G-quadruplex Research Articles

Target-catalyzed self-assembled spherical G-quadruplex/hemin DNAzymes for highly sensitive colorimetric detection of microRNA in serum.

The accurate and visual detection of circulating microRNA (miRNA) has attracted increasing interest due to its pivotal role in clinical disease diagnosis. Taking advantages of nucleic acid isothermal amplification and enzyme-catalyzed chromogenic reaction, here, a colorimetric sensing strategy was proposed for sensitive miRNA analysis. When the target miRNA was present, local catalytic hairpin assembly (CHA) would be triggered and proceed continuously to form dozens of double-stranded oligonucleotides with G-rich sticky ends on the gold nanoparticle, which could self-assemble into a spherical G-quadruplex (GQ)/hemin DNAzyme by binding with hemin and potassium ions. As a horseradish peroxidase-mimic, GQ/hemin DNAzyme could catalyze the redox reaction and color change of the substrates. Taking miRNA-21 as an example, the developed method exhibited satisfactory specificity as well as high sensitivity with a detection limit of 90.3fM. Furthermore, the sensing platform has been successfully employed to detect miRNA-21 in spiked serum, providing a promising tool for early diagnosis of cancers.

Structural Insights into the Antiparallel G-Quadruplex in the Presence of K+ and Mg2+ Ions.

G-Quadruplex (GQ) is a secondary structural unit of DNA, formed at the telomere region of the chromosome with a high guanine content. It is reported that the GQs can hinder many biological processes. Thus, research thrives to explore the structural stability of GQs. Studies based on circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments established the vital role of cations such as K+ and Mg2+ in the stability of antiparallel G-quadruplexes (AGQs). However, there is a need to understand how stability in AGQ is attained in the presence of cations. Here, we employed molecular dynamics (MD) simulations, steered MD (SMD) simulations, and QM/MM calculations to understand the biophysical and electronic bases of the stability imparted to AGQs via cation binding. Our results showed that Mg2+ prefers to bind in the plane with the guanine tetrad, whereas K+ binds in between the AGQ tetrads. Thus, three Mg2+ cations or two K+ ions are needed to stabilize an AGQ molecule, where each and every tetrad binds to Mg2+ more robustly with a higher binding affinity. SMD revealed that the traversal of K+ through the AGQ central channel required less force than that of Mg2+, illustrating the presence of more strong interactions between Mg2+ and AGQ tetrads compared to K+. The stabilization in the AGQ tetrads due to cation binding is reassessed by employing ab initio simulations. Mixed QM/MM calculations confirmed that Mg2+ binds strongly to AGQ compared to K+, and it induces higher interactions between the guanine tetrads. However, K+ binding to AGQ induces a higher stabilization energy than Mg2+ binding to AGQ tetrads. Despite the higher binding energy, Mg2+ binding imparts lower stabilization to AGQ due to its unfavorable fermionic quantum energy.

Dynamics and conformational heterogeneity of the PIM1 G-quadruplex from polarizable molecular dynamics simulations.

Reproductive cancers, such as triple-negative breast cancer (TNBC), are often challenging to treat due to a lack of specific chemotherapeutic targets. Overproduction of PIM1, an anti-apoptotic protein, is common in TNBC. Thus, counteracting this overproduction could serve as a viable therapeutic strategy against TNBC. The promoter region of the PIM1 gene can adopt two G-quadruplexes (GQs) with distinct topologies. GQs are higher-order, noncanonical nucleic acid structures that are rich in guanine and have become intensely explored as potential novel drug targets due to their proposed involvement in transcriptional regulation and other critical cellular processes. Two GQ-duplex hybrid structures have been found to exist in equilibrium in the promoter region of PIM1. The dominant GQ structure, form 1, contains conventional guanine tetrads and a duplex loop region of three Watson-Crick base pairs, while form 2 adopts a fold containing a mixed guanine-cytosine (GCGC) tetrad via slippage of two canonical Watson-Crick base pairs, and also contains a duplex loop consisting of two additional Watson-Crick pairs. We performed unbiased simulations and Gaussian-accelerated molecular dynamics simulations of both GQs with the Drude-2017 polarizable force field to better understand the properties of these GQs and how they might interconvert. Given the role of ion binding and noncanonical base pairing in stabilizing GQ structures, we monitored ion coordination and electronic properties (base dipole moments and electric fields). We observed an increased dipole moment for cytosine bases in the GCGC mixed tetrad of form 2, suggesting electronic plasticity as a factor in structural rearrangement between forms 1 and 2. We also report on conformational sampling in each structure to provide atomistic details that will be essential in the future design of more specific chemotherapeutic agents that target PIM1 on the nucleic acid level.

