G-quadruplex Stability Research Articles

A naphthyridine-indole ligand for selective stabilization of G-quadruplexes and conformational conversion of hybrid topology

The development of ligands to stabilize G-quadruplexes (G4s) or induce G4s to transition from metastable topology to stable topology is a potential strategy for inhibiting cancer cell proliferation. In this study, a novel G-quadruplex (G4) ligand based on a naphthyridine scaffold with two indole pendants, L5-DA, is reported to convert hybrid to the parallel topology. Circular dichroism (CD) and fluorescence spectroscopies were used to investigate the interactions between L5-DA and G4s. The CD spectra revealed that the L5-DA induced the conformational conversion from hybrid topologies to parallel topologies with a melting temperature increase of more than 30 °C. According to Förster resonance energy transfer assays, the presence of excess duplex competitor had no effect on the ligand-induced stabilization of the hybrid topology, confirming the L5-DA's selectivity for G4s over ds26. With IC50 values of 4.3 μM, the ligand showed significant cytotoxicity against HeLa cells and effectively induced growth inhibition and apoptosis in HeLa cells. Immunofluorescence microscopy revealed an increase in BG4 foci in the presence of the L5-DA, confirming ligand-induced G4s stabilization in HeLa cells. According to these results, the combination of naphthyridine and indole scaffold was an effective design strategy for G4s stabilization and conformational conversion of metastable G4 topology for inhibiting cancer cell growth.

Targeted Downregulation of MYC through G-quadruplex Stabilization by DNAi.

Modulating the expression or function of the enigmatic MYC protein has demonstrated efficacy in an array of cancer types and a marked potential therapeutic index and safety profile. Despite its high therapeutic value, specific and selective inhibitors or downregulating therapeutics have proven difficult to develop. In the current study, we expanded our work on a MYC promoter G-quadruplex (G4) stabilizing DNA clamp to develop an oligonucleotide interfering DNA (DNAi) therapeutic. We explored six DNAi for G4-stabilization through EMSA, DMS footprinting, and thermal stability studies, focusing on the DNAi 5T as the lead therapeutic. 5T, but not its scramble control 5Tscr, was then shown to enter the nucleus, modulate cell viability, and decrease MYC expression through G4-stabilization. DNAi 5T is thus described to be our lead DNAi, targeting MYC regulation through stabilization of the higher-order DNA G4 structure in the proximal promoter, and it is poised for further preclinical development as an anticancer therapeutic.

Guanine-tethered Oligonucleotides Restore Abnormal Protein Synthesis with a SNP Mutation in a 5′-UTR G-quadruplex of Human MSH2

Abstract Here we demonstrate that a guanine-to-adenine substitution within guanine repeat sequences in the MSH 5′-UTR weakens G-quadruplex stability, leading to increased protein synthesis. The abnormal protein synthesis was effectively restored to the original level with a guanine-tethered oligonucleotide by the sequence-specific reintroduction of a hetero RNA–DNA G-quadruplex into the SNP site of the 5′ UTR of MSH2. Our approach would provide a new insight into gene therapies for various diseases which are caused by gene mutations in biological relevant G-quadruplexes.

APTO-253 Interaction with G-Quadruplex DNA Is Linked to Inhibition of c-Myc Expression, Induction of DNA Damage, and Generation of Synthetic Lethality in Cells with BRCA1/2 Impairment

