G Quadruplex in DNA: Formation, Stability, and Roles

DNA is typically known for its double-helix structure, but under certain conditions, it can fold into alternative secondary structures. One such structure is the G-quadruplex (G4), which forms in guanine-rich sequences. These four-stranded configurations influence genomic stability and biological processes.

Researchers are exploring how G-quadruplexes contribute to cellular functions and disease mechanisms. Their presence in key genomic regions suggests a broader role beyond structural variation.

Structural Features In DNA

G-quadruplex structures adopt distinct topologies depending on strand orientation, loop arrangements, and the ionic environment. These variations influence their stability and function. The three primary conformations—parallel, antiparallel, and hybrid—exhibit unique structural characteristics based on guanine tetrad arrangements and connecting loops.

Parallel

In the parallel conformation, all four DNA strands run in the same 5′ to 3′ direction. This topology is commonly observed in sequences with short loops and is favored under physiological potassium concentrations. The guanine tetrads stack uniformly, stabilized by Hoogsteen hydrogen bonding and monovalent cations such as K⁺ or Na⁺. X-ray crystallography and NMR spectroscopy confirm that parallel G-quadruplexes often form in promoter regions of oncogenes like c-MYC, where they may act as regulatory elements. Studies published in Nucleic Acids Research (2021) show that small molecules stabilizing parallel G4s can modulate gene expression, highlighting their therapeutic potential.

Antiparallel

Antiparallel G-quadruplexes feature strands oriented in alternating 5′ to 3′ and 3′ to 5′ directions. This configuration arises when longer loops connect guanine tetrads, forming lateral or diagonal arrangements. Structural studies indicate that antiparallel G4s are prevalent in telomeric DNA, particularly in human sequences rich in TTAGGG repeats. Circular dichroism (CD) spectroscopy distinguishes antiparallel G4s by their characteristic spectral signatures. Research in The Journal of Biological Chemistry (2020) demonstrated that telomeric G-quadruplexes primarily adopt this topology in the presence of sodium ions, contributing to telomere maintenance.

Hybrid

Hybrid G-quadruplexes exhibit characteristics of both parallel and antiparallel structures, with strands adopting mixed orientations. These conformations are frequently seen in human telomeric DNA. The presence of different loop lengths and orientations results in structural polymorphism, as seen in NMR studies. A study in Nature Communications (2022) highlighted that hybrid G4s are influenced by protein interactions and cellular ionic balance, which dictate their folding preferences. Their adaptability suggests a role in fine-tuning cellular processes, making them a focus of ongoing research.

Formation And Stability

G-quadruplex formation is driven by guanine-rich sequences folding into stacked guanine tetrads through Hoogsteen hydrogen bonding. This structure is stabilized by monovalent cations, particularly potassium (K⁺), which enhances its integrity. Sodium (Na⁺) also supports G4 formation but favors antiparallel configurations. The ionic environment plays a key role, as physiological K⁺ concentrations promote stable G-quadruplexes, while lower salt conditions can lead to unfolding or structural transitions.

Loop length and sequence composition influence stability. Short loops, typically one to three nucleotides, enhance stability by minimizing entropic penalties. Longer loops introduce flexibility, leading to structural polymorphism. Differential scanning calorimetry (DSC) and UV melting assays reveal that sequences with shorter loops exhibit higher melting temperatures (Tm), indicative of greater stability. Human telomeric sequences, which commonly adopt hybrid G4 structures, demonstrate a Tm in the range of 60–70°C under physiological conditions, reinforcing their persistence in vivo.

Molecular crowding and cellular conditions also affect G-quadruplex stability. The intracellular environment, densely packed with macromolecules, promotes G4 folding by excluding water and increasing local ion concentrations. Crowding agents such as polyethylene glycol (PEG) enhance G4 stability in vitro. Additionally, post-translational modifications of G4-interacting proteins, such as helicases, modulate the equilibrium between folded and unfolded states. For example, the helicase WRN, mutated in Werner syndrome, unwinds G-quadruplexes, and its loss leads to increased G4 accumulation, implicating these structures in genome maintenance disorders.

Locations In The Genome

G-quadruplex structures are enriched in guanine-rich genomic regions. High-throughput sequencing techniques like G4-seq and ChIP-seq have mapped G-quadruplex motifs across various species, revealing conserved patterns that hint at their biological significance.