Determining the confromational ensemble of the HIV-1 LTR G-quadruplexes through gaussian-accelerated molecular dynamics using the drude polarizable force field.

Human immunodeficiency virus (HIV) impacts nearly 37.6 million people globally, but there is no effective cure for the infections it causes. HIV-1 is a retrovirus containing a single-stranded RNA genome. After reverse transcription, the 5’ long terminal repeat (LTR) acts as a promoter for viral replication. Two DNA G-quadruplexes (GQs), known as LTR-III and LTR-IV, form within this promoter and inversely regulate viral gene expression. The LTR-III GQ downregulates viral expression and is a quadruplex: duplex hybrid whereas LTR-IV upregulates viral expression and is a parallel GQ containing a single-nucleotide bulge. Their distinct topologies and functional roles suggest their potential to act as selective drug targets for HIV-1.To gain a greater understanding of structure and dynamics of the LTR-III and LTR-IV GQs, we performed conventional molecular dynamics simulations using the Drude polarizable force field. The loop nucleotides in both structures were highly flexible, sampling a variety of conformational states. The LTR-IV loop also formed base pair interactions that limited ion access to its loop cavity and further highlighted two distinct ion binding sites near the backbone tetrad guanines. Within the LTR-III duplex loop, an additional Watson-Crick base pair formed between Ade4:Thy14 and persisted for 40% of the snapshots collected in the total 4-μs simulation time. Analysis of the electric field exerted on Thy14 indicates a preference for base pairing with Ade4 compared to being unpaired as in the NMR ensemble. Gaussian-accelerated molecular dynamics simulations were also performed to identify high and low energy conformational states for subsequent drug targeting. Our results emphasize the importance of electronic polarization in GQ dynamics and can guide future development of antiviral therapeutics.

Characterization of a G-quadruplex structure at the 3' end of NEAT1_2 and its role in regulating its stability.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that causes deterioration of nerve cells, resulting in loss of muscle control and leads to paralysis. During the early stages of ALS, patients have an increased abundance of paraspeckles, membraneless organelles within nuclei that are comprised of RNA and proteins. The function of these subnuclear bodies is still largely unknown; however, they are created by a scaffold RNA, nuclear enriched abundant transcript 1 (NEAT1), a long noncoding RNA (lncRNA) that also has upregulated expression during ALS onset. Each paraspeckle may contain up to fifty NEAT1 RNAs. NEAT1 exists in two isoforms, NEAT1_1 of ∼3.7 kB and NEAT1_2 that is ∼22.7 kB, created by alternative 3’ splicing. Four G-rich regions common to both isoforms can fold into G-quadruplexes (GQ); however only NEAT1_2 contains a fifth GQ region near its 3’ end. At the 3’ terminus of NEAT1_2 there also exists a triple helix previously shown to affect its stability. Here we use different biophysical methods to analyze the role played by this fifth GQ upon the folding of the triple helix, and thus, the stability of NEAT1_2. Understanding the secondary structure of NEAT1 will help in expanding our breadth of knowledge surrounding neurodegenerative diseases like ALS by targeting the GQ and triple helix to disrupt their formation to alter its level of expression and its interaction with proteins necessary for paraspeckles.

