A quadruplex is a four-stranded structure that guanine-rich stretches of DNA or RNA can fold into, built from stacked layers of four guanines held together by hydrogen bonds and stabilized by metal ions at their core. Far from being a laboratory curiosity, quadruplexes form throughout the human genome and influence everything from how genes get switched on and off to how cells age and how viruses replicate. Their involvement in cancer, neurodegenerative disease, and viral infection has made them one of the more intensely studied non-standard DNA structures in modern molecular biology.
How a Quadruplex Forms
Standard DNA is a double helix, two strands wound around each other. A quadruplex breaks from that pattern entirely. When a stretch of DNA or RNA is rich in the base guanine, four guanines can arrange themselves into a flat, square-shaped unit called a G-tetrad, held together by a network of hydrogen bonds between the guanines. These tetrads then stack on top of each other like floors of a building, and metal ions, typically potassium or sodium, sit in the central channel between the stacked tetrads to hold the whole assembly together.1PubMed. Role of Alkali Metal Ions in G-Quadruplex Nucleic Acid Structure and Stability The result is a compact, remarkably stable structure that looks nothing like the familiar double helix.
Most G-quadruplexes are more stable in potassium than in sodium, since potassium fits snugly in the central channel between tetrads. But exceptions exist. One telomeric sequence that folds into two connected quadruplex units actually turns out to be more stable in sodium at body temperature, despite having a lower melting point overall.2PubMed Central. The exception that confirms the rule: a higher-order telomeric G-quadruplex structure more stable in sodium than in potassium Details like this matter because the ionic environment inside a cell is not uniform, and local conditions determine which quadruplexes actually fold and persist in living tissue.
The Mirror Image on the Other Strand
Where one strand of DNA is guanine-rich and can form a G-quadruplex, the complementary strand is typically cytosine-rich. That cytosine-rich strand can fold into its own four-stranded structure called an i-motif, built from pairs of cytosine bases where one member of each pair carries an extra proton. The requirement for that extra proton initially led researchers to assume i-motifs could only form in acidic conditions, with peak stability around pH 4 to 5.3PubMed Central. i-Motifs as regulatory switches: Mechanisms and implications for gene expression That would make them biologically irrelevant, since the interior of most cells sits near pH 7.
More recent work, though, has shown that i-motifs can form at neutral pH depending on the sequence and the surrounding environment.4Nucleic Acids Research. i-Motif DNA: structural features and significance to cell biology This has opened up the possibility that G-quadruplexes and i-motifs could form simultaneously on opposite strands of the same genomic region, each potentially serving as a regulatory switch. The pH sensitivity of i-motifs has also been exploited to design molecular sensors that respond to tiny shifts in acidity, with both the sensitivity and the transition point tunable through rational changes to the i-motif sequence.5PubMed. Rational design of highly responsive pH sensors based on DNA i-motif
Where Quadruplexes Sit in the Genome
Guanine-rich sequences with the potential to form quadruplexes are not randomly scattered across chromosomes. Genome-wide mapping has shown that these sequences cluster in specific functional regions. One study using an antibody-based pull-down approach found that the vast majority of enriched genomic regions, about three-quarters, contained sequences with quadruplex-forming potential, and the number of such regions was far higher than would be expected by chance.6Nature Communications. G-quadruplex structures are stable and detectable in human genomic DNA Quadruplex-forming sequences tend to concentrate in gene promoters (the regulatory regions that control when a gene turns on), at telomeres (the protective caps on chromosome ends), and in the first intron of many genes.
