The review organizes the sprawling literature into five distinct propositions: redox control of telomerase activity, the reverse control of redox metabolism by the telomerase reverse transcriptase TERT, oxidative damage to telomeric DNA itself, redox-associated alternative lengthening of telomeres (ALT), and the therapeutic translation of all of the above. Each proposition is then graded separately for causal strength and molecular directness — two qualities the authors emphasize can vary completely independently. A finding can be causally robust yet mechanistically indirect, resting on a chain of downstream events; or it can be chemically precise yet demonstrated only in a test tube. Recognizing this independence, they contend, is the first step toward cleaning up a literature that has often graded studies on a single, misleading scale.

One of the sharpest methodological criticisms in the review targets a design that appears throughout the redox–telomerase literature: pretreating cells with millimolar concentrations of a thiol antioxidant such as N-acetylcysteine or glutathione before exposing them to a thiol-reactive electrophile. The problem, the authors explain, is one of chemical ambiguity. At those concentrations, the added thiol can simply quench the electrophile directly in the culture medium, a brute-force chemical reaction that has nothing to do with restoring the cell’s own antioxidant capacity. A rescue observed under those conditions cannot distinguish between genuine redox regulation of telomerase and trivial chemical neutralization of the insult. Because so many headline claims about antioxidants modulating telomerase rest on exactly this design, the finding casts a long shadow over a substantial fraction of the field.

What survives the audit? The authors identify the peroxide-driven, Src kinase–dependent phosphorylation of TERT at tyrosine 707, which triggers export of the enzyme from the nucleus via the Ran transport pathway, as among the best-supported redox findings. Hydrogen peroxide activates Src family kinases, phosphorylation at that specific residue promotes nuclear export, and the pathway satisfies necessity tests — blocking the kinase or the export machinery prevents the effect. Similarly well-supported is the glutathione depletion-and-repletion paradigm, in which lowering cellular glutathione alters telomerase activity and restoring it rescues the phenotype. Yet even these strongest findings, the review notes, remain pathway-mediated rather than chemically direct, and several were established in transfected systems or non-cancer cells such as endothelial cells and fibroblasts, leaving open questions about how faithfully they recapitulate events in an intact tumor.

Perhaps the most striking conclusion of the entire appraisal is a negative one. Despite decades of work, the authors identified no study that causally links a residue-resolved oxidative modification of endogenous TERT — a specific cysteine or tyrosine chemically modified by a defined reactive species inside a living cancer cell — to an altered outcome in telomerase assembly, subcellular localization, or catalytic activity. The distinction matters because redox biology has matured into a chemistry-first discipline: modern chemoproteomic tools can now identify which cysteines in a proteome are reactive, which ones get oxidized under defined conditions, and what functional consequences follow. TERT, with its multiple regulatory phosphorylation sites and its dependence on assembly with the telomerase RNA component, dyskerin and other partners, is precisely the kind of protein where such residue-level resolution should be achievable — and has not yet been delivered.

In contrast, the most molecularly direct chemistry in the entire field sits not on the protein but on the telomeric DNA itself. Guanine-rich telomeric repeats are exquisitely vulnerable to oxidation, and the review assembles a compelling body of biochemical work showing that the identity and position of an oxidative lesion govern nearly everything that matters: binding by the shelterin proteins TRF1 and TRF2, folding of the telomeric G-quadruplex structures that regulate telomerase access, recruitment of repair glycosylases such as OGG1 and the NEIL family, and the ability of telomerase to extend the chromosome end. An 8-oxoguanine lesion at one position of a G-quadruplex can destabilize the structure and promote telomerase extension, while the same lesion elsewhere blocks it — a position-dependent logic revealed through single-molecule and biochemical assays.

Even more provocative are recent findings showing that telomeric oxidation can drive dysfunction without any detectable shortening of the telomere. In work highlighted by the review, a single acute burst of targeted 8-oxoguanine damage at telomeres was sufficient to trigger rapid premature senescence, with telomere length remaining essentially unchanged. This decouples telomere dysfunction from the classical model in which telomeres erode gradually with each cell division, suggesting that a discrete oxidative event can flip the shelterin-bound chromosome end into a damage-signaling state essentially overnight. For cancer biology, the implication is double-edged: oxidative stress can push premalignant cells toward senescence, but it can also, under other conditions, push telomeres toward the recombination-based ALT pathway that a subset of tumors uses to elongate their chromosome ends.

On the therapeutic front, the review delivers a sobering translational parallel to its mechanistic gap. Imetelstat, the lipid-conjugated oligonucleotide that directly targets the telomerase RNA template, has now established randomized phase 3 efficacy in lower-risk myelodysplastic syndromes and has earned regulatory approval in the United States and a positive assessment in Europe. Yet the review points out that its redox engagement — whether the drug’s effects intersect with the oxidative stress programs that co-occur in treated tumors — has never been measured, nor have telomere pharmacodynamics been tracked in a way that connects clinical response to telomere biology at the molecular level. The most clinically successful telomerase-targeting agent, in other words, is as much of a black box with respect to redox as endogenous TERT is with respect to residue-level oxidation.

