Diversity of Parallel Guanine Quadruplexes Induced by Guanine Substitutions

Abstract

Recently, we reported an inhibitory effect of guanine substitutions on the conformational switch from antiparallel to parallel quadruplexes (G4) induced by dehydrating agents. As a possible cause, we proposed a difference in the sensitivity of parallel and antiparallel quadruplexes to the guanine substitutions in the resulting thermodynamic stability. Reports on the influence of guanine substitutions on the biophysical properties of intramolecular parallel quadruplexes are rare. Moreover, such reports are often complicated by the multimerisation tendencies of parallel quadruplexes. To address this incomplete knowledge, we employed circular dichroism spectroscopy (CD), both as stopped-flow-assisted fast kinetics measurements and end-point measurements, accompanied by thermodynamic analyses, based on UV absorption melting profiles, and electrophoretic methods. We showed that parallel quadruplexes are significantly more sensitive towards guanine substitutions than antiparallel ones. Furthermore, guanine-substituted variants, which in principle might correspond to native genomic sequences, distinctly differ in their biophysical properties, indicating that the four guanines in each tetrad of parallel quadruplexes are not equal. In addition, we were able to distinguish by CD an intramolecular G4 from intermolecular ones resulting from multimerisation mediated by terminal tetrad association, but not from intermolecular G4s formed due to inter-strand Hoogsteen hydrogen bond formation. In conclusion, our study indicates significant variability in parallel quadruplex structures, otherwise disregarded without detailed experimental analysis.

Keywords: DNA secondary structure; circular dichroism; multimerisation; parallel guanine quadruplex; stopped-flow.

Figure 1

Model parallel quadruplexes with/without terminal overhangs: (A) CD spectra measured in 100K buffer at 20 °C; solid curves represent CD spectra, dashed curves represent difference to the Q spectrum; green line represents sum of differences of left and right overhang for AAT…TAA variant. (B) Native PAGE performed in 100K buffer at 20 °C. (C) Melting temperatures measured in 1K buffer, pH 7.

Figure 2

Different parameters of parallel WT sequence and all G/T mutants. (A) Schematic structure of the WT G4 with numbering of guanines used for labeling of G to T substituted variants. Green circles represent guanines, red ones adenines and blue circles represent thymines. Green trapezoids represent guanine tetrads. (B) Native PAGE. (C) The CD264 value (molar DNA strand circular dichroism (Δε) measured at 264 nm) before (black) and after (gray) thermal annealing. (D) T_m values observed from absorbance at 297 nm during renaturation (black) and denaturation (grey) processes. All data were observed in 100K buffer. Native PAGE and CD measurements were done at 23 °C. * T_m for WT could not be determined due to extreme thermal stability. Error bars in (C) and (D) represent standard deviations calculated from three independent measurements.

Figure 3

The relative portion of G4 folded calculated from circular dichroism for all G/T mutants in 10K (top) and 100K (bottom) buffer at various times after mixing. The values are based on Δε₂₆₅, normalized to value observed by stopped-flow experiment mixing DNA with 1NF buffer (0) and to value measured after annealing in 100K buffer (1). Error bars represent standard deviations calculated from three independent measurements.

Figure 4

Dependence of the G4 properties of WT and selected mutated sequences on DNA concentration in 100K buffer. (A) The CD spectra (B) T_m values from renaturation (black) and denaturation (gray) profile and (C) native PAGE o. Red CD spectra correspond to samples slowly annealed in 100K buffer at 100 µM DNA strand concentration. Blue CD spectra correspond to denatured samples measured at 98 °C in 1Nf buffer at 3 µM strand concentration.

Figure 5

The T_m of parallel (black) and antiparallel (gray) quadruplexes for WT sequences and all G/T substituted variants in 100K buffer. *—The T_m of parallel WT sequence could not be determined because of the extreme stability of the parallel WT quadruplex in 100K buffer.