Human Telomeric G-quadruplex DNA Research Articles

Electrochemical genosensor based on RNA-responsive human telomeric G-quadruplex DNA: A proof-of-concept with SARS-CoV-2 RNA

In this report, a facile and label-free electrochemical RNA biosensor is developed by exploiting methylene blue (MB) as an electroactive positive ligand of G-quadruplex. The electrochemical response mechanism of the nucleic acid assay was based on the change in differential pulse voltammetry (DPV) signal of adsorbed MB on the immobilized human telomeric G-quadruplex DNA with a loop that is complementary to the target RNA. Hybridization between synthetic positive control RNA and G-quadruplex DNA probe on the transducer platform rendered a conformational change of G-quadruplex to double-stranded DNA (dsDNA), and increased the redox current of cationic MB π planar ligand at the sensing interface, thereby the electrochemical signal of the MB-adsorbed duplex is proportional to the concentration of target RNA, with SARS-CoV-2 (COVID-19) RNA as the model. Under optimal conditions, the target RNA can be detected in a linear range from 1 zM to 1 μM with a limit of detection (LOD) obtained at 0.59 zM for synthetic target RNA and as low as 1.4 copy number for positive control plasmid. This genosensor exhibited high selectivity towards SARS-CoV-2 RNA over other RNA nucleotides, such as SARS-CoV and MERS-CoV. The electrochemical RNA biosensor showed DPV signal, which was proportional to the 2019-nCoV_N_positive control plasmid from 2 to 200000 copies (R2 = 0.978). A good correlation between the genosensor and qRT-PCR gold standard was attained for the detection of SARS-CoV-2 RNA in terms of viral copy number in clinical samples from upper respiratory specimens.

Spectroscopy and calorimetry evidence of binding-induced thermal stabilization of human telomeric G-quadruplex DNA by 1,5-disubstituted morpholino-amido anthraquinone: Molecular basis of anticancer action

We report interaction of 1,5-Bis (3-morpholinopropionamido) anthracene-9,10-dione with Human Telomeric G4 DNA sequences, wHTel26 and HTel22 in presence of K+ and Na+ ions, respectively. Surface Plasmon Resonance yields association constant Kb = 1.0–3.8x105 M−1 with HTel22 in K+-rich solution. Significant hypochromism (46–79 %) and fluorescence quenching (83–92 %), no change in absorption and emission maxima, unaltered fluorescence lifetime of ligand, change in conformation of G4 DNA with no overall disruption of G-quartets and absence of induced Circular Dichroism band, suggest external groove binding of ligand. Molecular docking shows short contacts of 9O, 15N, 18O, 11NH, 12CO atoms of ligand with A3, G4, G22, G23 and A1, G3, G14 bases of 3 + 1 hybrid-2 and 2 + 2 anti-parallel conformers, respectively without making any contact with loops, to an energetically more favourable conformation. Ligand-influenced thermal stabilization, up to ΔTm = 37/48 °C, may disrupt access of telomerase enzyme to telomere and promote apoptosis in malignant tissues.

Recognition of human telomeric G-quadruplex DNA by 1,5-disubstituted diethyl-amido anthraquinone derivative in different ion environments causing thermal stabilization and apoptosis