A G-quadruplex structure within the 3'-untranslated region of hnRNP K affects its translation by controlling the accessibility of miR-1249-3p to its binding site.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in motor neuron loss in the brain and spinal cord. Mutations of the DNA/RNA binding proteins Tar-DNA binding protein 43 (TDP-43) and fused in sarcoma/translocated in liposarcoma protein (FUS/TLS) are causative of ALS, and these two RNA-binding proteins are the major protein components of the pathological inclusions observed in over 90% of ALS cases. Moreover, a hexanucleotide GGGGCC (G4C2) expansion in the C9ORF72 gene is a common genetic cause of ALS, being found in at least 8% of sporadic and more than 40% of familial ALS. Thus, it is proposed that alterations to RNA metabolism play a pathogenic role in ALS. This hypothesis is supported by a genome-wide study of microRNAs expression in serum from ALS patients that revealed a significant deregulation of miR-142-3p and miR-1249-3p. Interestingly, miR-1249-3p has been shown to regulate the translation of the heterogenous nuclear protein K (hnRNP K), a DNA/RNA binding protein implicated in transcription and translation regulation, as well as in several diseases, including multiple cancers. In ALS, hnRNP K has been shown to associate with TDP-43 and to bind to the RNA C9ORF72 (G4C2) repeat expansion. In this study we identified and characterized by biophysical methods a G-quadruplex (GQ) structure in the 3’-untranslated region of hnRNP K mRNA, which encompasses the miR-1249-3p binding site. We show that this GQ is recognized by the Fragile X Messenger Ribonucleoprotein 1 (FMRP), suggesting a possible mechanism by which the GQ structure affects miR-1249-3p to its binding site accessibility, therefore, regulating the levels the hnRNP K protein.

Folylpolyglutamate synthetase mRNA G-quadruplexes regulate its cell protrusion localization and enhance a cancer cell invasive phenotype upon folate repletion

BackgroundFolates are crucial for the biosynthesis of nucleotides and amino acids, essential for cell proliferation and development. Folate deficiency induces DNA damage, developmental defects, and tumorigenicity. The obligatory enzyme folylpolyglutamate synthetase (FPGS) mediates intracellular folate retention via cytosolic and mitochondrial folate polyglutamylation. Our previous paper demonstrated the association of the cytosolic FPGS (cFPGS) with the cytoskeleton and various cell protrusion proteins. Based on these recent findings, the aim of the current study was to investigate the potential role of cFPGS at cell protrusions.ResultsHere we uncovered a central role for two G-quadruplex (GQ) motifs in the 3′UTR of FPGS mediating the localization of cFPGS mRNA and protein at cell protrusions. Using the MBSV6-loop reporter system and fluorescence microscopy, we demonstrate that following folate deprivation, cFPGS mRNA is retained in the endoplasmic reticulum, whereas upon 15 min of folate repletion, this mRNA is rapidly translocated to cell protrusions in a 3′UTR- and actin-dependent manner. The actin dependency of this folate-induced mRNA translocation is shown by treatment with Latrunculin B and inhibitors of the Ras homolog family member A (RhoA) pathway. Upon folate repletion, the FPGS 3′UTR GQs induce an amoeboid/mesenchymal hybrid cell phenotype during migration and invasion through a collagen gel matrix. Targeted disruption of the 3′UTR GQ motifs by introducing point mutations or masking them by antisense oligonucleotides abrogated cell protrusion targeting of cFPGS mRNA.ConclusionsCollectively, the GQ motifs within the 3′UTR of FPGS regulate its transcript and protein localization at cell protrusions in response to a folate cue, inducing cancer cell invasive phenotype. These novel findings suggest that the 3′UTR GQ motifs of FPGS constitute an attractive druggable target aimed at inhibition of cancer invasion and metastasis.