APTO-253 is a Phase 1 clinical stage small molecule that selectively kills acute myeloid leukemia (AML) cells in the absence of toxicity to normal peripheral blood mononuclear (PBMCs) and bone marrow cells in various animal species and humans. Previously, APTO-253 was reported to exhibit concentration and time dependent inhibition of c-MYC oncogene expression in AML cells and consequently to induce G0/G1 cell cycle arrest and apoptosis, but not affect normal PBMCs (58th ASH abstract #1716). Current mechanistic studies demonstrated that APTO-253 modulates c-MYC at the transcriptional level by decreasing acetylated H3K27 at its promoters and additionally by destabilizing c-MYC mRNA. In addition, differential gene expression analysis of RNA-seq and reverse phase protein array (RPPA) data highlighted a role for c-MYC in the mechanism of APTO-253 (GO terms - Down regulated by c-MYC p-value 6E-26, Gene promoters bound by c-MYC p-value 4.2 E-10, ChIP targets of c-MYC p-value 3.3E-8). Furthermore, from the RPPA data we observed an increase in p-Chk1, p-Chk2, γH2Ax, and total p53 and E2F1, all of which are indicative of activation of DNA damage response pathways. This was accompanied by elevated levels of XBP1, GRP78, and p-p38 that point towards cellular stress response signaling (GO term Regulation of Cell Stress, p-value 1.89E-8). Subsequent work demonstrated that APTO-253 induced DNA damage as documented by gH2AX foci formation and the neutral COMET assay, and exhibited synthetic lethality in cells with BRCA1/2 mutations that have impaired ability to repair DNA damage by homologous recombination (abstract submitted to 59th ASH annual meeting). This amalgam of data, coupled with structural informatics analyses, suggested a mechanism of action involving the binding and/or stabilization of G-quadruplex (G4) DNA motifs. Intracellular pharmacokinetic studies revealed that APTO-253 converts from a monomer to a ferrous complex. PCR stop and modified FRET assays demonstrated that the APTO-253 ferrous complex Fe(253)3 has activity similar to that of TMPyP4, a well-established G4 ligand, to bind/stabilize G4 structures in telomere sequences (∆T1/2 vs concentration slope 7.7 ±2.7 and 10.67 ±1.23 for Fe(253)3 and TMPyP4, respectively), in MYC promoter sequences (5.07 ±1.56 and 5.62 ±0.75, respectively) and in rRNA sequences (4.6 ±1.1 and 5.6 ±0.88, respectively). Although APTO-253 may participate in multiple mechanistic events, the effect of APTO-253 on c-MYC expression, cell cycle arrest and DNA damage, as well as synthetic lethality in cells with compromised DNA repair mechanisms, can be explained by the action of the Fe(253)3 complex on G-quadruplex DNA motifs. DisclosuresLocal:Aptose Biosciences, Inc.: Employment. Zhang:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

Differential responses of neurons, astrocytes, and microglia to G-quadruplex stabilization.

The G-quadruplex (G4-DNA or G4) is a secondary DNA structure formed by DNA sequences containing multiple runs of guanines. While it is now firmly established that stabilized G4s lead to enhanced genomic instability in cancer cells, whether and how G4s contribute to genomic instability in brain cells is still not clear. We previously showed that, in cultured primary neurons, small-molecule G4 stabilizers promote formation of DNA double-strand breaks (DSBs) and downregulate the Brca1 gene. Here, we determined if G4-dependent Brca1 downregulation is unique to neurons or if the effects in neurons also occur in astrocytes and microglia. We show that primary neurons, astrocytes and microglia basally exhibit different G4 landscapes. Stabilizing G4-DNA with the G4 ligand pyridostatin (PDS) differentially modifies chromatin structure in these cell types. Intriguingly, PDS promotes DNA DSBs in neurons, astrocytes and microglial cells, but fails to downregulate Brca1 in astrocytes and microglia, indicating differences in DNA damage and repair pathways between brain cell types. Taken together, our findings suggest that stabilized G4-DNA contribute to genomic instability in the brain and may represent a novel senescence pathway in brain aging.

G-quadruplex targeting chemical nucleases as a nonperturbative tool for analysis of cellular G-quadruplex DNA

SummaryG-quadruplex structures are associated with various biological activities, while in vivo evidence is essential to confirm the formation of G-quadruplexes inside cells. Most conventional agents that recognize G-quadruplex, including antibodies and small-molecule G-quadruplex ligands, either stabilize the G-quadruplex or prevent G-quadruplex unfolding by helicase, thereby artificially increasing the G-quadruplex levels in cells. Unambiguous study of G-quadruplexes at natural cellular levels requires agents that do not enhance the stability of G-quadruplex. Herein, we report the first example of nonperturbative chemical nucleases that do not influence the stability of G-quadruplex telomeric DNA but can selectively cleave G-quadruplex DNA over duplex DNA. These chemical nucleases can be readily taken up by cells and promote selective cleavage of telomeric DNA with low levels of nonselective DNA cleavage of other regions of the genome. The cleavage of G-quadruplex telomeric DNA by nonperturbative chemical nucleases confirms the formation of G-quadruplex telomeric DNA in live cells.

