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PubMed2025

G4STAB: a multi-input deep learning model to predict G-quadruplex thermodynamic stability based on sequence and salt concentration.

MOTIVATION: G-quadruplexes (G4s) are non-canonical nucleic acid structures formed in guanine-rich regions that modulate gene regulation and genomic stability. The thermodynamic stability of G4s directly influences their biological functions and potential as therapeutic targets. However, current quantitative frameworks for predicting G4 stability rely on predetermined structural features, limiting their effectiveness for diverse G4 topologies, and fail to account for environmental factors such as ion concentration and pH that significantly modulate G4 stability in cellular contexts. RESULTS: We present G4STAB, a multi-input deep learning neural network that accurately predicts DNA G4 melting temperatures based on sequence features, salt concentration, and pH. Trained on 2382 diverse DNA G4 sequences, our model achieves high accuracy (R 2=0.8) without relying on predetermined G4 structural features. G4STAB successfully captures established G4 stability determinants and proposes previously unobserved sequence-stability relationships. Analysis of 391 502 experimentally validated G4s reveals that cancer-like ionic environments alter G4 stability profiles, with a 13.5-fold increase in the number of structures exhibiting physiological melting temperatures (36-42°C). These findings suggest systematic genomic patterns in G4 stability responses across chromosomes and gene types. AVAILABILITY AND IMPLEMENTATION: G4STAB is available at https://github.com/donn-liew/G4STAB; G4STAB web database interface is available at https://donn-liew.github.io/g4stab-web-database/.

G-Quadruplexes↗
PubMed2025

RNA G-quadruplexes emerge from a compacted coil-like ensemble via multiple pathways.

RNA G-quadruplexes (rG4s) are emerging as vital structural elements involved in processes like gene regulation, translation, and genome stability. Found in untranslated regions of messenger RNAs (mRNAs), they influence translation efficiency and mRNA localization. Additionally, rG4s of long noncoding RNAs and telomeric RNA play roles in RNA processing and cellular aging. Despite their significance, the atomic-level folding mechanisms of rG4s remain poorly understood due to their complexity. We studied the folding of the r(GGGA)3GGG and r(GGGUUA)3GGG (TERRA) sequences into parallel-stranded rG4 using all-atom enhanced-sampling molecular dynamics simulations, applying well-tempered metadynamics coupled with solute tempering. The obtained folding pathways suggest that RNA initially adopts a compacted coil-like ensemble characterized by dynamic guanine stacking and pairing. The three-quartet rG4 gradually forms from this compacted coil ensemble via diverse routes involving strand rearrangements and guanine incorporations. While the folding mechanism is multipathway, various two-quartet rG4 structures appear to be a common transitory ensemble along most routes. Thus, the process seems more complex than previously predicted, as G-hairpins or G-triplexes do not act as distinct intermediates, even though some are occasionally sampled. We also discuss the challenges of applying enhanced sampling methodologies to such a multidimensional free-energy surface and address the force-field limitations.

G-Quadruplexes↗
PubMed2025

Post-transcriptional control of KRAS: functional roles of 5'UTR RNA G-quadruplexes, long noncoding RNA, and hnRNPA1.

Previous studies have shown that human KRAS expression is regulated at the transcriptional level by G-quadruplex DNA structures within its promoter. Here we show an additional level of regulation involving a post-transcriptional mechanism centred on the 5'-untranslated region (5'UTR) of the messenger RNA (mRNA) characterized by G4 structures (rG4s). Long noncoding RNAs (lncRNAs) and the protein hnRNPA1 are also involved in this mechanism. RIP-seq confirmed the presence of rG4s in the 5'UTR. Deletion of the rG4 region using CRISPR/Cas9 resulted in a significant increase in KRAS mRNA levels, indicating the role of the 5'UTR in controlling mRNA levels. RIP shows that hnRNPA1 is recruited to the 5'UTR, where it unfolds the rG4 structures and potentially affects mRNA stability. In addition, lncRNAs transcribed from the LINC01750 locus can hybridize to the rG4 region of 5'UTR and form RNA duplexes leading to RNase III-assisted degradation of the targeted mRNA. Activation of the LINC01750 locus with dCas9-VP64 resulted in downregulation of KRAS mRNA, whereas its suppression with dCas9-KRAB led to upregulation of both KRAS mRNA and protein. Since lncRNA-mediated regulation of mRNA appears to be a crucial aspect of cellular homeostasis and its disruption contributes to various diseases, understanding these mechanisms may reveal promising new therapeutic targets.