Promoter regions frequently contain G-quadruplexes, particularly in genes regulating the cell cycle, transcription, and signal transduction. The promoters of oncogenes such as c-MYC, KRAS, and BCL-2 harbor G4-forming sequences that can modulate gene activity by altering RNA polymerase accessibility. Experimental validation using G4-specific ligands and mutagenesis approaches has shown that stabilizing these structures can lead to transcriptional repression.

Beyond promoters, G-quadruplexes are often found in untranslated regions (UTRs) of mRNAs, influencing post-transcriptional regulation. The 5′ UTR, in particular, is a hotspot for G4 formation, affecting translation efficiency. Ribosome profiling studies show that stable G-quadruplexes in this region can impede translation initiation. However, some RNA-binding proteins resolve these structures, allowing controlled translation under specific conditions.

Role In Gene Regulation

G-quadruplexes regulate gene expression by altering DNA accessibility and modulating protein-DNA interactions. In promoters, they can act as molecular switches, either facilitating or hindering transcription depending on stability and interacting factors. When a G4 forms, it can impede RNA polymerase progression, leading to transcriptional repression. Conversely, some transcription factors bind G-quadruplexes to enhance gene activation.

Beyond transcriptional control, G-quadruplexes influence chromatin remodeling and epigenetic regulation. Their ability to recruit chromatin-modifying proteins affects nucleosome positioning and histone modifications. For instance, G4 formation has been linked to histone acetyltransferase recruitment, promoting an open chromatin state conducive to gene activation. Helicases such as PIF1 and WRN unwind G-quadruplexes to restore normal transcriptional dynamics, maintaining gene expression homeostasis.

Telomeric Function

G-quadruplex structures are abundant in telomeres, where they contribute to chromosome stability and replication dynamics. Human telomeres, composed of TTAGGG repeats, readily form G-quadruplexes, influencing telomerase activity. When a G-quadruplex forms at the 3′ overhang, it can hinder telomerase, which extends telomeric DNA. This regulatory mechanism has been explored as a potential cancer therapy, with small-molecule stabilizers designed to trap G4s and inhibit telomerase activity.

Beyond telomerase regulation, G-quadruplexes protect chromosome ends from being recognized as DNA damage. Shelterin, a protein complex, interacts with these structures to maintain end stability and prevent erroneous DNA repair activation. Disruptions in G4 dynamics have been linked to genomic instability and premature aging syndromes such as dyskeratosis congenita. Single-molecule imaging has shown that telomeric G-quadruplexes undergo structural transitions in response to cellular signals, suggesting they integrate environmental and metabolic cues into telomere maintenance.

Laboratory Methods Of Detection

The study of G-quadruplexes relies on specialized techniques to identify their presence, characterize structure, and assess function.

Spectroscopic methods such as circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy characterize G-quadruplex conformations. CD spectroscopy detects absorbance patterns distinguishing parallel, antiparallel, and hybrid topologies. NMR provides atomic-level resolution of G4 folding, revealing interactions with small molecules or proteins. Förster resonance energy transfer (FRET) tracks folding and unfolding kinetics in real time.

Genome-wide approaches like G4-seq and BG4-ChIP map G-quadruplexes across the genome. G4-seq stabilizes G-quadruplexes during DNA processing, revealing their distribution with single-nucleotide resolution. BG4-ChIP uses a G4-specific antibody for chromatin immunoprecipitation followed by sequencing (ChIP-seq), identifying G4-enriched regions in native chromatin contexts.

Links To Oncogenesis And Other Conditions

G-quadruplexes play a role in cancer by regulating oncogene expression, telomere maintenance, and genome stability. Many cancer-associated genes contain G4-prone sequences in their promoters. Small molecules such as pyridostatin and BRACO-19 selectively bind and stabilize G-quadruplexes, downregulating oncogenic transcription programs.

Beyond cancer, G-quadruplex dysregulation is implicated in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and fragile X syndrome. In ALS, RNA G-quadruplexes formed by repeat expansions in the C9orf72 gene contribute to toxic protein aggregation. In fragile X syndrome, G4 structures within the FMR1 gene lead to transcriptional silencing, causing cognitive impairment.