The review also surveys a crowded pipeline of redox-active agents with claimed telomerase effects: disulfiram and its copper diethyldithiocarbamate complex, which has been reported to induce both mitochondrial and telomerase dysfunction in lung cancer models; glutathione synthesis inhibitors such as butionine sulfoximine tested in combination regimens; the nucleoside analog 6-thio-deoxyguanosine, now in a phase 3 trial in non-small cell lung cancer, which recent structural work suggests stalls telomerase in a non-productive complex; and G-quadruplex ligands whose mechanisms have required substantial reevaluation. The authors’ framework provides a way to grade each of these claims, and the grades are frequently humbling: many rest on pharmacological exposures at concentrations far from physiological, in cell lines of uncertain provenance, with telomerase activity measured by the TRAP assay, which is itself known to be vulnerable to artifacts from redox-active compounds in the lysates.

What would close the gaps? The authors set out a concrete measurement agenda. For the protein side, it means deploying residue-resolved chemoproteomics and structural methods such as cryo-electron microscopy to catch endogenous TERT in the act of being oxidatively modified, and then testing causality with precisely engineered non-oxidizable mutants. For the telomere side, it means mapping lesion identity and position in cells, not just in synthetic oligonucleotides, and tracking how those lesions propagate through shelterin binding, repair, and telomerase extension. For the clinic, it means measuring telomere pharmacodynamics and redox biomarkers in imetelstat-treated patients. Until those measurements exist, the review concludes, the field should resist the temptation to narrate a tidy story of oxidants and telomeres — because the honest story, carefully audited, is one of strong chemistry at the chromosome end, plausible but unproven signaling at the enzyme, and a translational success whose deepest mechanisms remain to be written.

Subject of Research: Redox regulation of telomerase and telomeres in cancer

Article Title: Redox regulation of telomerase and telomeres in cancer: a critical appraisal of exposure, causality and molecular directness

Article References: Tatar, M., Khorrami, M., Kazemi, F., Masoumi, M., Babaei, S., & Khorrami, M. (2026). Redox regulation of telomerase and telomeres in cancer: a critical appraisal of exposure, causality and molecular directness. Molecular Biology Reports, 53(1), Article 1638. https://doi.org/10.1007/s11033-026-12826-3

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12826-3

Keywords: telomerase, TERT, telomeres, redox biology, oxidative stress, cancer, 8-oxoguanine, glutathione, ALT, imetelstat, DNA damage, G-quadruplex

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

The structures in question are G-quadruplexes, or G4s, four-stranded nucleic acid architectures that form in guanine-rich sequences. In a G4, four guanine bases pair through Hoogsteen hydrogen bonding to create a planar G-quartet, and the stacking of at least two such quartets, stabilized by pi-pi interactions and by monovalent cations such as potassium, produces a compact and remarkably stable fold. Depending on the orientation of the four guanine tracts that form the core, G4s can adopt parallel, antiparallel or hybrid topologies. In human cells, G4s are known to regulate telomere maintenance, DNA replication, transcription, mRNA processing and chromatin remodeling, and conserved potential G-quadruplex sequences have been documented in the genomes of Epstein-Barr virus, hepatitis C virus, HIV and SARS-CoV-2, where they influence replication and infection.

Researchers led by a team at Anhui Medical University systematically searched full-length hepatitis B virus genomes from genotypes A through H for sequences capable of forming G4s, using the QGRS Mapper algorithm to score the likelihood of quadruplex formation. High-scoring motifs clustered at positions 1204, 1732, 1886 and 3021 in a representative genotype B genome. Multiple sequence alignment and WebLogo analysis revealed that three of these motifs, at positions 1204, 1732 and 1886, are highly conserved across genotypes, with the exception of genotype G, while the 3021 motif is not evolutionarily conserved. Crucially, the 1204 motif lies within the region overlapping the X promoter and Enhancer I, the 1732 motif sits in the overlap between the core promoter and Enhancer II, and the 1886 motif falls within the epsilon RNA element of the precore and core gene.

To confirm that these predicted sequences actually fold, the team synthesized the corresponding oligonucleotides and subjected them to a battery of biophysical tests. Native polyacrylamide gel electrophoresis showed that the wild-type sequences migrated faster than guanine-mutated counterparts, consistent with the formation of compact intramolecular quadruplexes. Proton nuclear magnetic resonance spectroscopy detected the characteristic imino proton signals of Hoogsteen base pairing in the 10 to 12.5 parts per million range for all three sequences, signals that shifted toward Watson-Crick pairing signatures when guanines were mutated. Circular dichroism spectroscopy then resolved the topologies: the 1732 sequence adopts a parallel G4, while 1204 and 1886 fold into antiparallel conformations. Potassium ions proved preferred for stabilization, and the G4-stabilizing ligand BRACO-19 raised the melting temperatures of the structures, with 1732’s melting temperature exceeding the detectable range after ligand addition, underscoring the exceptional stability of these folds under physiological conditions.

