, and NaClO₄.