A quadruplex, most commonly called a G-quadruplex or G4, is a four-stranded structure that forms in DNA and RNA when sequences rich in the nucleotide guanine fold onto themselves. Unlike the familiar double helix, which pairs two strands, a quadruplex stacks flat layers of four guanine bases on top of each other, creating a compact, unusually stable structure. These structures are found throughout the human genome and play active roles in how genes are switched on or off, how chromosomes are maintained, and how cells divide.
How the Structure Forms
In a normal DNA double helix, bases pair across two strands (A with T, G with C) using what’s called Watson-Crick bonding. A G-quadruplex works differently. Four guanine bases arrange themselves in a flat square called a G-quartet, connected by a distinct type of hydrogen bonding known as Hoogsteen pairing. Each guanine in the quartet forms two hydrogen bonds with its neighbor, locking the four bases into a planar ring.
These flat quartets then stack on top of one another like floors in a building. The stacking is stabilized by interactions between the electron clouds of the guanine bases at different layers. At the center of the stacked quartets sits a positively charged metal ion, typically potassium or sodium, which neutralizes the negative charges pointing inward and holds the whole structure together. Without these metal ions, the quadruplex won’t form. This is why researchers preparing quadruplexes in the lab add potassium chloride to the solution, heat the DNA, then slowly cool it to let the structure fold.
Where Quadruplexes Appear in the Genome
G-quadruplex-forming sequences are not scattered randomly across human DNA. They cluster in two especially important locations: telomeres and gene promoters.
Telomeres are the repetitive caps at the ends of chromosomes that protect genetic information during cell division. Human telomeres consist of repeating guanine-rich sequences that readily fold into quadruplexes. Promoter regions, the stretches of DNA just upstream of a gene that control when and how much the gene is active, are the other major hotspot. Computational analysis has found that guanine-rich sequences capable of forming quadruplexes are especially concentrated in the promoters of proto-oncogenes, the genes that can drive cancer when improperly activated.
Beyond these two locations, quadruplex-forming sequences also appear in the untranslated regions of messenger RNA, in replication origins where DNA copying begins, and within the coding sequences of genes themselves.
What Quadruplexes Do in Cells
Quadruplexes act as structural switches that can turn biological processes up or down depending on where they form and on which strand of DNA.
In gene promoters, a quadruplex on the template strand (the strand the cell’s machinery reads to build RNA) can physically block RNA polymerase from moving forward, effectively silencing the gene. On the other hand, genes with more quadruplexes on the non-template strand tend to have higher-than-average activity. The likely explanation is that quadruplexes on the non-template strand help keep the DNA in an open, accessible state, making it easier for the cell to re-initiate transcription.
During DNA replication, quadruplexes can be problematic. When the double helix is unwound for copying, the transiently single-stranded DNA can fold into quadruplexes that stall the replication machinery. If the cell’s specialized enzymes can’t resolve these structures, the replication fork collapses, causing double-strand breaks. Mutations in the helicases responsible for unwinding quadruplexes have been directly linked to genome instability, certain cancers, and genetic disorders.
At telomeres, the story is especially interesting. Telomerase, the enzyme that lengthens chromosome ends, needs a free single-stranded tail of at least eight nucleotides to do its job. When a quadruplex forms at the very tip of a telomere, the remaining tail is only about five nucleotides long, too short for telomerase to grab. This means quadruplex formation at chromosome ends can naturally block telomere extension.
RNA Quadruplexes
Quadruplexes aren’t limited to DNA. RNA molecules also form G-quadruplexes, and because RNA is already single-stranded, it can fold into these structures more readily. RNA quadruplexes have been detected in the cytoplasm of human cells using a specially engineered antibody called BG4, which binds G-quadruplex structures with nanomolar affinity.
These RNA quadruplexes influence several stages of protein production. When they form in the 5′ untranslated region of a messenger RNA (the stretch before the protein-coding sequence), they block ribosomes from scanning along the RNA, reducing protein output. They can even redirect ribosomes to alternative start sites, changing which protein gets made. In the coding sequence itself, ribosomes stall about six to seven nucleotides before a quadruplex, disrupting translation elongation. Evolution has partially dealt with this by selecting for synonymous codons (different DNA spellings that code for the same amino acid) that avoid forming quadruplexes in coding regions.
RNA quadruplexes in the 3′ untranslated region play yet another role, influencing alternative splicing and polyadenylation. G4 structures in the 3′ UTR of the tumor suppressor TP53 and the telomerase gene hTERT, for instance, regulate how their transcripts are spliced into different forms.
Quadruplexes as Cancer Drug Targets
Because quadruplexes in oncogene promoters can silence those genes, researchers have been developing small molecules that lock quadruplexes into their folded state, preventing the DNA from returning to its normal double-helix form. The logic is straightforward: if you can trap a quadruplex in the promoter of a cancer-driving gene, you can shut that gene down.
The oncogene c-MYC has been one of the primary targets. c-MYC is overactive in many cancers, and its promoter contains a guanine-rich sequence called Pu-27 that forms intramolecular quadruplexes. When these quadruplexes are stabilized, they physically prevent transcription factors from accessing the promoter, reducing c-MYC expression. The protein nucleolin naturally promotes this process in cells, binding to the G4 structure and stabilizing it to arrest c-MYC transcription.
Many synthetic small molecules that stabilize quadruplexes in lab assays also show antiproliferative activity in cancer cell cultures, triggering DNA damage and programmed cell death. Some have reduced tumor volume in animal models. However, only three compounds have advanced to clinical trials so far. Quarfloxin (CX-3543), which targets quadruplexes in ribosomal DNA, reached Phase II trials for carcinoid and neuroendocrine tumors. CX-5461, targeting ribosomal DNA quadruplexes in BRCA1/2-deficient tumors, entered Phase I trials. APTO-253, which targets the c-MYC promoter quadruplex in acute myeloid leukemia, also reached Phase I trials, where it was shown to inhibit c-MYC expression and induce cell cycle arrest.
The i-Motif: A Complementary Structure
On the opposite strand from a G-quadruplex, the cytosine-rich complementary sequence can form its own four-stranded structure called an i-motif (also known as a C-quadruplex). Instead of guanine quartets, the i-motif is built from pairs of cytosine bases where one cytosine in each pair picks up an extra proton. These hemiprotonated cytosine pairs then intercalate, zipping together in an unusual interlocking pattern.
The key difference from G-quadruplexes is pH sensitivity. i-Motifs are most stable between pH 4.0 and 5.0, and they tend to unfold at the physiological pH of 7.0 to 7.4 found inside most cells. At pH 8.0, the structure collapses entirely into a random coil. This pH dependence initially made researchers skeptical that i-motifs could exist in living cells, though molecular crowding inside the nucleus may partially compensate. Where G-quadruplexes and i-motifs do co-exist on complementary strands, they may act as coordinated regulatory switches, with changes in local pH toggling one structure on while the other unfolds.