Functional relevance was established with reporter systems. In a luciferase construct in which expression is driven by the hepatitis B virus core promoter together with Enhancer I and Enhancer II, disrupting the G4 at 1204, 1732 or 1886 significantly reduced reporter activity, with the 1732 mutation producing the strongest effect. In a more complete HBV 1.3-mer plasmid system, destroying the 1732 G4 markedly lowered the levels of hepatitis B surface antigen, e antigen and core antigen. Intriguingly, total viral RNA and the 3.5 kilobase pregenomic RNA were not significantly affected, pointing to a post-transcriptional role for the 1732 structure, possibly through modulation of translation efficiency, RNA conformation or transcript processing, rather than a direct effect on transcription itself. The 1204 mutation, constrained by the overlapping polymerase reading frame to a single G-to-A substitution that only partially weakened the fold, produced no significant changes in the 1.3-mer model, consistent with residual G4 signatures still visible in its circular dichroism spectra.

The next question was which host proteins these viral structures recruit. Using biotinylated 1204 and 1732 oligonucleotides as bait in pull-down experiments from HepG2.2.15 cell lysates, followed by data-independent acquisition mass spectrometry, the researchers identified 619 proteins enriched more than 1.5-fold over a non-G4 control, of which 34 participate in DNA repair, transcriptional regulation, RNA processing or G4 binding. The cellular nucleic acid-binding protein CNBP, a CCHC-type zinc finger protein, emerged as the most highly enriched G4-binding partner, while the hepatocyte nuclear factor 4 alpha, HNF4A, a master transcriptional activator of the hepatitis B virus core promoter, had never previously been reported to bind G4 structures. RNA interference against five candidate proteins, including CNBP and HNF4A, significantly reduced viral antigen and RNA levels, confirming their positive contribution to viral gene expression.

Surface plasmon resonance quantified the physical interactions. Both the 1204 and 1732 quadruplexes bound HNF4A directly, with equilibrium dissociation constants of 2.552 times ten to the minus five molar and 1.991 times ten to the minus six molar respectively, meaning the 1732 structure binds roughly an order of magnitude more tightly. Neither the mutated sequences nor a non-G4 control showed any detectable binding, and replacing potassium with lithium, which disrupts G4 folding, abolished the interaction entirely, demonstrating that the folded structure itself, not the primary sequence, is what HNF4A recognizes. BRACO-19, by further stabilizing the quadruplexes, actively enhanced HNF4A binding in pull-down assays. AlphaFold3-based structural predictions supported these findings, revealing hydrogen bonds between both G4 elements and specific HNF4A residues, with partially overlapping binding interfaces.

Functional assays then separated the two host factors mechanistically. Overexpression of HNF4A markedly increased activity of the core promoter-enhancer reporter, but when the 1732 G4 was destroyed, HNF4A could no longer fully restore reporter output, indicating that an intact 1732 quadruplex is required for optimal HNF4A-dependent activation. The authors propose a dual regulatory model in which HNF4A activates the core promoter-enhancer region primarily through its classical DNA binding sites, while a secondary, G4-dependent mechanism involving direct engagement of the 1732 structure potentiates maximal promoter output, likely facilitated by the genomic proximity of the two elements. CNBP, by contrast, boosted viral antigen and RNA levels when overexpressed and reduced them when knocked down, yet it did not enhance, and even slightly decreased, core promoter-enhancer reporter activity, indicating that CNBP promotes hepatitis B virus expression through a mechanism independent of this promoter region, perhaps by stabilizing or unwinding RNA G4s within viral transcripts, as it has been shown to do with SARS-CoV-2 genomic G4s.

The study’s implications extend beyond basic virology. Because properly folded G4s in the core promoter-enhancer region support, rather than repress, viral gene expression, these structures and the proteins that bind them represent candidate targets for new anti-hepatitis B strategies, complementing G4-directed approaches already explored against SARS-CoV-2 and other viruses. The authors caution that their experiments relied largely on plasmid-based reporter and 1.3-mer systems, and that future work using authentic infection models and fully chromatinized cccDNA templates will be needed to confirm whether HNF4A associates directly with viral cccDNA in living cells. Quantifying effects on nascent RNA synthesis, RNA polymerase II recruitment and liquid-liquid phase separation, a process recently implicated in G4-driven cccDNA transcription, could further clarify how these unusual DNA folds choreograph the life of a virus that has evaded elimination for millennia.

Subject of Research: Conserved G-quadruplex structures in the hepatitis B virus core promoter and enhancer regions and their host protein partners regulate viral gene expression

Article Title: Regulatory roles of G-quadruplexes and G-quadruplex-binding proteins across the enhancer and promoter of the HBV genome

Article References: He, L., Huang, B., Ma, H., Wang, L., Wu, Q., Zhang, J., Yu, L., & Lv, S. (2026). Regulatory roles of G-quadruplexes and G-quadruplex-binding proteins across the enhancer and promoter of the HBV genome. Virology Journal, 23(1), Article 218. https://doi.org/10.1186/s12985-026-03132-2

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03132-2

Keywords: hepatitis B virus, G-quadruplex, core promoter, enhancer, G4-binding proteins, HNF4A, CNBP, BRACO-19, viral gene expression, cccDNA, surface plasmon resonance, antiviral therapy