Four-Stranded DNA Shapes Plant and Insect Biology and Could Transform Pest Control

Deep inside the genomes of plants and insects, stretches of DNA are quietly folding into shapes that defy the textbook double helix. These structures, known as G-quadruplexes, are four-stranded architectures built from guanine-rich sequences, and a new review published in Crop Health argues that they are far more than molecular curiosities. According to researchers led by Xiaojuan Zhang and Kangkang Niu of South China Normal University, G-quadruplexes act as regulatory switches that influence how plants respond to cold and drought, how silkworms grow and metamorphose, and how crop pests such as the fall armyworm detoxify insecticides. The work suggests that these folded DNA structures could become entirely new molecular targets for protecting crops, offering a strategy that is more species-specific and environmentally friendly than conventional pesticides.

To understand why this matters, it helps to start with the chemistry. Since Watson and Crick described the double helix in 1953, DNA has been imagined as a linear molecule in which adenine pairs with thymine and cytosine pairs with guanine. But guanine-rich sequences can do something unusual: four guanine bases can associate in a square, coplanar arrangement called a G-quartet, held together by eight Hoogsteen hydrogen bonds, a bonding pattern first described by biochemist Arthur Hoogsteen in 1963. When two or more of these G-quartets stack on top of one another, they form a G-quadruplex. Monovalent cations such as potassium, sodium and ammonium nestle in the central channel of the stack and stabilize the structure. Depending on the number of stacked quartets, the length of the connecting loops and the polarity of the strands, G-quadruplexes adopt parallel, antiparallel or hybrid conformations, giving them a remarkable structural versatility.

The evidence that these structures exist inside living cells is now strong. Using the G4-specific antibody BG4, researchers visualized G-quadruplex foci in human cell lines, and the signals vanished after DNase treatment and multiplied when cells were exposed to pyridostatin, a small molecule that stabilizes G4s. G4-binding proteins such as the helicase DHX36, the fly protein LARK and the yeast helicase Pif1 provide further indirect proof of their presence in vivo. Genomewide, the picture is striking. Roughly 700,000 potential G4-forming sequences have been predicted in the human genome, and across 37 surveyed species the number and density of these motifs increased with evolutionary complexity. Crucially, the sequences are not scattered randomly: they cluster at replication origins, promoters, telomeres and untranslated regions, exactly the places where a regulatory element would be expected to sit.

That nonrandom distribution underpins the biological roles of G-quadruplexes. In mammalian genomes, 80 to 90 percent of replication origins are GC-rich and capable of forming G4s, and these structures can act both as barriers that helicases must unwind to prevent fork collapse and as recognition sites that recruit initiation factors such as the origin recognition complex and Rif1. In promoters, G4s can either boost or suppress transcription by altering how transcription factors bind. The classic example is the human oncogene c-Myc, whose expression dropped when cells were treated with the G4 ligand TMPyP4 and fell dramatically when the promoter G4 was disrupted by CRISPR-Cas9 editing. Recent work has even shown that promoter G4s serve as common binding hubs for many transcription factors. At telomeres, the single-stranded G-rich overhang folds into G4s that modulate telomerase activity and telomere length, while RNA G-quadruplexes in untranslated regions regulate translation, splicing and mRNA stability.

Plants have their own distinctive G4 landscape. An analysis of 15 sequenced plant genomes, from Arabidopsis thaliana and rice to mosses and lycophytes, revealed that more than 90 percent of plant G4s contain only two G-quartets, a profile quite different from the human genome, where three-quartet structures dominate. The density of predicted G4-forming sequences in monocotyledons was five to ten times higher than in dicotyledons, and G4 motifs were even found in chloroplast and mitochondrial DNA, with mitochondrial DNA showing roughly three times the G4 frequency of nuclear and chloroplast genomes. In Arabidopsis, rice and maize, these motifs concentrate around transcription start sites, 5-prime untranslated regions and other regulatory zones, hinting at cis-regulatory functions in gene expression.

Functional studies in plants have delivered some of the most vivid demonstrations of G4 biology. The first RNA G4 identified in a living plant cell sat in the 5-prime untranslated region of the DNA damage response gene ATR, where it acted as a translational repressor. Another RNA G4 in the untranslated region of SMXL4/5 suppresses translation and restricts phloem differentiation, directly linking a folded RNA structure to vascular development. Perhaps most strikingly, the RNA G4 in the 3-prime untranslated region of the drought-induced dehydrin gene HIRD11 inhibits its translation; when the G4 motif was mutated, Arabidopsis roots grew significantly longer. Even more intriguingly, when researchers compared the nucleotide composition of transcriptomes across 1,000 plant species, they found that plants from cold climates carried G-rich transcriptomes prone to forming RNA G4s, and in Arabidopsis these cold-responsive G4s stabilized mRNAs and helped regulate growth at low temperatures. G4s in maize have also been tied to hypoxia, oxidative stress and energy status, suggesting that these structures are deeply woven into how crops cope with a changing environment.