Ligand binding to G-quadruplex (G4) structures at human telomeric DNA ends promotes thermal stabilization, disrupting the interaction of the telomerase enzyme, which is found active in 80–85% of cancers and serves as a molecular marker. Anthraquinone compounds are well-known G-quadruplex (G4) binders that inhibit telomerase and induce apoptosis in cancer cells. Our current investigation is based on 1,5-bis[3-(diethylamino)propionamido]anthracene-9,10-dione, a derivative of anthraquinone and its binding characterization with two different human telomeric DNA structures, wHTel26 and HTel22, in the effect of K+ and Na+ by using an array of biophysical, calorimetry, molecular docking and cell viability assay techniques. Binding constants (K b) in the range of ∼105–107 M−1 and stoichiometries of 1:1, 2:1 & 4:1 were obtained from the absorbance, fluorescence, and circular dichroism study. Remarkable hypochromism (55, 97%) and ∼17 nm shift in absorbance, fluorescence quenching (95, 97%), the unaltered value of fluorescence lifetime, restoration of Circular Dichroism bands, absence of ICD band, indicated the external groove binding/binding somewhere at loops. This is also evident in molecular docking results, the ligand binds to groove forming base (G4, G5, G24, T25) and in the vicinity to TTA loop (G14, G15, T17) bases of wHTel26 and HTel22, respectively. Thermal stabilization induced by ligand was found greater in Na+ ion (27.5 °C) than (19.1 °C) in K+ ion. Ligand caused cell toxicity in MCF-7 cancer cell lines with an IC50 value of ∼8.4 µM. The above findings suggest the ligand, 1,5-bis[3-(diethylamino)propionamido]anthracene-9,10-dione could be a potent anticancer drug candidate and has great therapeutic implications. Binding of disubstituted amido anthraquinone derivative, 1,5-bis[3-(diethylamino)propionamido]anthracene-9,10-dione to human telomere HTel22 antiparallel conformation induced thermal stabilization. Communicated by Ramaswamy H. Sarma

The mechanism of UP1 binding and unfolding of human telomeric DNA G-quadruplex

The human telomere contains multiple copies of the DNA sequence d(TTAGGG) which can fold into higher order intramolecular G-quadruplexes and regulate the maintenance of telomere length and chromosomal integrity. The nucleic acid binding protein heteronuclear ribonucleoprotein A1 (hnRNP A1) and its N-terminus proteolytic product UP1 have been shown to efficiently bind and unfold telomeric DNA G-quadruplex. However, the understanding of the molecular mechanism of the UP1 binding and unfolding telomeric G-quadruplexes is still limited. Here, we performed biochemical and biophysical characterizations of UP1 binding and unfolding of human telomeric DNA G-quadruplex d[AGGG(TTAGGG)3], and in combination of systematic site-direct mutagenesis of two tandem RNA recognition motifs (RRMs) in UP1, revealed that RRM1 is responsible for initial binding and unfolding, whereas RRM2 assists RRM1 to complete the unfolding of G-quadruplex. Isothermal titration calorimetry (ITC) and circular dichroism (CD) studies of the interactions between UP1 and DNA G-quadruplex variants indicate that the “TAG” binding motif in Loop2 of telomeric G-quadruplex is critical for UP1 recognition and G-quadruplex unfolding initiation. Together we depict a model for molecular mechanism of hnRNP A1 (UP1) binding and unfolding of the human telomeric DNA G-quadruplex.

Post- and Pre-Radiolabeling Assays for anti Thymidine Cyclobutane Dimers as Intrinsic Photoprobes of Various Types of G-Quadruplexes, Reverse Hoogsteen Hairpins, and Other Non-B DNA Structures.

G-quadruplexes are thought to play an important role in gene regulation and telomere maintenance, but developing probes for their presence and location is challenging due to their transitory and highly dynamic nature. The majority of probes for G-quadruplexes have relied on antibody or small-molecule binding agents, many of which can also alter the dynamics and relative populations of G-quadruplexes. Recently, it was discovered that ultraviolet B (UVB) irradiation of human telomeric DNA and various G-quadruplex forming sequences found in human promoters, as well as reverse Hoogsteen hairpins, produces a unique class of non-adjacent anti cyclobutane pyrimidine dimers (CPDs). Therefore, one can envision using a pulse of UVB light to irreversibly trap these non-B DNA structures via anti CPD formation without perturbing their dynamics, after which the anti CPDs can be identified and mapped. As a first step toward this goal, we report radioactive post- and pre-labeling assays for the detection of non-adjacent CPDs and illustrate their use in detecting trans,anti T=(T) CPD formation in a human telomeric DNA sequence. Both assays make use of snake venom phosphodiesterase (SVP) to degrade the trans,anti T=(T) CPD-containing DNA to the tetranucleotide pTT=(pTT) corresponding to CPD formation between the underlined T's of two separate dinucleotides while degrading the adjacent syn TT CPDs to the trinucleotide pGT=T. In the post-labeling assay, calf intestinal phosphodiesterase is used to dephosphorylate the tetranucleotides, which are then rephosphorylated with kinase and [32P]-ATP to produce radiolabeled mono- and diphosphorylated tetranucleotides. The tetranucleotides are confirmed to be non-adjacent CPDs by 254 nm photoreversion to the dinucleotide p*TT. In the pre-labeling assay, radiolabeled phosphates are introduced into non-adjacent CPD-forming sites by ligation prior to irradiation, thereby eliminating the dephosphorylation and rephosphorylation steps. The assays are also demonstrated to detect the stereoisomeric cis,anti T=(T) CPD.