Humans↗
PubMed2003

Specific interactions of distamycin with G-quadruplex DNA.

Distamycin binds the minor groove of duplex DNA at AT-rich regions and has been a valuable probe of protein interactions with double-stranded DNA. We find that distamycin can also inhibit protein interactions with G-quadruplex (G4) DNA, a stable four-stranded structure in which the repeating unit is a G-quartet. Using NMR, we show that distamycin binds specifically to G4 DNA, stacking on the terminal G-quartets and contacting the flanking bases. These results demonstrate the utility of distamycin as a probe of G4 DNA-protein interactions and show that there are (at least) two distinct modes of protein-G4 DNA recognition which can be distinguished by sensitivity to distamycin.

Base Sequence↗
PubMed2006

A real-time assay for monitoring nucleic acid cleavage by quadruplex formation.

Direct and straightforward methods to follow nucleic acid cleavage are needed. A spectrophotometric quadruplex formation assay (QFA) was developed, which allows real-time monitoring of site-specific cleavage of nucleic acids. QFA was applied to study both protein and nucleic acid restriction enzymes, and was demonstrated to accurately determine Michaelis-Menten parameters for the cleavage reaction catalyzed by EcoRI. QFA can be used to study the mechanisms of protein-nucleic acid recognition. QFA is also a useful tool for dissecting individual nicking rates of a double-stranded cleavage.

Aptamers, Nucleotide↗
PubMed2006

Molecular modeling of anti-parallel G-quadruplex DNA/TMPyP complexes.

We carried out the molecular modeling of anti-parallel G-quadruplex/TMPyP complex, molecular dynamics simulation and estimation of binding free energy using MM-PBSA method to validate groove binding model in addition to external stacking one. We found that not total electrostatic but van der Waals energy contributes to the negative binding free energies in both models.

DNA↗
PubMed2025

G4SNVHunter: An R/Bioconductor Package for Evaluating SNV-Induced Disruption of G-Quadruplex Structures Leveraging the G4Hunter Algorithm.

G-quadruplexes (G4s) are nucleic acid secondary structures with important regulatory functions. Single-nucleotide variants (SNVs), one of the most common forms of genetic variation, can potentially impact the formation of G4 structures if they occur within G4 regions. However, there is currently a lack of software tools specifically designed to assess such effects. Here, we present an R/Bioconductor package named G4SNVHunter, which enables rapid detection of variants that may disrupt G4 structures. This tool, based on the core principles of the G4Hunter algorithm, can provide precise quantitative assessment of the propensity for G4 formation within genomic sequences. Specialized experimental methods can then be designed based on the results provided by G4SNVHunter to further verify the specific functions of the affected G4 structures, facilitating deeper insights into the biological impacts of genetic variants from the perspective of G4 structures. To showcase the functionality of the G4SNVHunter package, we analyzed the Neandertal and Denisovan archaic introgressed variants detected by the Sprime software, and identified approximately 5,800 variants located within G4 regions, among which around 230 may impair G4 structure formation propensity. The source code for the G4SNVHunter package has been publicly released under the MIT license at https://github.com/rongxinzh/G4SNVHunter and https://bioconductor.org/packages/devel/bioc/html/G4SNVHunter.html.

G-Quadruplexes↗
PubMed2024

Quadruplex-duplex junction in LTR-III: A molecular insight into the complexes with BMH-21, namitecan and doxorubicin.