Characterization of In Vitro G-Quadruplex Formation of Imetelstat Telomerase Inhibitor

Imetelstat (GRN163L) is a potent and specific telomerase inhibitor currently in clinical development for the treatment of hematological malignancies such as myelofibrosis and myelodysplastic syndrome. It is a 13-mer N3'-P5' thio-phosphoramidate oligonucleotide covalently functionalized at the 5'-end with a palmitoyl lipid moiety through an aminoglycerol linker. As a competitive inhibitor of human telomerase, imetelstat directly binds to the telomerase RNA component sequence (hTR) in the catalytic site of the enzyme and acts as a direct competitor of human telomere binding. Administration of imetelstat causes progressive shortening of the telomeres, thereby inhibiting malignant cells' proliferation. We report here the ability of imetelstat to form stable, parallel, intermolecular G-quadruplex structures in vitro. The impact of the ionic environment on the formation and stability of imetelstat higher-order structure was investigated through circular dichroism spectroscopy, thermal denaturation analysis, and size-exclusion chromatography. We demonstrated that different structural elements, such as the 5'-palmitoyl linker and the thio-phosphoramidate backbone, critically contribute to G-quadruplex stability. Experiments further showed that G-quadruplex formation does not hamper binding to the hTR oligonucleotide sequence in vitro.

Encounters between Cas9/dCas9 and G-Quadruplexes: Implications for Transcription Regulation and Cas9-Mediated DNA Cleavage.

Using the nuclease-dead Cas9 (dCas9), we targeted in cellulo a G-rich sequence, which contains multiple potentially G-quadruplex (GQ) forming sites, within the human tyrosine hydroxylase (TH) promoter. We demonstrate that transcription can be up or down regulated by targeting different parts of this G-rich sequence. Our results suggest that TH transcription levels correlate with stability of different GQs formed by this sequence and targeting them with dCas9 can modulate their stability. Unlike alternative approaches, regulating TH expression by targeting the promoter GQs with dCas9 enables a specific and potentially transient control and does not require mutations in the sequence. We also investigated whether the presence of GQs in target sequences impacts DNA cleavage activity of Cas9. We discovered significant reduction in cleavage activity when the vicinity of a high-stability GQ was targeted. Furthermore, this reduction is significantly more prominent for the G-rich strand compared to the complementary C-rich strand.

G-quadruplex regulation of neural gene expression.

G-quadruplexes are four-stranded helical nucleic acid structures characterized by stacked tetrads of guanosine bases. These structures are widespread throughout mammalian genomic DNA and RNA transcriptomes, and prevalent across all tissues. The role of G-quadruplexes in cancer is well-established, but there has been a growing exploration of these structures in the development and homeostasis of normal tissue. In this review, we focus on the roles of G-quadruplexes in directing gene expression in the nervous system, including the regulation of gene transcription, mRNA processing, and trafficking, as well as protein translation. The role of G-quadruplexes and their molecular interactions in the pathology of neurological diseases is also examined. Outside of cancer, there has been only limited exploration of G-quadruplexes as potential intervention targets to treat disease or injury. We discuss studies that have used small-molecule ligands to manipulate G-quadruplex stability in order to treat disease or direct neural stem/progenitor cell proliferation and differentiation into therapeutically relevant cell types. Understanding the many roles that G-quadruplexes have in the nervous system not only provides critical insight into fundamental molecular mechanisms that control neurological function, but also provides opportunities to identify novel therapeutic targets to treat injury and disease.

Ubiquitin-mediated DNA damage response is synthetic lethal with G-quadruplex stabilizer CX-5461

CX-5461 is a G-quadruplex (G4) ligand currently in trials with initial indications of clinical activity in cancers with defects in homologous recombination repair. To identify more genetic defects that could sensitize tumors to CX-5461, we tested synthetic lethality for 480 DNA repair and genome maintenance genes to CX-5461, pyridostatin (PDS), a structurally unrelated G4-specific stabilizer, and BMH-21, which binds GC-rich DNA but not G4 structures. We identified multiple members of HRD, Fanconi Anemia pathways, and POLQ, a polymerase with a helicase domain important for G4 structure resolution. Significant synthetic lethality was observed with UBE2N and RNF168, key members of the DNA damage response associated ubiquitin signaling pathway. Loss-of-function of RNF168 and UBE2N resulted in significantly lower cell survival in the presence of CX-5461 and PDS but not BMH-21. RNF168 recruitment and histone ubiquitination increased with CX-5461 treatment, and nuclear ubiquitination response frequently co-localized with G4 structures. Pharmacological inhibition of UBE2N acted synergistically with CX-5461. In conclusion, we have uncovered novel genetic vulnerabilities to CX-5461 with potential significance for patient selection in future clinical trials.