This distribution is not accidental. Across dozens of species, from yeast to mammals, quadruplex-forming motifs are more conserved than surrounding sequences, meaning evolution has preserved them rather than letting them drift randomly. A genome-wide analysis across 37 species found that short-loop G-quadruplexes were conserved in most organisms, and that the proportion of genes harboring these motifs increased over evolutionary time, with growing enrichment among transcription factor genes in particular.7PubMed Central. Genome-wide analysis of DNA G-quadruplex motifs across 37 species provides insights into G4 evolution In yeast, conservation patterns at the nucleotide level suggest that natural selection is maintaining the ability to form the four-stranded structure itself, not just the underlying sequence.8PLoS Computational Biology. G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae
Controlling Genes Like a Structural Switch
One of the clearest examples of a quadruplex influencing gene activity involves MYC, an oncogene whose overactivation drives many cancers. The MYC promoter contains a well-studied G-quadruplex, and researchers recently used CRISPR to eliminate the ability of that specific region to fold into a quadruplex inside living cells. When the quadruplex could no longer form, MYC transcription from one of its promoters dropped, a new nucleosome deposited itself over the site, and the recruitment of the enzyme that reads DNA into RNA was altered. Crucially, when the team replaced the original MYC quadruplex sequence with an entirely different quadruplex-forming sequence from the KRAS oncogene, MYC transcription was restored. The structure mattered; the exact DNA letters did not.9PubMed Central. G-quadruplex DNA structure is a positive regulator of MYC transcription
The MYC quadruplex also physically recruits proteins. A 2025 structural study showed that nucleolin, a protein abundant in the cell nucleus, directly recognizes and binds this quadruplex to help regulate MYC transcription.10PubMed Central. Structural basis for nucleolin recognition of MYC promoter G-quadruplex Because MYC is overexpressed in so many tumor types, the MYC quadruplex has become a prime drug target. A small molecule called DC-34, designed to bind the MYC quadruplex preferentially, reduced MYC RNA levels and decreased cell viability in myeloma cells, with weaker effects on other genes.11Nature Communications. Chemical and structural studies provide a mechanistic basis for recognition of the MYC G-quadruplex
Quadruplexes at Telomeres
Telomeres, the repetitive sequences capping the ends of chromosomes, are among the most guanine-rich regions in the genome. In humans, the telomeric repeat (TTAGGG) readily folds into G-quadruplexes, and this has complex consequences. There is evidence, at least in yeast, that telomeric quadruplexes can play a protective capping role and may positively affect telomere maintenance by the enzyme telomerase or by recombination-based pathways. At the same time, quadruplex formation at telomeres can block the replication machinery and become a source of genome instability.12PubMed Central. G-Quadruplexes at Telomeres: Friend or Foe?
The dual nature of telomeric quadruplexes has attracted drug developers. Telomestatin, a natural compound isolated from a soil bacterium, stabilizes telomeric G-quadruplexes and is one of the most potent known telomerase inhibitors, working at concentrations as low as 5 nanomolar.13PubMed. Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular g-quadruplex The idea is simple: lock the telomeric DNA into its quadruplex form and telomerase cannot extend it, which should limit the immortality of cancer cells. But detailed analysis of how quadruplex-binding molecules actually work revealed something more nuanced. Most such ligands do lock quadruplex-prone sequences into the folded state, inhibiting the initial step of telomerase elongation, yet only a few interfere with the enzyme’s ongoing processivity. These molecules also turned out to be more effective at blocking telomeric DNA amplification than at inhibiting telomerase extension per se, suggesting they are not simple telomerase inhibitors but a distinct class of biologically active compounds.14PubMed Central. Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action
When Quadruplexes Threaten Genome Stability
Every time a cell divides, the replication machinery must copy the entire genome. A quadruplex sitting on the template strand is a physical roadblock: the replication fork stalls when it hits the compact four-stranded structure. Cells deal with this by deploying specialized helicase enzymes that unfold quadruplexes ahead of the fork. Several helicases, including BLM, WRN, and FANCJ, have been shown to resolve these structures, and single-molecule imaging revealed that they share a common strategy of repetitively unfolding the quadruplex multiple times in succession, providing robust resolving power.15PubMed Central. Single-molecule imaging reveals a common mechanism shared by G-quadruplex-resolving helicases
When these helicases fail, the consequences are serious. FANCJ deficiency leads to quadruplex accumulation on replicating DNA, DNA damage at replication forks that encounter quadruplexes, and suppression of the normal stress-response signaling that would alert the cell to trouble.16PubMed Central. Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling Stable quadruplexes can cause double-strand DNA breaks during replication, and these breaks are processed by a nuclease called Mus81, producing patterns of instability that resemble a repair pathway associated with chromosomal rearrangements.17PubMed Central. Stable G-quadruplex DNA structures promote replication-dependent genome instability Mutations in the genes encoding these helicases underlie several human genetic syndromes characterized by cancer predisposition and premature aging, connecting quadruplex biology directly to human disease.