Quadruplex-Duplex (Q-D) junctions are unique structural motifs garnering increasing interest as drug targets, due to their frequent occurrence in genomic sequences. The viral HIV LTR-III sequence was chosen as a Q-D junction model to study the affinity of the selected compounds BMH-21, namitecan (ST-1968), and doxorubicin (DOXO), all containing a planar polycyclic aromatic moiety, linked to either one short aminoalkyl or an aminoglycosyl group. A multidisciplinary approach that combines NMR spectroscopy, molecular modelling, circular dichroism (CD) and fluorescence spectroscopy was employed. The studied ligands induced moderate but clear stabilization to the Q-D junction by interacting with the interfacial tetrad. DOXO was found to be the best Q-D junction binder. Interestingly, the removal of the aminoglycosyl group significantly changed the pattern of the interactions, indicating that highly polar substituents have a stronger affinity with the exposed regions of the Q-D junction, particularly at the level of the interfacial tetrad.

Doxorubicin↗
PubMed2025

Viral hijacking of host DDX60 promotes Crimean-Congo haemorrhagic fever virus replication via G-quadruplex unwinding.

Crimean-Congo haemorrhagic fever virus (CCHFV) is the most prevalent tick-borne zoonotic bunyavirus, causing severe hemorrhagic fever and fatality in humans. Currently, the absence of approved vaccines or therapeutics for CCHFV infection necessitates the development of innovative therapeutic strategies. Here, we identify a guanine (G)-rich sequence located within the mRNA of the glycoprotein precursor in the medium (M) segment of the CCHFV genome, designated as M-PQS-1664(+). M-PQS-1664(+) can form stable G-quadruplex (G4) structure and functions as a negative regulatory element for viral replication. Host DDX60 is up-regulated in response to CCHFV infection, thereby it is hijacked to unwind M-PQS-1664(+) G4 for facilitating viral replication. The FDA-approved drug Cepharanthine (CEP), which competes with DDX60 to specifically stabilize M-PQS-1664(+) G4 without a global induction of host cellular G4s formation, exhibits remarkable antiviral activity in vitro and in vivo. More importantly, CEP possesses antiviral activity (50% inhibitory concentration ~ 0.2 μM) that having ~ 88 × the potency of ribavirin. Our findings underscore the CCHFV G4s as a promising target for drug development and highlight the significant potential of CEP in combating CCHFV.

Hemorrhagic Fever Virus, Crimean-Congo↗
PubMed2004

A G-quadruplex telomere targeting agent produces p16-associated senescence and chromosomal fusions in human prostate cancer cells.

The trisubstituted acridine derivative BRACO-19 has been designed to interact with and stabilize the quadruplex DNA structures that can be formed by folding of the single-stranded repeats at the 3' end of human telomeres. We suggest that the BRACO-19 complex inhibits the catalytic function of telomerase in human cancer cells and also destabilizes the telomerase-telomere capping complex so that cells enter senescence. Here, we present evidence showing that the inhibition of cell growth caused by BRACO-19 in DU145 prostate cancer cells occurs more rapidly than would be expected solely by the inhibition of the catalytic function of telomerase, and that senescence is accompanied by an initial up-regulation of the cyclin-dependent kinase inhibitor p21, with subsequent increases in p16(INK4a) expression. We also show that treatment with BRACO-19 causes extensive end-to-end chromosomal fusions, consistent with telomere uncapping.

3T3 Cells↗
PubMed2006

[Generation and characterization of the monoclonal antibody and scFv against yeast telomeric guanine-quadruplex DNA].

Most eukaryotic telomeres contain many tandem repeats of G-rich sequence. In budding yeast, Sacchromyces cerevisiae, the G-rich sequence can form parallel-stranded quadruplex comformation (G4-DNA) in vitro. Whether this structure exsits in vivo is unknown. To address this question, we generated the antibodies against the G4-DNA of the S. cerevisiae by immunizing the BALB/c mouse with in vitro synthesized G4-DNA oligonucleotides. The antibodies recognize G4-DNA, as well as G-rich DNA sequence in vitro. In order to improve the affinity to G4-DNA substrate, we cloned V(H) and V(L) genes of the antibodies and constructed the single-chain antibody fragment (scFv) with a peptide linker. The recombinated scFv was successfully expressed in E. coli and purified by the affinity chromotography. Based on the sequence of the scFv, we proposed the structure of the antibody by computer-remodeling. The engineered antibodies will be used to detect the existence of the existence of the G4-DNA structure in vivo.

