Insight into Tetramolecular DNA G-Quadruplexes Associated with ALS and FTLD: Cation Interactions and Formation of Higher-Ordered Structure

Abstract

The G₄C₂ hexanucleotide repeat expansion in the c9orf72 gene is a major genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), with the formation of G-quadruplexes directly linked to the development of these diseases. Cations play a crucial role in the formation and structure of G-quadruplexes. In this study, we investigated the impact of biologically relevant potassium ions on G-quadruplex structures and utilized ¹⁵N-labeled ammonium cations as a substitute for K⁺ ions to gain further insights into cation binding and exchange dynamics. Through nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we demonstrate that the single d(G₄C₂) repeat, in the presence of ¹⁵NH₄⁺ ions, adopts a tetramolecular G-quadruplex with an all-syn quartet at the 5'-end. The movement of ¹⁵NH₄⁺ ions through the central channel of the G-quadruplex, as well as to the bulk solution, is governed by the vacant cation binding site, in addition to the all-syn quartet at the 5'-end. Furthermore, the addition of K⁺ ions to G-quadruplexes folded in the presence of ¹⁵NH₄⁺ ions induces stacking of G-quadruplexes via their 5'-end G-quartets, leading to the formation of stable higher-ordered species.

Keywords: ALS; DNA; FTLD; NMR; c9orf7; cations; quadruplex.

Figure 1

Imino and aromatic regions of the ¹H NMR spectrum of d(G₄C₂) in the absence (A) and the presence (B) of 100 mM of ¹⁵NH₄Cl. Spectra were recorded in 90% H₂O and 10% ²H₂O at 25 °C, pH 6.0, on a 600 MHz NMR spectrometer. The oligonucleotide concentration was 2.0 mM per strand. The doublet signal (J = 74 Hz) denoted with asterisks corresponds to ammonium ions bound to binding site O₅ within the G-quadruplex.

Figure 2

(A) Folding topology and cation binding sites within G-quadruplex adopted by d(G₄C₂) in the presence of ¹⁵NH₄⁺ ions, with the three ion binding sites shown. Syn- and anti-conformations of guanines are represented with grey and white rectangles, respectively. The arrows represent the observed movements between cation binding sites, with apparent rate constants depicted next to them. (B) Imino-aromatic region of the NOESY spectrum (left), showing cross-peaks between bound ¹⁵NH₄⁺ ions and nearby imino protons, and the 2D ¹H-¹⁵N HSQC spectrum (right) exhibiting cross-peaks corresponding to ¹⁵NH₄⁺ ions in different chemical environments within [d(G₄C₂)]₄. The vertical and horizontal dashed lines connect resonances of guanine imino and ¹⁵NH₄⁺ ion protons. (C) The 2D ¹H-¹⁵N NzExHSQC spectrum at a mixing time (τ_m) of 1.1 s (left) and the 2D ¹H-¹⁵N HzExHSQC spectrum at a mixing time (τ_m) of 40 ms (right). Annotations of autocorrelation and exchange cross-peaks are shown next to the cross-peaks.

Figure 3

(A) Lowest energy structure of the [d(G₄C₂)]₄ G-quadruplex in the presence of ¹⁵NH₄⁺ ions (PDB ID 8C7B) and (B) of the dimeric form of the [d(G₄C₂)]₄ G-quadruplex in the presence of K⁺ ions (PDB ID 8C7A). Residues G1, G2, G3, G4, C5, and C6 are shown in magenta, blue, green, violet, orange, and red, respectively. Cations within both structures are shown. Ion binding sites in (A) and (B) are marked with O₅, I, O₃, and O_GC. O_GC is the binding site between G4 and C5 residues. In (B) an additional binding site, O_GG, is present. It is located between G1 and G1 residues, formed by 5′ stacking of two [d(G₄C₂)]₄ G-quadruplexes. Stacking of G-quartets and G-quartets and cytosines are shown.

Figure 4

The projection of the center of a G-quartet composed of G4 residues (marked as G4-quartet) to the adjacent G-quartet composed of G3 residues (marked as G3-quartet) within [d(G₄C₂)]₄ in the presence of ¹⁵NH₄⁺ ions. Due to clarity, the ¹⁵NH₄⁺ ion between G-quartets is not present. The O6 atoms of the G-quartets are shown as red sphere models, and centers of the G-quartets are shown in blue. d is the distance between the centers of two adjacent G-quartets.

Figure 5

(A) Free-energy profile for NH₄⁺ ion movements through G-quartets of [d(G₄C₂)]₄ G-quadruplex and the pulling direction of ¹⁵NH₄⁺ ions, as well as (B) the representation of the reaction coordinate. G-quartets composed of G1, G2, G3, and G4 residues are marked as G1-, G2-, G3-, and G4-quartets, respectively.

Figure 6

Imino regions of ¹H NMR and corresponding ¹H-¹⁵N HSQC spectra of [d(G₄C₂)]₄ at 25 °C, pH 6.0, in 10% ²H₂O in the presence of 100 mM of ¹⁵NH₄Cl and gradual addition of KCl. The oligonucleotide concentration was 2.0 mM per strand. Assignments of imino resonances at the beginning and end of titration are indicated above the signals. Annotations of cross-peaks are shown next to the peaks.

Figure 7

Gradual exchange of ¹⁵NH₄⁺ by K⁺ ions and the formation of dimeric species as the concentration of K⁺ ions is increased from 0 to 80 mM.