GW-2974 and SCH-442416 modulators of tyrosine kinase and adenosine receptors can also stabilize human telomeric G-quadruplex DNA.

GW-2974 is a potent tyrosine kinase receptor inhibitor while SCH-442416 is a potent adenosine receptors' antagonist with high selectivity towards human adenosine A2A receptor over other adenosine receptors. The two compounds were reported to possess anti-cancer properties. This study aimed to investigate whether stabilization of human telomeric G-quadruplex DNA by GW-2974- and SCH-442416 is a plausible fundamental mechanism underlying their anti-cancer effects. Human telomeric G-quadruplex DNA with sequence AG3(TTAGGG)3 was used. The study used ultraviolet-visible (UV-Vis), fluorescence, fluorescence quenching, circular dichroism (CD), melting temperatures (Tm) and molecular docking techniques to evaluate interactions. The results showed that GW-2974 and SCH-442416 interacted with G-quadruplex DNA through intercalation binding into two types of dependent binding sites. Binding affinities of 1.3 × 108-1.72 × 106 M-1 and 1.55 × 107-3.74 × 105 M-1 were obtained for GW-2974 and SCH-442416, respectively. An average number of binding sites between 1 and 2 was obtained. Additionally, the melting temperature curves indicated that complexation of both compounds to G-quadruplex DNA provided more stability (ΔTm = 9.9°C and 9.6°C, respectively) compared to non-complexed G-quadruplex DNA. Increasing the molar ratios over 1:1 (drug:G-quadruplex) showed less stabilization effect on DNA. Furthermore, GW-2974 and SCH-442516 have proven ≥ 4.0 folds better selective towards G-quadruplex over double-stranded ct-DNA. In silico molecular docking and dynamics revealed favorable exothermic binding for the two compounds into two sites of parallel and hybrid G-quadruplex DNA structures. The results supported the hypothesis that GW-2974 and SCH-442416 firmly stabilize human telomeric G-quadruplex DNA in additions to modulating tyrosine kinase and adenosine receptors. Consequently, stabilizing G-quadruplex DNA could be a mechanism underlying their anti-cancer activity.

Human telomeric G-quadruplex DNA enabled preferential recognition of copper (II) and Iron (III) ions sensed by a red emissive probe

Metal ions regulate several cellular processes in humans and other life forms and their balance in the body is essential for a healthy life. Specific detection of metal ions is important from both physiological and environmental perspectives. We report here the discovery of human telomeric G-quadruplex DNA enabled selective recognition of Cu2+ and Fe3+ ions using a molecular rotor that displays emission in the red region of the spectrum. This probe selectively senses Cu2+ and Fe3+ ions among a pool of other metal cations (monovalent, divalent, trivalent) and its sensing efficiency is maintained even at nanomolar levels of the DNA-probe complex. Circular dichroism and UV–visible spectroscopy-based studies revealed that the metal recognition abilities are linked to selective interaction of compound 1 with the human telomeric G-quadruplex DNA and its subsequent displacement upon binding of certain metal ions. The probe also shows robust sensing performance in the presence of other metal ions. Between Cu2+ and Fe3+, the recognition of Cu2+ is more sensitive with 3 ppb limit of detection.

Preferential Recognition of Human Telomeric G-Quadruplex DNA by a Red-Emissive Molecular Rotor.