Amino Acid Sequence↗
PubMed2025

Whole-Genome Conservation Analysis for the Specific and Accurate Detection of Influenza A and B Viruses and Respiratory Syncytial Virus by Quadruplex RT-qPCR.

Influenza virus (Flu) and respiratory syncytial virus (RSV) are the primary pathogens responsible for acute respiratory infections. Both viruses are prone to mutations due to the seasonal epidemic, leading to an increasing rate of false-negative results. In this study, comprehensive meta-analyses of the genomes focusing on most conserved fragments have been performed for the four seasonal influenza viruses (two subtypes of Flu A: H1N1 and H3N2; two subtypes of Flu B: Yamagata and Victoria) and the two types of RSV: RSVA and RSVB), respectively. The most conserved sequences of 200 bp were identified as targets of the designed primer/probe sets for RT-qPCR were screened and optimized. Good sensitivities of the optimized primer/probe sets were obtained with the limits of detections of 2.95, 2.82, 1.57, 2.8, 1.19, and 2.12 copies/reaction for H1N1, H3N2, Yamagata, Victoria, RSVA and RSVB, respectively. Eventually, quadruplex qPCR using the four designed primer/probe sets can achieve simultaneous screening of the four viruses at a single tube. Furthermore, the assay's good performance in detecting target viruses from clinical throat swab samples demonstrated its potential for diagnosis of these viruses. The method, based on the identified conserved sequences and primer/probe sets, can effectively reduce false-negative results and rapidly respond to these viruses during respiratory disease outbreaks, or even before their widespread emergence, which aid in preventing outbreaks and guiding clinical treatment.

Humans↗
PubMed1998

Genetic data obtained for two Chinese Han populations with a quadruplex fluorescent STR typing system (HUMVWA, HUMTH01, D21S11 and HPRT).

Abstract DNA typing of four tetrameric repeat loci (HUMVWA, HUMTH0I, D21SII and HPRT) was carried out in a Chinese Han population from Shanghai (East China) and one from Guangzhou (South-East China) using a quadruplex PCR amplification and detection of the fluorescent-labeled alleles on the ALF DNA sequencer. All loci were in accordance with Hardy-Weinberg equilibrium except for D21S11 in the Guangzhou population. A test for population differentiation showed no statistical difference in the allele frequency distribution between the two populations. Comparison of the allele frequency data with other Chinese Han populations from North and South-West China for the STR loci HUMVWA and HUMTH01 revealed heterogeneity between Northern Chinese Han and Southern Chinese Han, which is in accordance with previous studies on the basis of protein markers.

China↗
PubMed2002

STM1, a gene which encodes a guanine quadruplex binding protein, interacts with CDC13 in Saccharomyces cerevisiae.

The CDC13 gene encodes a protein that binds to the G-rich single-strand at yeast telomeres, and serves as a regulator of telomere replication. Cdc13 interacts with Est1 and DNA polymerase alpha, and cells carrying the temperature-sensitive allele cdc13-1 cannot complete telomere replication at the restrictive temperature and possess long telomeres. We attempted to isolate and characterize genes that interact with CDC13, in order to clarify the molecular mechanisms of telomere replication. A STM1 cDNA was isolated in a two-hybrid screen using CDC13 as a bait. The temperature-sensitive growth phenotype and the alteration in telomere size in cdc13-1 cells were corrected by introduction of the STM1 gene on a multicopy vector, but the extended G-rich single-strand overhangs which are also characteristic in the cdc13-1 mutant were not affected. Furthermore, we found that multiple copies of SGS1, a gene encoding a helicase that can unwind guanine quadruplexes, inhibited suppression of the cdc13-1 phenotype by STM1. We also demonstrate that a fusion protein consisting of the N-terminal region of Cdc13 and the C-terminal region of Stm1 (which shows similarity to the beta-subunit of the telomere binding complex in Oxytricha) could complement a cdc13 disruptant. Although STM1 itself is not essential for telomere replication, our findings suggest that STM1 genetically interacts with CDC13 to maintain telomere structure.