Imide Condensation as a Strategy for the Synthesis of Core-Diversified G-Quadruplex Ligands with Anticancer and Antiparasitic Activity*.

A facile imide coupling strategy for the one‐step preparation of G‐quadruplex ligands with varied core chemistries is described. The G‐quadruplex stabilization of a library of nine compounds was examined using FRET melting experiments, and CD, UV‐Vis, fluorescence and NMR titrations, identifying several compounds that were capable of stabilizing G‐quadruplex DNA with interesting selectivity profiles. The best G4 ligand was identified as compound 3, which was based on a perylene scaffold and exhibited 40‐fold selectivity for a telomeric G‐quadruplex over duplex DNA. Surprisingly, a tetra‐substituted flexible core, compound 11, also exhibited selective stabilization of G4 DNA over duplex DNA. The anticancer and antiparasitic activity of the library was also examined, with the lead compound 3 exhibiting nanomolar inhibition of Trypanosoma brucei with 78‐fold selectivity over MRC5 cells. The cellular localization of this compound was also studied via fluorescence microscopy. We found that uptake was time dependant, with localization outside the nucleus and kinetoplast that could be due to strong fluorescence quenching in the presence of small amounts of DNA.

Characterization of G‐quadruplex Structures in Serum Amyloid A Gene as a Novel Approach for Systemic Amyloidosis Therapy

Transcriptional initiation is an important step in the regulation of transcription. When transcriptional initiation is dysfunctional, it will lead to abnormal gene expression. Overexpression of Serum Amyloid A (SAA) and self-assembly into fibrils have been associated with chronic inflammatory conditions and certain neoplasms, which lead to the deposition of AA amyloid in organs such as the liver, spleen, and kidney (systemic amyloidosis). For this reason, SAA is a highly significant molecular target. Insight into the structural biology of the promoter element that determines the combinatorial control of gene expression by different transcriptional factors is essential for understanding the dysfunction of SAA gene expression. The highly GC-rich region of the human SAA promoter is crucial for basal promoter activity and likely to assume a DNA secondary structure, such as G-quadruplex (GQ). GQs often serve as transcriptional silencer elements. Stabilization of GQ is possible with small molecule therapeutics to modulate gene expression. In the present study, G-quadruplex formation has been isolated from the GC-rich region of the 5’-untranslated region (5’UTR) of SAA2. An electrophoretic mobility shift and thioflavin T fluorescence assays were used to confirm the formation of a biologically relevant intramolecular structure. The structure of the G-quadruplex is currently under investigation by circular dichroism and DMS footprinting. These data characterizing the formation of a unique secondary structure in SAA gene are promising to develop novel anti-amyloid therapy for the treatment of systemic amyloidosis.

Human telomerase is directly regulated by non-telomeric TRF2-G-quadruplex interaction.

SummaryHuman telomerase reverse transcriptase (hTERT) remains suppressed in most normal somatic cells. Resulting erosion of telomeres leads eventually to replicative senescence. Reactivation of hTERT maintains telomeres and triggers progression of >90% of cancers. However, any direct causal link between telomeres and telomerase regulation remains unclear. Here, we show that the telomere-repeat-binding-factor 2 (TRF2) binds hTERT promoter G-quadruplexes and recruits the polycomb-repressor EZH2/PRC2 complex. This is causal for H3K27 trimethylation at the hTERT promoter and represses hTERT in cancer as well as normal cells. Two highly recurrent hTERT promoter mutations found in many cancers, including ~83% glioblastoma multiforme, that are known to destabilize hTERT promoter G-quadruplexes, showed loss of TRF2 binding in patient-derived primary glioblastoma multiforme cells. Ligand-induced G-quadruplex stabilization restored TRF2 binding, H3K27-trimethylation, and hTERT re-suppression. These results uncover a mechanism of hTERT regulation through a telomeric factor, implicating telomere-telomerase molecular links important in neoplastic transformation, aging, and regenerative therapy.