The development of new fluorescent molecules for the recognition of specific G-quadruplex DNA structures has attracted wide attention due to their diverse roles in drug design, sensing, and cellular probing. In this work, we report the discovery of a red-emissive styryl quinolinium-based molecular rotor (compound 1), which recognizes human telomeric G-quadruplex with a distinct preference over DNA duplexes. Optical spectroscopy (UV-vis and circular dichroism)-based experiments indicated discernible interaction of compound 1 with the human telomeric DNA G-quadruplex with features of stacking interactions. Fluorescence-based Job's plot revealed a 1:1 binding stoichiometry between compound 1 and the human telomeric DNA G-quadruplex, and subsequent titration experiments showed micromolar affinities (Ka = 0.51 × 106 M-1). Molecular docking experiments showed interactions of compound 1 in the grooves of the quadruplex. Finally, we provide the application of compound 1 as a reporter molecule in the fluorescence displacement experiments, which showed its ability to act as a fluorescent probe compatible with ligands having aromatic cores.

Phen-DC3 Induces Refolding of Human Telomeric DNA into a Chair-Type Antiparallel G-Quadruplex through Ligand Intercalation.

Human telomeric G-quadruplex DNA structures are attractive anticancer drug targets, but the target's polymorphism complicates the drug design: different ligands prefer different folds, and very few complexes have been solved at high resolution. Here we report that Phen-DC3 , one of the most prominent G-quadruplex ligands in terms of high binding affinity and selectivity, causes dTAGGG(TTAGGG)3 to completely change its fold in KCl solution from a hybrid-1 to an antiparallel chair-type structure, wherein the ligand intercalates between a two-quartet unit and a pseudo-quartet, thereby ejecting one potassium ion. This unprecedented high-resolution NMR structure shows for the first time a true ligand intercalation into an intramolecular G-quadruplex.

Computationally enhanced X-ray diffraction analysis of a gold(III) complex interacting with the human telomeric DNA G-quadruplex. Unravelling non-unique ligand positioning

The crystal structure of the human telomeric DNA Tel24 G-quadruplex (Tel24 = TAG3(T2AG3)3T) in complex with the novel [AuL] species (with L = 2,4,6-tris(2-pyrimidyl)-1,3,5-triazine - TPymT-α) was solved by a novel joint molecular mechanical (MM)/quantum mechanical (QM) innovative approach. The quantum-refinement crystallographic method (crystallographic refinement enhanced with quantum mechanical calculation) was adapted to treat the [AuL]/G-quadruplex structure, where each gold complex in the binding site was found spread over four equally occupied positions. The four positions were first determined by docking restrained to the crystallographically determined metal ions' coordinates. Then, the quantum refinement method was used to resolve the poorly defined density around the ligands and improve the crystallographic determination, revealing that the binding preferences of this metallodrug toward Tel24 G-quadruplex arise from a combined effect of pyrimidine stacking, metal–guanine interactions and charge–charge neutralizing action of the π-acid triazine. The occurrence of interaction in solution with the Tel24 G-quadruplex DNA was further proved through DNA melting experiments, which showed a slight destabilisation of the quadruplex upon adduct formation.

Single-molecule displacement assay reveals strong binding of polyvalent dendrimer ligands to telomeric G-quadruplex

Binding between a ligand and a receptor is a fundamental step in many natural or synthetic processes. In biosensing, a tight binding with a small dissociation constant (Kd) between the probe and analyte can lead to superior specificity and sensitivity. Owing to their capability of evaluating competitors, displacement assays have been used to estimate Kd at the ensemble average level. At the more sensitive single-molecule level, displacement assays are yet to be established. Here, we developed a single-molecule displacement assay (smDA) in an optical tweezers instrument and used this innovation to evaluate the binding of the L2H2-6OTD ligands to human telomeric DNA G-quadruplexes. After measuring Kd of linear and dendrimer L2H2-6OTD ligands, we found that dendrimer ligands have enhanced binding affinity to the G-quadruplexes due to their polyvalent geometry. This increased binding affinity enhanced inhibition of telomerase elongation on a telomere template in a Telomerase Repeated Amplification Protocol (TRAP). Our experiments demonstrate that the smDA approach can efficiently evaluate binding processes in chemical and biological processes.