Amino Acid Sequence↗
PubMed1995

Ammonium ion at low concentration stabilizes the G-quadruplex formation by telomeric sequence.

A synthetic analog of telomeric DNA, d(T6G16) or d(G4T2G4T2G4T2G4) was found to form G-quadruplex structure in NH4-ion environments as low as 1 mM. Other counter ions like K(I) or Sr(II) known to have a stabilizing effect on G-tetrad require much higher concentrations. The multiconformational form of G-tetrad could be identified in NH4(I) upon heating the sample up to 100 degrees C. Circular dichroism spectral studies indicated that on thermally treating the complex NH4(I) ion helps in intramolecular G-tetrad formation over intermolecular association. However the G-tetrad structure formed in NH4(I) was found to be unstable in the presence of intercalator Actinomycin D or Tb(III) ion. It appears that the ionic radius of NH4(I) influence tight fitting of this ion inside the cavity of the tetrad. The stability of G-tetrad in the presence of very low concentration of NH4(I) may have potential applications in future.

DNA↗
PubMed1995

Further validation of a quadruplex STR DNA typing system: a collaborative effort to identify victims of a mass disaster.

The relatively new, PCR-based technique of Short Tandem Repeat (STR) profiling has been used in the identification of the victims of a mass disaster. The analysis relied upon a recently developed multiplex reaction and the use of automated fluorescence technology to simulataneously analyse four tetrameric STR loci. The performance of the 'quadruplex' test was assessed by use of a collaborative study incorporating a blind trial and was demonstrated to be accurate, reliable and robust. Furthermore, the system proved to be highly successful despite the fact that many of the samples from the mass disaster scene were extremely degraded. The high success rate coupled with the discrimination power of the system enabled many severely decomposed human remains to be positively identified.

DNA↗
PubMed2005

Effects of 8-methyl-2'-deoxyadenosine incorporation into quadruplex forming oligodeoxyribonucleotides.

In this paper we report the synthesis and the structural characterization of two modified oligodeoxyribonucleotides (ODNs), namely d(A8MeGGGT) and d(TA8MeGGGT), where A8Me represents a 8-methyl-2'-deoxyadenosine. Both ODNs have been studied by 1H NMR, CD spectroscopy and molecular modelling and shown to form fourfolds symmetric G-quadruplex structures, with all strands parallel and equivalent to each other. The complexes are characterized by thermal stabilities comparable to that of their natural counterparts. NOE patterns involving 8-methyl group in A8Me residues allowed us to define the main structural features at the 5'-end of the complexes. Particularly, inter- and intrastrand NOEs show a syn-orientation and a symmetrical arrangement of A8Me bases stacking on the adjacent G-tetrad.

Circular Dichroism↗
PubMed2004

Synthesis, biophysical and biological evaluation of 3,6-bis-amidoacridines with extended 9-anilino substituents as potent G-quadruplex-binding telomerase inhibitors.

Telomerase and telomere maintenance are emerging targets for the treatment of human cancers. We report here on the targeting of the telomere-telomerase complex with a series of small molecules based on an acridine platform. A series of 3,6-bisamidoacridines with extended 9-anilino sidechains were designed and synthesised as potential telomeric G-quadruplex DNA (G4) interacting compounds. G4-stabilisation was assessed using a high-throughput FRET (fluorescence resonance energy transfer) assay and telomerase inhibition quantified by a modified TRAP (telomerase repeat amplification protocol) method. Within the series, the compounds showed significant G4-stabilising ability (Delta T(m) values of 25-36 degrees C at 1 microM concentration) and telomerase inhibition in the nanomolar region ((tel)EC(50) values of 80-318 nM). Furthermore, a direct correlation between the FRET and TRAP assays was observed, supporting the use of the rapid screening FRET assay for early assessment of potential G4-stabilising telomerase inhibitors.

Acridines↗