RNA Quadruplexes and Neurodegenerative Disease
Quadruplexes are not confined to DNA. Guanine-rich stretches of messenger RNA can fold into equivalent structures, and these RNA quadruplexes influence how efficiently a transcript gets translated into protein. In the cell, RNA helicases and binding proteins work together to manage them. One well-characterized mechanism involves the helicase DHX36 first unwinding the RNA quadruplex, followed by the binding of proteins that keep the RNA in its unfolded state, regulating translation of messenger RNAs involved in aggressive brain tumors.18Trends in Biochemical Sciences. What Is a Quadruplex and Its Role in Biology and Disease?
The most striking disease connection for RNA quadruplexes involves amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The most common known genetic cause of both diseases is an expansion of a six-letter repeat (GGGGCC) in the C9orf72 gene. The transcribed RNA from these expanded repeats forms stable G-quadruplexes, as demonstrated by multiple biophysical methods.19Scientific Reports. C9orf72 hexanucleotide repeat associated with amyotrophic lateral sclerosis and frontotemporal dementia forms RNA G-quadruplexes These RNA quadruplexes assemble into toxic foci inside the cell nucleus, where they trap RNA-binding proteins and disrupt normal RNA processing, ultimately contributing to neurodegeneration.20Nucleic Acids Research. Crystal structure of a tetrameric RNA G-quadruplex formed by hexanucleotide repeat expansions of C9orf72 in ALS/FTD Atomic force microscopy has shown that these RNA repeats can even assemble into extended wire-like higher-order structures under physiologically relevant conditions, suggesting that quadruplex-mediated aggregation may be part of the disease mechanism.21PubMed. Formation of RNA G-wires by G(4)C(2) repeats associated with ALS and FTD
Quadruplexes in Viral Genomes
Viruses are not immune to quadruplex biology. Quadruplex-forming sequences have been identified in genomes across most major virus groups, and they tend to sit in regulatory regions that control critical stages of the viral life cycle.22PubMed Central. G-Quadruplexes in the Viral Genome: Unlocking Targets for Therapeutic Interventions and Antiviral Strategies Stabilizing these viral quadruplexes with small molecules could, in principle, jam the virus’s own regulatory machinery.
A concrete example comes from hepatitis B virus (HBV). The virus maintains a persistent DNA form inside liver cells called cccDNA, which serves as the template for all viral gene expression. Researchers found that G-quadruplexes form in a key regulatory region of this cccDNA, and that mutations disrupting two of these quadruplexes altered viral transcription and replication. The study also showed, for the first time, that cccDNA undergoes a form of liquid-liquid phase separation in a quadruplex-dependent manner to promote its own transcription inside infected liver cells.23PubMed Central. G-quadruplexes control hepatitis B virus replication by promoting cccDNA transcription and phase separation in hepatocytes Targeting viral quadruplexes is still early-stage, but it offers a fundamentally different angle of attack compared to conventional antivirals.
Quadruplexes and the Epigenome
The relationship between quadruplexes and DNA methylation, one of the cell’s primary tools for silencing genes, turns out to run in both directions. On one hand, G-quadruplex structures physically block the major maintenance methylation enzyme (DNMT1) from doing its job. Although DNMT1 actually binds quadruplex DNA more tightly than it binds normal double-stranded DNA, the quadruplex structure itself inhibits the enzyme’s catalytic activity. The net effect is that quadruplexes protect nearby regions from being methylated, keeping associated genes in an active, readable state.24PubMed Central. DNA G-quadruplex structures mould the DNA methylome
On the other hand, methylation suppresses quadruplex formation. When researchers knocked out DNMT1 or treated cells with a methylation inhibitor, genomic quadruplex abundance rose significantly, and restoring DNMT1 reversed the increase. The methylation of cytosines within or near guanine-rich sequences appears to directly discourage the DNA from folding into a quadruplex. Combined analysis showed that reduced methylation enhances quadruplex formation, which in turn promotes transcription of nearby genes.25PubMed Central. DNA 5mC methylation inhibits the formation of G-quadruplex structures in the genome This reciprocal antagonism creates a feedback loop: methylation keeps quadruplexes from forming, and quadruplexes keep methylation from spreading, with gene activity as the ultimate output.