Oxidative Probing of the G4 DNA Structure by Znp1 Porphyrin within Sequences of MYC and TERT Promotors

The formation of G4 structures in a DNA double helix competes with the complementary strand interaction. The local environment in DNA can change equilibrium of G4 structures, which are studied on single-stranded (ss) models by classical structural methods. A relevant task is to develop methods for detecting and localizing G4 structures in extended native double-stranded (ds) DNA in the promoter regions of the genome. The ZnP1 porphyrin derivative selectively binds to G4 structures and leads to photo-induced oxidation of guanine in ssDNA and dsDNA model systems. We have shown the oxidative effect of ZnP1 on native sequences of MYC and TERT oncogene promoters, which can form G4 structures. Single-strand breaks in the guanine-rich sequence because of ZnP1 oxidation and subsequent cleavage of the DNA strand with Fpg glycosylase have been identified and assigned to the nucleotide sequence. The detected break sites have been shown to correspond to sequences capable of forming G4 structures. Thus, we have demonstrated the possibility of using porphyrin ZnP1 for the identification and localization of G4 quadruplexes in extended regions of the genome. Here we have shown the novel data on a possibility of folding G4 structures in the presence of complementary strand in native DNA double helix.

Identification and characterization of a flexile G-quadruplex in the distal promoter region of stemness gene REX1

We have identified a parallel G-quadruplex (R1WT) in the distal promoter region (−821 base-pairs upstream of the TSS) of the pluripotent gene REX1. Through biophysical and biochemical approach, we have characterized the G-quadruplex (GQ) as a potential molecular switch that may control REX1 promoter activity to determine the transcriptional fate. Small- molecule interactive study of the monomeric form of R1WT (characterized as R1mut2) with TMPyP4 and BRACO-19 revealed GQ destabilization upon interaction with TMPyP4 and stabilization upon interaction with BRACO-19. This distinctive drug interactivity suggests the in cellulo R1WT to be a promising drug target. The endogenous existence of R1WT was confirmed by BG4 antibody derived chromatin immunoprecipitation experiment. Here in, we also report the endogenous interaction of GQ specific transcription factors (TFs) with R1WT region in the human chromatin of cancer cell. The wild-type G-quadruplex was found to interact with four important transcription factors, (i) specificity protein (Sp1) (ii) non-metastatic cell 2 (NM23-H2): a diphosphatase (iii) cellular nucleic acid binding protein (CNBP) and (iv) heterogenous nuclear ribonucleoprotein K (hnRNPK) in the REX1 promoter. In contrast, nucleolin protein (NCL) binding was found to be low to the said G-quadruplex. The flexibility of R1WT between folded and unfolded states, obtained from experimental and computational analysis strongly suggests R1WT to be an important gene regulatory element in the genome. It controls promoter DNA relaxation with the coordinated interaction of transcription factors, the deregulation of which seeds stemness characteristic in cancer cells for further metastatic progression.

Unlocking the power of G-quadruplex for anti-HIV1 development

The role of G-quadruplexes (GQP) in the life cycle of other viruses has led to the speculation that they may also have a function in the HIV genome. This paper aims to write-up on the potential of G-quadruplex as a potential anti-viral development. This paper narrates on (1) innovation of G-quadruplex detector, (2) location of G-quadruplex in HIV genome, (3) evidence and therapeutic approach using GQP as anti-HIV, and (4) contradiction on G-quadruplex utilization. The results of this investigation offer a quick insight on the use of G-quadruplex as a target and highlighting the potential of such intervention for antiviral treatments.

G-quadruplex DNA selective targeting for anticancer therapy: a computational study of a novel PtII monofunctional complex activated by adaptive binding.

Targeting of G-quadruplex (G-Q) nucleic acids, which are helical four-stranded structures formed from guanine-rich nucleic acid sequences, has emerged in recent years as an appealing opportunity for drug intervention in anticancer therapy. Small-molecule drugs can stabilize quadruplex structures, promoting selective downregulation of gene expression and telomerase inhibition and also activating DNA damage responses. Thus, rational design of small molecular ligands able to selectively interact with and stabilize G-Q structures is a promising strategy for developing potent anti-cancer drugs with selective toxicity towards cancer cells over normal ones. Here, the outcomes of a thorough computational investigation of a recently synthesized monofunctional PtII complex (Pt1), whose selectivity for G-Q is activated by what is called adaptive binding, are reported. Quantum mechanics and molecular dynamics calculations have been employed for studying the classical key steps of the mechanism of action of PtII complexes, the conversion of the non-charged and non-planar Pt1 complex into a planar and charged PtII (Pt2) complex able to play the role of a G-Q binder and, finally, the interaction of Pt2 with G-Q. The information obtained from such an investigation allows us to rationalize the behavior of the novel PtII complex proposed to be activated by adaptive binding toward selective interaction with G-Q or similar molecules and can be exploited for designing ligands with more effective recognition ability toward G-quadruplex DNA.

