DNA is most famously known for its double helix structure, a twisted ladder of two strands that stores the genetic blueprint for life. However, certain regions of DNA and RNA can fold into an entirely different, non-canonical shape called a quadruplex structure. These formations are built from sequences unusually rich in the guanine base. The resulting four-stranded “G-quadruplex” (G4) is a compact, globular arrangement that departs significantly from the linear, two-stranded architecture of the classic double helix. The existence and function of these G-quadruplexes have emerged as a significant area of modern genetic research.
The Unique Structure of Quadruplex DNA
The physical architecture of a G-quadruplex is centered on a unique molecular arrangement known as the G-quartet. A G-quartet is a planar structure formed when four guanine bases associate together using Hoogsteen hydrogen bonding. This bonding pattern allows the four bases to align in a nearly square plane, contrasting with the Watson-Crick pairing found in the double helix. The stability of the entire quadruplex structure is achieved by stacking at least two of these G-quartets directly on top of one another.
These stacked G-quartets create a central channel, and the presence of specific ions is necessary to stabilize this architecture. Positively charged metal ions, particularly potassium ($K^+$), are drawn into this central pore where they coordinate with the oxygen atoms of the guanine bases. This coordination provides the electrostatic balance required to hold the negatively charged phosphate backbone and the stacked quartets together. The overall topology of a quadruplex is diverse, depending on whether the four guanine-rich segments come from a single DNA strand (unimolecular) or multiple strands (bi- or tetramolecular).
The way the four guanine-rich segments are connected by intervening nucleotides, called loops, dictates the final shape and orientation of the quadruplex. These loops can be lateral (connecting adjacent G-runs), diagonal (connecting opposite G-runs), or propeller-like, leading to various three-dimensional forms. Different loop arrangements result in distinct topologies, such as parallel, anti-parallel, or hybrid folding patterns. This structural diversity alters how the quadruplex interacts with cellular proteins.
Genomic Locations and Biological Presence
G-quadruplex-forming sequences are concentrated in areas with specific functional relevance. The most well-known location for these structures is in the telomeres, the protective caps found at the ends of linear chromosomes. Human telomeres are composed of thousands of repetitive TTAGGG sequences. The single-stranded, guanine-rich overhang at the end is highly prone to folding into a G-quadruplex, which plays a role in regulating the activity of the telomerase enzyme.
G-quadruplex sequences are frequently found in the promoter regions of numerous genes. The promoter is the stretch of DNA immediately upstream of a gene where the cellular machinery initiates transcription. The presence of G4 motifs in these regulatory regions suggests a mechanism for switching gene activity on or off. Bioinformatic analyses estimate that hundreds of thousands of sequences with the potential to form G-quadruplexes exist across the human genome.
Guanine-rich sequences also exist in RNA molecules, particularly within the untranslated regions (UTRs) of messenger RNA (mRNA). These structures, known as RNA G-quadruplexes, regulate processes after the genetic code has been transcribed from DNA. The existence of both DNA and RNA G-quadruplexes confirms that this four-stranded structure is a widespread feature of nucleic acid biology, affecting function at multiple levels of genetic expression.
Role in Gene Regulation and Cellular Processes
The formation of a G-quadruplex in a regulatory region acts as a physical barrier or a molecular switch. During DNA replication, the quadruplex structure is a potent obstacle that can stall the progression of DNA polymerases, the enzymes responsible for synthesizing new DNA strands. The replication fork can collapse or become unstable when a polymerase encounters a tightly folded G4 structure. Cells have evolved specialized DNA unwinding enzymes, known as G4-resolving helicases—such as FANCJ, BLM, and WRN—that are dedicated to rapidly resolving these structures to ensure smooth replication.
The effect of G-quadruplex formation can be either inhibitory or stimulatory, depending on its precise location and orientation within the gene. If a G4 forms on the template strand of a gene’s promoter, it can physically stall or block the movement of RNA polymerase. This blockage acts as a transcriptional repressor, shutting down the expression of that gene. This mechanism is thought to regulate many oncogenes, where stabilizing the G4 structure can suppress the overproduction of cancer-promoting proteins.
Conversely, a G-quadruplex forming on the non-template strand can sometimes enhance gene expression. Such a G4 can stabilize the formation of a three-stranded structure called an R-loop, which involves the newly synthesized RNA annealing back to the template DNA strand. This alternative structure can promote the recruitment of transcription factors or facilitate the re-initiation of RNA polymerase. The regulatory role extends to the RNA level, where G-quadruplexes in the 5’ untranslated region of an mRNA molecule act as translational switches, regulating protein synthesis.
Quadruplexes as Targets for Therapeutic Intervention
The involvement of G-quadruplexes in cellular processes linked to uncontrolled cell growth has positioned them as targets for therapeutic strategies. Since G4 sequences are highly enriched in the regulatory regions of many oncogenes, such as c-MYC and Bcl-2, manipulating these structures offers a pathway to selectively inhibit cancer cell proliferation. The strategy is to exploit the structural difference between the four-stranded quadruplex and the two-stranded double helix to achieve high drug specificity.
Researchers are developing small molecules, referred to as G-quadruplex ligands, designed to bind tightly and specifically to the G4 structure. Ligands can be engineered to either stabilize the G-quadruplex in its folded state or to destabilize it. In cancer therapy, the goal is often to stabilize the G4 in the promoter of an oncogene, such as c-MYC. This action locks the gene in an “off” position, repressing the production of the cancer-driving protein.
Therapeutic focus is also placed on the telomeric G-quadruplex, which can be stabilized by ligands like telomestatin. Stabilizing the G4 at the end of the chromosome inhibits the action of telomerase, the enzyme that maintains telomere length. By blocking telomerase and preventing telomere extension, these ligands cause telomere shortening, eventually leading to cell death in malignant cells. This approach aims to exploit the dependency of cancer cells on telomere maintenance.