Promoter G-quadruplex favours epigenetic reprogramming-induced atypical expression of ZEB1 in cancer cells

BackgroundAberrant expression of Zinc-finger E-box binding homeobox 1 (ZEB1), which remains repressed in normal cells, is frequently associated with cancer aggressiveness. However, transcriptional mechanism underlying such atypical ZEB1 expression in cancer is not yet well-understood. MethodsZEB1 promoter G-quadruplexes were studied and modeled extensively using circular dichroism, fluorescence spectroscopy, ITC and DMS protection assay. Luciferase assay, qPCR, FAIRE, ChIP, western blotting, confocal microscopy was used to access the regulation of ZEB1 transcription. ResultsOur study unravels the occupancy of nucleolin to ZEB1 promoter as a crucial determinant which facilitates the binding of SP1 transcription factor to chromatin, by locally remodelling the region. SP1, subsequently, recruits P300 acetyl transferase leading to enriched acetyl-histone H3 at promoter and activates ZEB1 transcription. ZEB1 promoter analysis identifies presence of four putative G-quadruplex (G4) forming motifs within 700 bp of TSS; each quadruplex is characterized structurally in details with an array of biophysical techniques. Surprisingly, stabilization of G4 with cationic porphyrin TMPyP4 represses its transcription and eventually impedes cell invasiveness. ConclusionsTMPyP4 binding to a selected G4 motif (5′ -534/−511–3′ from TSS), where nucleolin/SP1/P300 co-occupies, prevents the association of nucleolin which consequently hinders SP1 binding, leading to chromatin compactness and transcriptional repression. General significanceOur findings demonstrate an epigenetic mechanism of ZEB1 reactivation where dynamic occupancy of transcription regulators encompassing a G4 motif is crucial and thus, small molecule induced G-quadruplex stabilization may act as a potential molecular switch to turn-off gene expression.

Improving Strategies in the Development of Protein‐Downregulation‐Based Antiandrogens

AR plays a crucial role in the occurrence and development of prostate cancer (PCa) and its signaling pathway remains active in castration-resistant prostate cancer (CRPC) patients. The drug resistance of current clinical used antiandrogens is a major challenge for the treatment of PCa, thus the development of new generations of antiandrogens is highly demanded. Recently, strategies of the downregulation of AR protein have been attracted the huge attention due to its potential in the discovery and development of new antiandrogens, including the stabilizers of G-Quadruplex, inhibitors of ROR-γ, AR targeting PROTACs, and other selective AR degraders (SRADs), which are able to conquer current resistances such as the acquired mutation of AR, the expression of AR variable splices, or over-expression of AR. This review summarized the various strategies for down-regulating AR protein, at either mRNA or protein level, thus providing new ideas for the development of the promising antiandrogens.

Discovery of MTR-106 as a highly potent G-quadruplex stabilizer for treating BRCA-deficient cancers.

G-quadruplexes (G4s) are DNA or RNA structures formed by guanine-rich repeating sequences. Recently, G4s have become a highly attractive therapeutic target for BRCA-deficient cancers. Here, we show that a substituted quinolone amide compound, MTR-106, stabilizes DNA G-quadruplexes in vitro. MTR-106 displayed significant antiproliferative activity in homologous recombination repair (HR)-deficient and PARP inhibitor (PARPi)-resistant cancer cells. Moreover, MTR-106 increased DNA damage and promoted cell cycle arrest and apoptosis to inhibit cell growth. Importantly, its oral and i.v. administration significantly impaired tumor growth in BRCA-deficient xenograft mouse models. However, MTR-106 showed modest activity against talazoparib-resistant xenograft models. In rats, the drug rapidly distributes to tissues within 5min, and its average concentrations were 12-fold higher in the tissues than in the plasma. Overall, we identified MTR-106 as a novel G-quadruplex stabilizer with high tissue distribution, and it may serve as a potential anticancer agent.

An atomistic investigation on the interaction of distamycin A and its derivative with the telomeric G-Quadruplex as anticancer agents by molecular dynamics simulation