Structured Waters Mediate Small Molecule Binding to G-Quadruplex Nucleic Acids.

The role of G-quadruplexes in human cancers is increasingly well-defined. Accordingly, G-quadruplexes can be suitable drug targets and many small molecules have been identified to date as G-quadruplex binders, some using computer-based design methods and co-crystal structures. The role of bound water molecules in the crystal structures of G-quadruplex-small molecule complexes has been analyzed in this study, focusing on the water arrangements in several G-quadruplex ligand complexes. One is the complex between the tetrasubstituted naphthalene diimide compound MM41 and a human intramolecular telomeric DNA G-quadruplex, and the others are in substituted acridine bimolecular G-quadruplex complexes. Bridging water molecules form most of the hydrogen-bond contacts between ligands and DNA in the parallel G-quadruplex structures examined here. Clusters of structured water molecules play essential roles in mediating between ligand side chain groups/chromophore core and G-quadruplex. These clusters tend to be conserved between complex and native G-quadruplex structures, suggesting that they more generally serve as platforms for ligand binding, and should be taken into account in docking and in silico studies.

QSAR, pharmacophore modeling and molecular docking studies to identify structural alerts for some nitrogen heterocycles as dual inhibitor of telomerase reverse transcriptase and human telomeric G-quadruplex DNA

BackgroundTelomerase reverse transcriptase (TERT) and human telomeric G-quadruplex DNA are amongst the favorable target for researchers to discover novel and more effective anticancer agents. To understand and elucidate structure activity relationship and mechanism of inhibition of telomerase reverse transcriptase (TERT) and human telomeric G-quadruplex DNA, a QSAR modeling and molecular docking were conducted.ResultsTwo robust QSAR model were obtained which consist of full set QSAR model (R2: 0.8174, CCCtr: 0.8995, Q2loo: 0.7881, Q2LMO: 0.7814) and divided set QSAR model (R2: 0.8217, CCCtr: 0.9021, Q2loo: 0.7886, Q2LMO: 0.7783, Q2-F1: 0.7078, Q2-F2: 0.6865, Q2-F3: 0.7346) for envisaging the inhibitory activity of telomerase reverse transcriptase (TERT) and human telomeric G-quadruplex DNA. The analysis reveals that carbon atom exactly at 3 bonds from aromatic carbon atom, nitrogen atom exactly at six bonds from planer nitrogen atom, aromatic carbon atom within 2 A0 from the center of mass of molecule and occurrence of element hydrogen within 2 A0 from donar atom are the key pharmacophoric features important for dual inhibition of TERT and human telomeric G-quadruplex DNA. To validate this analysis, pharmacophore modeling and the molecular docking is performed. Molecular docking analysis support QSAR analysis and revealed that, dual inhibition of TERT and human telomeric DNA is mainly contributed from hydrophobic and hydrogen bonding interactions.ConclusionThe findings of molecular docking, pharmacophore modelling, and QSAR are all consistent and in strong agreement. The validated QSAR analyses can detect structural alerts, pharmacophore modelling can classify a molecule's consensus pharmacophore involving hydrophobic and acceptor regions, whereas docking analysis can reveal the mechanism of dual inhibition of telomerase reverse transcriptase (TERT) and human telomeric G-quadruplex DNA. The combination of QSAR, pharmacophore modeling and molecular docking may be useful for the future drug design of dual inhibitors to combat the devastating issue of resistance.Graphical abstract

Spectroscopic and molecular docking studies for the binding and interaction aspects of curcumin-cysteine conjugate and rosmarinic acid with human telomeric G-quadruplex DNA

