Quadruplex DNA: Formation, Roles, and Relevance

DNA can adopt secondary structures beyond the double helix, including the G-quadruplex. These non-canonical conformations arise in guanine-rich sequences and play roles in gene regulation and genome stability. Their stable four-stranded structure makes them potential therapeutic targets.

Understanding their formation and distribution is key to uncovering their biological significance. Researchers are investigating their roles in telomere maintenance, transcriptional control, and genomic integrity while developing methods for detection and characterization.

Structural Motifs and Formation

G-quadruplex DNA forms through the stacking of guanine residues into planar tetrads, stabilized by Hoogsteen hydrogen bonding. Each tetrad consists of four guanine bases arranged in a square planar configuration, with each base contributing two hydrogen bonds. These tetrads stack, creating a stable four-stranded structure. Monovalent cations, particularly potassium (K⁺) and sodium (Na⁺), enhance stability by coordinating with guanine carbonyl groups. Potassium ions provide stronger stabilization due to their optimal ionic radius, fitting well within the quadruplex’s central channel.

G-quadruplexes adopt diverse topologies influenced by sequence composition, strand orientation, and loop arrangements. They can form from a single DNA strand (intramolecular) or multiple strands (intermolecular), resulting in parallel, antiparallel, or hybrid conformations. In parallel structures, all strands run in the same direction, while antiparallel structures have alternating strand orientations. Hybrid forms combine these features, influenced by the molecular environment. Loop regions connecting guanine tracts vary in length and sequence, affecting stability and folding kinetics.

Environmental factors such as molecular crowding, pH, and specific ligands influence quadruplex formation and stability. Physiological conditions, including intracellular ionic concentrations and chaperone proteins, modulate their folding dynamics. Small molecules like telomestatin and pyridostatin selectively bind to G-quadruplexes, stabilizing them and altering biological functions. These ligands interact through π-π stacking with terminal guanine tetrads or electrostatic interactions with the phosphate backbone, making them promising drug targets, particularly in oncogenic pathways.

Genomic Distribution

G-quadruplex structures are enriched in functionally significant genomic regions, including gene promoters, untranslated regions (UTRs), and replication origins, where they influence transcription and replication. Computational analyses have identified thousands of potential quadruplex-forming sequences (PQS) in the human genome, particularly in guanine-rich regions, suggesting evolutionary selection for their regulatory roles.

Promoters of proto-oncogenes and housekeeping genes, such as c-MYC, KRAS, and VEGF, contain PQS that regulate transcription by affecting RNA polymerase activity and transcription factor binding. Stabilizing these structures with small molecules can suppress gene expression, highlighting their regulatory potential. Their asymmetric distribution within promoters suggests they act as molecular switches responsive to cellular conditions.

G-quadruplexes are also prevalent in replication origins, where they influence DNA synthesis. High-throughput sequencing and chromatin immunoprecipitation assays reveal PQS enrichment in replication initiation sites, particularly under replication stress. These structures can impede replication fork progression, necessitating helicases like PIF1 and WRN to resolve them. Mutations in these helicases, linked to genomic instability disorders such as Bloom and Werner syndromes, underscore the importance of quadruplex unwinding in maintaining replication fidelity.

In untranslated regions, particularly the 5′ UTR, G-quadruplexes regulate translation by affecting ribosome scanning and initiation. Their presence in these regions is linked to protein synthesis control, especially in stress response and proliferation-related genes. RNA-binding proteins and helicases such as DHX36 modulate their stability and function, influencing gene expression in response to environmental cues.

Telomeric Regions

Telomeres, the repetitive sequences capping chromosomes, are rich in guanine residues and prone to forming G-quadruplex structures. These (TTAGGG)n repeats in vertebrates naturally fold into stable quadruplexes under physiological conditions, affecting telomere function. Their structural stability is reinforced by potassium ions, which enhance guanine tetrad stacking.

Telomeric G-quadruplexes regulate telomerase, the enzyme that extends telomeric DNA to counteract shortening during cell division. In somatic cells, telomerase is tightly controlled, but in many cancers, it is upregulated, allowing unchecked proliferation. Quadruplex formation obstructs telomerase access to the 3′ overhang, limiting elongation. Small-molecule ligands like BRACO-19 and RHPS4 selectively stabilize telomeric quadruplexes, inducing telomere dysfunction, growth arrest, and apoptosis in cancer cells with high telomerase activity.

Beyond telomerase regulation, telomeric quadruplexes influence shelterin complex binding, which protects chromosome ends from being recognized as DNA damage. The shelterin subunit POT1 binds single-stranded telomeric DNA, competing with quadruplex formation and maintaining structural equilibrium. Disrupting this balance, through mutations or chemical stabilization, can trigger DNA damage responses and impact cellular aging. Quadruplex stabilization in telomeres induces replication stress, leading to telomere fragility and genomic instability, particularly in rapidly dividing cells.

Contrasting DNA With RNA G-Quadruplex

Both DNA and RNA form G-quadruplex structures, but differences in molecular composition and function lead to distinct properties. The 2’-hydroxyl group in RNA increases stability and folding rigidity, making RNA quadruplexes more thermodynamically stable than DNA counterparts. This stability arises from enhanced hydrogen bonding and structural constraints. Unlike DNA quadruplexes, which adopt various topologies, RNA quadruplexes predominantly form parallel structures due to steric hindrance from the ribose sugar.

These structural differences translate into distinct biological roles. DNA quadruplexes primarily regulate genomic processes, while RNA quadruplexes influence post-transcriptional regulation. In messenger RNA (mRNA), quadruplex formation in the 5′ UTR affects translation efficiency, either promoting or inhibiting ribosome binding. RNA quadruplexes also modulate alternative splicing by influencing exon inclusion or exclusion, with implications for neurodegenerative disorders.

Detection And Characterization Approaches

Studying G-quadruplex structures requires biophysical, biochemical, and computational techniques to determine their formation, stability, and biological relevance. Given their dynamic nature, accurate detection methods must distinguish quadruplexes from other DNA or RNA secondary structures while maintaining physiological sensitivity. Advances in high-resolution imaging and molecular probes have improved in vitro and in-cell analysis.

Spectroscopic techniques such as circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy help determine quadruplex topology. CD spectroscopy rapidly assesses folding by analyzing absorption spectra in circularly polarized light, distinguishing parallel, antiparallel, and hybrid structures. NMR spectroscopy provides atomic-level resolution, mapping guanine tetrad stacking and loop conformations, particularly in telomeric and oncogene promoter regions.

Fluorescent and small-molecule probes enhance in-cell detection. Thioflavin T (ThT) and N-methyl mesoporphyrin IX (NMM) selectively bind quadruplexes, producing fluorescence signals visible through confocal microscopy. G-quadruplex-specific antibodies enable chromatin-based detection, revealing genome-wide distribution patterns via chromatin immunoprecipitation sequencing (ChIP-seq). Mass spectrometry and single-molecule force spectroscopy further refine quadruplex stability assessments under physiological conditions, enhancing understanding of their biological function.