Human telomerase that activates within cancer cells has a telomeric sequence at the 3′ end. Each factor that stabilizes the G-quadruplex in guanine-rich telomeric sequences can inhibit the regular telomerase activity. Therefore, the telomeric G-quadruplex is known as a promising target in cancer treatment. In this work, we studied the binding of positively charged distamycin A and its uncharged derivative to the G-quadruplex in a solution environment by Molecular Dynamics (MD) simulation. The binding mechanism and subtle conformational changes were investigated as a result of the ligand attachment. Moreover, binding free energy and clustering analysis describe the stability and flexibility of G-quadruplexes upon ligand binding. Structural analyses displayed that the favorable binding of both ligands imposes significant stability and rigidity in G-quadruplex conformation compared to free G-quadruplex, especially charged distamycin. Hydration pattern and ion distribution were different for free G-quadruplex and both of the ligand complexes. Energy decomposition reveals the electrostatic effect on the stability of G-quadruplex. The radial distribution function displayed the solvent shell and ion moving away from the groove. The hydrogen bond played an essential role in the binding of both ligands, especially for the charged derivative. van der Waals interaction is the only factor that is more important in binding uncharged distamycin into G-quadruplex than the charged one. The calculated ΔGbind showed the stability of both ligands within grooves and good agreement with the experimental binding free energy data. Finally, the results suggest that ligand modification improves the binding mode toward stabilizing G-quadruplexes.

G-quadruplex binding properties of a potent PARP-1 inhibitor derived from 7-azaindole-1-carboxamide

Poly ADP-ribose polymerases (PARP) are key proteins involved in DNA repair, maintenance as well as regulation of programmed cell death. For this reason they are important therapeutic targets for cancer treatment. Recent studies have revealed a close interplay between PARP1 recruitment and G-quadruplex stabilization, showing that PARP enzymes are activated upon treatment with a G4 ligand. In this work the DNA binding properties of a PARP-1 inhibitor derived from 7-azaindole-1-carboxamide, (2-[6-(4-pyrrolidin-1-ylmethyl-phenyl)-pyrrolo[2,3-b]pyridin-1-yl]-acetamide, compound 1) with model duplex and quadruplex DNA oligomers were studied by NMR, CD, fluorescence and molecular modelling. We provide evidence that compound 1 is a strong G-quadruplex binder. In addition we provide molecular details of the interaction of compound 1 with two model G-quadruplex structures: the single repeat of human telomeres, d(TTAGGGT)4, and the c-MYC promoter Pu22 sequence. The formation of defined and strong complexes with G-quadruplex models suggests a dual G4 stabilization/PARP inhibition mechanism of action for compound 1 and provides the molecular bases of its therapeutic potential.

Learning to Model G-Quadruplexes: Current Methods and Perspectives.

G-quadruplexes have raised considerable interest during the past years for the development of therapies against cancer. These noncanonical structures of DNA may be found in telomeres and/or oncogene promoters, and it has been observed that the stabilization of such G-quadruplexes may disturb tumor cell growth. Nevertheless, the mechanisms leading to folding and stabilization of these G-quadruplexes are still not well established, and they are the focus of much current work in this field. In seminal works, stabilization was observed to be produced by cations. However, subsequent studies showed that different kinds of small molecules, from planar and nonplanar organic molecules to square-planar and octahedral metal complexes, may also lead to the stabilization of G-quadruplexes. Thus, the comprehension and rationalization of the interaction of these small molecules with G-quadruplexes are also important topics of current interest in medical applications. To shed light on the questions arising from the literature on the formation of G-quadruplexes, their stabilization, and their interaction with small molecules, synergies between experimental studies and computational works are needed. In this review, we mainly focus on in silico approaches and provide a broad compilation of different leading studies carried out to date by different computational methods. We divide these methods into twomain categories: (a) classical methods, which allow for long-timescale molecular dynamics simulations and the corresponding analysis of dynamical information, and (b) quantum methods (semiempirical, quantum mechanics/molecular mechanics, and density functional theory methods), which allow for the explicit simulation of the electronic structure of the system but, in general, are not capable of being used in long-timescale molecular dynamics simulations and, therefore, give a more static picture of the relevant processes.

A stable uncompleted tetramolecular G-quadruplex formed by d(AGnA) under acidic condition

In this work, the effects of terminal adenines on the formation and stability of tetramolecular G-quadruplexes (G4s) have been studied by electrospray ionization mass spectrometry (ESI-MS), UV, CD and NMR spectroscopy. Several evidences suggested that the sequences d(AGnA) (n = 4 or 5) form stable uncompleted tetramolecular G4 at acidic condition which is different from the canonical one in the neutral condition. In addition, hydrolysis of guanine has also been observed in acidic condition that may occur for unpaired strands rather than in complete G4. Thus, a new G4 topology containing incomplete G-quartet is proposed that is very stable and particularly presents in acidic ammonium ions solution. The information presented in this study provides the new insight on the polymorphism of G4s in acidic environment, which may help understand of the special role of adenines on the formation of G4s.