Insights into the Molecular Structure, Stability, and Biological Significance of Non-Canonical DNA Forms, with a Focus on G-Quadruplexes and i-Motifs

Abstract

This article provides a comprehensive examination of non-canonical DNA structures, particularly focusing on G-quadruplexes (G4s) and i-motifs. G-quadruplexes, four-stranded structures formed by guanine-rich sequences, are stabilized by Hoogsteen hydrogen bonds and monovalent cations like potassium. These structures exhibit diverse topologies and are implicated in critical genomic regions such as telomeres and promoter regions of oncogenes, playing significant roles in gene expression regulation, genome stability, and cellular aging. I-motifs, formed by cytosine-rich sequences under acidic conditions and stabilized by hemiprotonated cytosine-cytosine (C:C+) base pairs, also contribute to gene regulation despite being less prevalent than G4s. This review highlights the factors influencing the stability and dynamics of these structures, including sequence composition, ionic conditions, and environmental pH. Molecular dynamics simulations and high-resolution structural techniques have been pivotal in advancing our understanding of their folding and unfolding mechanisms. Additionally, the article discusses the therapeutic potential of small molecules designed to selectively bind and stabilize G4s and i-motifs, with promising implications for cancer treatment. Furthermore, the structural properties of these DNA forms are explored for applications in nanotechnology and molecular devices. Despite significant progress, challenges remain in observing these structures in vivo and fully elucidating their biological functions. The review underscores the importance of continued research to uncover new insights into the genomic roles of G4s and i-motifs and their potential applications in medicine and technology. This ongoing research promises exciting developments in both basic science and applied fields, emphasizing the relevance and future prospects of these intriguing DNA structures.

Keywords: G-quadruplex; i-motif; ligands; molecular dynamics; multi-stranded DNA structures; stability.

Figure 8

G-quadruplex folding model proposed in the study by Kim et al. [213] This model accounts for all possible folding pathways, viz. chair (C), basket (B), hybrids (H1 and H2), and propeller (P), and explicitly shows their interconversion mechanisms. G-stacking in each layer is classified as diad (d), triad (t), tetrad (q), or none (n) depending on the number status of guanine-aggregation through hydrogen bonds. Thus, three consecutive letters in subscripts in the generic notation indicate G-aggregation numbers pertaining to three layers of G-stacking in the G1 to G3 direction. Superscript “*” indicates the existence of non-native G-pairing. A single-hairpin is denoted as “hp1” and a double-hairpin as hp2a or hp2b. (Reprinted with permission from Ref. [213] Copyright 2023 American Chemical Society).

Figure 1

Watson–Crick hydrogen bonding interactions between complementary nitrogenous bases: A:T and G:C.

Figure 2

Models of the canonical forms of A-DNA, B-DNA, and Z-DNA using the sequence d (GC)₁₂. View along the helix axis with distinct major and minor grooves, and top view showing the bases arranged in the center surrounded by the phosphate–sugar backbone.

Figure 3

Watson–Crick and Hoogsteen base pairs. On the left are the traditional Watson–Crick AT and GC base pairs. On the right are the Hoogsteen TA and C+G base pairs. For cytosine to pair with guanine in a Hoogsteen bond, the N3 position of cytosine in the third strand must be protonated. The base pairing sites involved in Hoogsteen bonds in a purine molecule differ from those involved in Watson–Crick pairing (Hoogsteen pairing involves the N7 position in the imidazole ring) [22].

Figure 4

(A) H-DNA, intramolecular triple-stranded DNA. Within the polypurine–polypyrimidine helix with mirror symmetry of repeats, one of the single strands (marked in red) bends and forms a triple structure, while the other strand (blue) remains unpaired [37]. (B) Molecular conformation of a parallel DNA triple helix with 5′ and 3′ triplex–duplex junctions [38].

Figure 5

Schematic structures of G-quadruplexes. These structures are represented by three stacks of guanine quartets and are categorized into three types: parallel, antiparallel, and hybrid, according to strand orientation. In the quartet shown on the left, the symbol M+ indicates the position of a monovalent metal ion, and the dotted lines represent Hoogsteen hydrogen bonds between guanines [71]. (Adapted with permission from Ref. [71] Copyright 2020 Elsevier).

Figure 6

Schematic comparison of the Watson–Crick double helix with the i-motif structure [12]. (Adapted with permission from Ref. [12], Copyright 2014 Elsevier).

Figure 7

Schematic illustration of i-motif conformations: (A) hemi-protonated cytosine–cytosine+ base pair; (B) tetramolecular i-motif structure; (C) dimeric i-motif structure; (D) intramolecular i-motif structure [121]. (Adapted with permission from Ref. [121], Copyright 2014, American Chemical Society).

Figure 9

Contour maps of the potential of mean force (PMF) accompanied the biased unfolding of i-motifs within the i-motif+G-quadruplex structure at neutral pH, i.e., with the unprotonated i-motif. The collective variables rC13-T34 and rC13/T34-A23 are defined as distances between centers of masses of C13 and T34 bases and C13 and T34 taken together and A23, respectively. State A is the initial configuration, which is reference point (zero) in the PMF map. State B is the lowest energy configuration found, while states C and D are unfolded states close to the hairpin (C) or the random coil (D) configuration of the i-motif. (E)–(H) Contour maps of the potential of mean force (PMF) accompanied the biased unfolding of i-motifs within the i-motif+G-quadruplex structure at acidic pH, i.e., with the protonated i-motif (Adapted with permission from Ref. [234], Copyright 2019 American Chemical Society).