Phase Separation and Cellular Compartments
One of the more surprising recent findings is that quadruplexes can trigger liquid-liquid phase separation, the process by which molecules in the cell spontaneously separate into droplet-like compartments without a surrounding membrane. RNA quadruplexes were shown to drive phase separation under physiological conditions, with the quadruplex structure itself being essential: when the guanine-rich sequence was mutated so it could no longer fold, the droplets did not form.26Nucleic Acids Research. G-quadruplex structures trigger RNA phase separation
The connection between quadruplexes and phase separation extends to disease-relevant proteins. FUS, a protein whose aggregation is linked to ALS and FTD, interacts with RNA quadruplexes in a way that depends on the quadruplex’s length and stability. Short RNA quadruplexes inhibit FUS assembly into droplets, while longer ones switch to promoting it. Chemical modifications that stabilize the quadruplex enhance the inhibitory function and make it resistant to changes in salt concentration.27PubMed Central. RNA G-quadruplexes function as a tunable switch of FUS phase separation In the nucleus, quadruplex-dependent condensates have been observed at telomeres, transcription start sites, and within nucleoli and other nuclear bodies.28PubMed Central. G-Quadruplexes in Nuclear Biomolecular Condensates The picture that emerges is of quadruplexes serving as structural seeds that help organize specific compartments within cells.
Seeing Quadruplexes in Living Cells
For years, the criticism of quadruplex biology was that these structures formed readily in a test tube but might not exist inside living cells. That skepticism has been largely put to rest by multiple visualization techniques. Fluorescent probes that light up specifically when they bind quadruplex structures have been used to image them in real time in living cells, revealing concentrations of quadruplexes in the nucleolus, the ribosome-building factory of the cell.29PubMed. Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe Beyond fluorescence microscopy, researchers have applied transmission electron microscopy and atomic force microscopy to visualize quadruplexes and i-motifs with high spatial resolution.30PubMed Central. Visualization of G-Quadruplexes, i-Motifs and Their Associates The combination of antibody-based detection, structure-specific fluorescent dyes, and high-resolution imaging has built a strong case that quadruplexes are genuine features of the living genome, not artifacts of bench chemistry.
The Drug Development Landscape
Because quadruplexes sit in the promoters of many oncogenes and at telomeres, they have attracted heavy interest as anticancer drug targets. The strategy is conceptually elegant: a small molecule that stabilizes a quadruplex in a cancer gene’s promoter could shut down that gene’s expression, while a molecule that locks telomeric quadruplexes could block telomerase and limit cancer cell immortality.31PubMed Central. G-quadruplexes: a promising target for cancer therapy Natural products with quadruplex-stabilizing activity are also being explored, particularly for breast cancer, where they may offer multi-target activity with good biocompatibility.32Drug Design, Development and Therapy. Natural G-Quadruplex Stabilizers: A Targeted Strategy for Breast Cancer Therapy
The honest status of the field, however, is that no quadruplex-targeting drug has yet been approved for clinical use.33PubMed. G-quadruplex ligands in cancer therapy: Progress, challenges, and clinical perspectives The main challenge is selectivity. The human genome contains hundreds of thousands of potential quadruplex-forming sequences, and most existing ligands do not distinguish well between different quadruplexes. Stabilizing a quadruplex in a cancer gene’s promoter is only useful if you are not simultaneously disrupting quadruplex dynamics everywhere else in the genome. The field has been moving toward more selective molecules, like DC-34 for the MYC quadruplex, but the gap between test-tube potency and a safe, effective drug in a patient remains wide. The excitement is real, but so are the obstacles.