Insects, meanwhile, offer both the best model systems and the most direct agricultural stakes. In Drosophila, the first predicted G4 was found in the HeT-A retrotransposon at chromosome ends, and subsequent work showed G4 motifs overlapping replication origins, common in all centromeres, and enriched in long intergenic noncoding RNAs, introns and promoters, with an improved G4-seq method detecting 22,511 such sequences genomewide. G4 signals localize to heterochromatin in salivary gland polytene chromosomes and are weaker in germline stem cells, implying a role in cell differentiation. The Drosophila homolog of the helicase DHX36 has been crystallized bound to a G4, revealing at atomic resolution how the enzyme unfolds the structure, and mutations in pif1 cause chromosome segregation defects consistent with unresolved G4s stalling replication forks.

Outside the fruit fly, the silkworm Bombyx mori has become the showcase for insect G4 biology. Its telomeric repeat d[TAGG(TTAGG)3], conserved across many insects, folds into a chair-type intramolecular G4 that is more stable in sodium than in potassium solution. The silkworm genome harbors nearly 24,000 predicted G4s, and the first insect G4 shown to regulate transcription was found in the promoter of BmPOUM2, a gene governing development. The G4-binding protein LARK binds this motif to boost transcription, and CRISPR-Cas9 knockout of LARK causes embryonic lethality with broad changes in cuticle and pigment gene expression. A G4 in the promoter of the acyl-CoA binding protein gene BmACBP regulates lipid metabolism: when fifth-instar larvae were treated with the G4-stabilizing ligand pyridostatin, BmACBP expression and triacylglycerol levels dropped, fat body mass shrank, and larval growth and metamorphosis slowed. A G4 in the promoter of the silk gland factor SGF1 acts as a positive regulator of silk protein production, and its knockout reduced silk output in mutant larvae.

The pest control implications come into sharpest focus in the fall armyworm, Spodoptera frugiperda, a globally destructive crop pest. Genomewide analysis identified 387,875 predicted G4-forming sequences, nearly 67 percent of them upstream of start codons, and the genes carrying promoter G4s were enriched for metabolic pathways, especially xenobiotic metabolism by cytochrome P450 enzymes. Treatment with the G4 ligand N-methyl mesoporphyrin IX suppressed P450 expression and enzyme activity and increased larval mortality. In the corn earworm Helicoverpa zea, a transposon-inserted G4 in the promoter of the detoxification gene CYP321A1 acts as a silencer, and destroying it or stabilizing it with NMM reduced the promoter’s response to plant toxins. Because insects rely on P450s, glutathione-S-transferases and carboxylesterases to survive both plant allelochemicals and pesticides, these findings open a concrete path: G4 ligands could be deployed either as standalone insecticides that disrupt the G4s of development genes, producing abnormal larvae, or as pesticide sensitizers that knock down detoxification genes and restore the potency of existing chemicals.

The review’s authors sketch three strategies for turning this biology into practice. First, cell-permeable G4 ligands could be screened or designed to target the specific structures of pest development genes, exploiting the fact that G4 structural polymorphism offers ligand specificity. Second, G4 ligands could accompany conventional insecticides, either destabilizing the G4s that drive detoxification gene expression or stabilizing them so that transcription factors cannot bind, thereby synergizing with the pesticide. Third, and perhaps most elegant, plants themselves could be engineered to produce higher levels of natural G4-binding compounds, so that feeding pests ingest ligands that disrupt their own gene regulation. Plant-derived molecules such as the flavonoids fisetin and kaempferol and the alkaloids chelerythrine and berberine already bind G4s and are being explored as low-toxicity anticancer leads, and the same chemistry could be redirected toward agriculture. No G4-based pesticide has yet reached the field, and large-scale screening of plant extracts has only just begun, but the underlying logic is compelling: unlike organophosphates or hormone analogs that hit broad physiological processes, a molecule aimed at a single G4 structure in a pest’s DNA or RNA could, in principle, be exquisitely species-specific, sparing beneficial insects and leaving the environment largely untouched. As more G4s are mapped and their regulatory functions confirmed, the humble four-stranded guanine quartet may prove to be one of the most unexpected weapons in the fight to protect the world’s crops.

Subject of Research: G-quadruplex nucleic acid structures and their regulatory roles in plants and insects with applications in pest control

Article Title: G-quadruplex structure in plants and insects and potential applications in pest control

Article References: G-quadruplex structure in plants and insects and potential applications in pest control. (n.d.). https://doi.org/10.1007/s44297-025-00047-2

Image Credits: AI Generated

DOI: 10.1007/s44297-025-00047-2

Keywords: G-quadruplex, DNA structure, RNA G-quadruplex, plants, insects, pest control, silkworm, fall armyworm, cytochrome P450, G4 ligands, gene regulation, crop protection