The binding and interaction aspects of potential anticancer ligands like: curcumin-cysteine (CC) and rosmarinic acid (RA) with human telomeric G-quadruplex DNA, a novel anticancer target, have been probed by spectroscopic and molecular docking approach. The circular dichroism study unravels the conformational switching from mixed hybrid to parallel structure for the short sequence of human telomeric G-quadruplex structure in the presence of both the ligands. Further a good correlation for binding affinity has been established from the emission and absorption binding spectrum analysis. Further our spectroscopic and molecular docking studies have suggested that the CC having better binding capability than RA to human telomeric G-quadruplex. The presence of L-cysteine moiety in CC ligand is responsible factor for its binding via both minor as well as major groove of human telomeric G-quadruplex DNA where-as RA binds only via minor groove of telomeric G-DNA.

The Folding Landscapes of Human Telomeric RNA and DNA G-Quadruplexes are Markedly Different.

We investigated the folding kinetics of G‐quadruplex (G4) structures by comparing the K+‐induced folding of an RNA G4 derived from the human telomeric repeat‐containing RNA (TERRA25) with a sequence homologous DNA G4 (wtTel25) using CD spectroscopy and real‐time NMR spectroscopy. While DNA G4 folding is biphasic, reveals kinetic partitioning and involves kinetically favoured off‐pathway intermediates, RNA G4 folding is faster and monophasic. The differences in kinetics are correlated to the differences in the folded conformations of RNA vs. DNA G4s, in particular with regard to the conformation around the glycosidic torsion angle χ that uniformly adopts anti conformations for RNA G4s and both, syn and anti conformation for DNA G4s. Modified DNA G4s with 19F bound to C2′ in arabino configuration adopt exclusively anti conformations for χ. These fluoro‐modified DNA (antiTel25) reveal faster folding kinetics and monomorphic conformations similar to RNA G4s, suggesting the correlation between folding kinetics and pathways with differences in χ angle preferences in DNA and RNA, respectively.

Molecular Recognition and Imaging of Human Telomeric G-Quadruplex DNA in Live Cells: A Systematic Advancement of Thiazole Orange Scaffold To Enhance Binding Specificity and Inhibition of Gene Expression.

A series of fluorescent ligands, which were systematically constructed from thiazole orange scaffold, was investigated for their interactions with G-quadruplex structures and antitumor activity. Among the ligands, compound 3 was identified to exhibit excellent specificity toward telomere G4-DNA over other nucleic acids. The affinity of 3-Htg24 was almost 5 times higher than that of double-stranded DNA and promoter G4-DNA. Interaction studies showed that 3 may bind to both G-tetrad and the lateral loop near the 5'-end. The intracellular colocalization with BG4 and competition studies with BRACO19 reveal that 3 may interact with G4-structures. Moreover, 3 reduces the telomere length and downregulates hTERC and hTERT mRNA expression in HeLa cells. The cytotoxicity of 3 against cancer cells (IC50 = 12.7-16.2 μM) was found to be generally higher than noncancer cells (IC50 = 52.3 μM). The findings may support that the ligand is telomere G4-DNA specific and may provide meaningful insights for anticancer drug design.

Highly-sensitive and fast detection of human telomeric G-Quadruplex DNA based on a hemin-conjugated fluorescent metal-organic framework platform

The formation of G-quadruplex (G4) structures in Human telomeric DNA (H-Telo) has been demonstrated to inhibit the activity of telomerase enzyme that is associated with the proliferation of many cancer cells. Accordingly, G-quadruplex structures have become one of the well-established targets in anticancer therapeutic strategies. And, the development of simple and selective detection platforms for G4 structures has become a significant focus of research in recent years. In this study, a simple “off-on” fluorometric method was developed for the selective detection of picomolar quantities of H-Telo G4 DNA based on a fluorescent cerium-based metal organic framework (Ce-MOF) conjugated with hemin to form the sensing probe, Hemin@Ce-MOF. The solvothermal synthesis of the Ce-MOF took advantage of 5-aminoisophtlalic acid (5AIPA) as the organic bridging ligand, (Ce2(5AIPA)3(DMF)2). Characterization of Ce-MOF and Hemin@Ce-MOF was performed by XRD, XPS, TEM, SEM, BET and FTIR techniques. The detection and quantification of the H-Telo was carried out through the adsorption/incorporation of hemin molecules on the pores and surface of Ce-MOF resulting in the fluorescent quenching of the system followed by the restoration of the fluorescence upon addition of H-Telo probably due to a competition between H-Telo and Ce-MOF to bind to hemin. The impact of the key variables including MOF quantity, hemin concentration and detection time was investigated and optimized. Under the optimized conditions, the developed probe provides a limit of detection (LOD) of 665 pM, linear dynamic range (LDR) of 1.6–39.7 nM and excellent selectivity towards H-Telo. Taken together, these results present a simple, novel and superior platform for the selective detection of H-Telo G4 DNA.

Theoretical investigation of conformational deviation of the human parallel telomeric G-quadruplex DNA in the presence of different salt concentrations and temperatures under confinement.

Various experimental reports address the stability of G-quadruplex DNA inside a close confinement such as α-hemolysin, nanocavity water pool and different metal-organic-frameworks (MOFs). To understand the conformational change of G-quadruplex DNA at the atomistic level, we have carried out a total of 40 μs simulation run under both non-polar and polar confinement conditions. To investigate the dynamics, we have considered two different KCl salt concentrations, i.e., 0.47 M (minimal salt concentration) and higher than 2 M (higher salt concentration), at two distinct temperatures, 300 K and 350 K. Here, we have observed that the human telomeric G-quadruplex DNA deviates more from its crystal structure at minimal salt concentration under both non-polar and polar confinement conditions. Besides, the loop regions deviate and fluctuate more compared to the other regions, i.e., sugar-phosphate backbone and tetrad regions. The presence of K+ ions is found to be primarily responsible for this phenomenon. From the spatial density function (SDF) plots, a higher density of K+ ions is observed in the backbone region. Furthermore, from the residue-wise first solvation shell estimation, we have noticed that the K+ ions mainly accumulate in the tetrad region under both non-polar and polar confinement conditions due to which the tetrad regions are more rigid than the loop regions. Higher salt concentration results in increased rigidity of the G-quadruplex DNA. Our study provides valuable insight into the conformational deviation of the G-quadruplex DNA under nanoconfinement conditions.

Tandem Mass Spectrometry Reveals Preferential Ruthenation of Thymines in Human Telomeric G-Quadruplex DNA by an Organometallic Ruthenium Anticancer Complex

The interaction of the organometallic ruthenium(II) anticancer complex [(η6-biphenyl)Ru(en)Cl][PF6] (1; en = ethylenediamine) and a 22-mer human telomeric G-quadruplex (G4) DNA (sequence 5′-A1GGGTT...

Insights into the Kinetic Partitioning Folding Dynamics of the Human Telomeric G-Quadruplex from Molecular Simulations and Machine Learning.

The human telomeric DNA G-quadruplex follows a kinetic partitioning folding mechanism. The underlying folding landscape potentially has many minima separated by high free-energy barriers. However, using current theoretical models to characterize this complex folding landscape has remained a challenging problem. In this study, by developing a hybrid atomistic structure-based model that merges structural information on the hybrid-1, hybrid-2, and chair-type G-quadruplex topologies, we investigated a kinetic partitioning folding process of human telomeric DNA involving three native folds. The model was validated as it reproduced the experimental observation that the hybrid-1 conformation is the major fold and the hybrid-2 conformation is kinetically more accessible. A three-step mechanism was revealed for the formation of the hybrid-1 conformation, while a two-step mechanism was demonstrated for the formation of hybrid-2 and chair-type conformations. Likewise, a class of state in which structures adopted inappropriate combinations of syn/anti guanine nucleotides was found to greatly slow down the folding process. In addition, by employing the XGBoost machine learning algorithm, three interatom distances and six dihedral angles were identified as essential internal coordinates to represent the low-dimensional folding landscape. The strategy of coupling the multibasin model and the machine learning algorithm may be useful to investigate the conformational dynamics of other multistate biomolecules.