Association of Augmented Immune-Staining of G-Quadruplex Tertiary DNA Structure in Chemo-Tolerant TNBC with Downregulation of WNT/Epidermal Growth Factor Receptor Pathway receptor Genes: A Pilot Clinicopathological Study

Purpose: The aim of the study is to understand the involvement of G-Quadruplex (G-Q) structures in altering the expression profile of WNT/epidermal growth factor receptor (EGFR) pathway receptor genes in chemo-tolerant Triple Negative Breast Cancer (TNBC) samples. Materials and Methods: At first, Gene Expression Omnibus datasets were mined where the expression profile of WNT/EGFR pathway genes in TNBC samples and MDA-MB-231, a TNBC cell line, were checked in response to doxorubicin, a chemotherapeutic drug. Next, to unveil the probable mechanism of regulation, the presence of G-Q structure was checked in in silico study and later validated by immunohistochemical analyses in our pool of sample. These observed results were correlated with patient's demography and survival status. Results: Expression of the receptors (FZD7, LRP6, EGFR) of the WNT/EGFR pathway were found to be differentially expressed in TNBC samples; further emphasized in our samples (n = 61). Notably, these G-Q structures were found in the promoter region of the WNT pathway receptor genes (FZD7, LRP6, and EGFR). Validating in our patient sample pool, a significant increase in G-Q immunostaining was observed in samples, after neoadjuvant chemotherapy (NACT) samples (n = 17) than the pretherapeutic samples (n = 44). Similar pattern of G-Q immunostaining was noticed in doxorubicin-treated MDA-MB-231 cell line. Intriguingly, low staining of G-Q among the pretherapeutic samples, but NACT TNBC samples, was found to be significantly correlated with lymph node metastasis. Conclusions: This study showed that the augmented immunostaining of G-Q structure might have an important involvement in regulating the expression pattern of the WNT/EGFR pathway genes in response to doxorubicin treatment of TNBC.

Optimization of an aptamer against Prorocentrum minimum – A common harmful algae by truncation and G-quadruplex-forming mutation

Harmful algal blooms (HABs) caused by Prorocentrum minimum have seriously posed economic losses and ecological disasters. To reduce these losses, aptamers are used as a new molecular probe to establish rapid methods. Herein, to improve the affinity and application of aptamers in the detection of harmful algae, the optimization was performed on the previously reported aptamers against P. minimum. First, a total of seven candidate aptamers, including three truncated aptamers (TA1, TA2 and TA3) and four mutant aptamers (MA1, MA2, MA3 and MA4), were obtained by truncation and G-quadruplex (GQ)-forming mutation. Next, the specificity and affinity test by flow cytometry revealed that except for TA1 and TA2, all of the candidate aptamers are specific with the equilibrium dissociation constant of (40.4 ± 5.5) nM for TA3, (63.3 ± 24.0) nM for MA1, (71.7 ± 14.6) nM for MA2, (365.9 ± 74.4) nM for MA3, and (21.1 ± 0.5) nM for MA4, respectively. The circular dichroism analysis of the mutant aptamers demonstrated that the GQ structures formed by MA1/MA2, MA3 and MA4 were antiparallel, mixed parallel and parallel, respectively. The affinity of aptamers with various GQ is in the order of parallel structure > antiparallel structure > mixed parallel structure. In addition, to further improve binding ability, the binding conditions of MA4 were optimized as follows: binding time, 60 min; binding temperature, 37 °C; pH of the binding buffer, 7.5; and Na+/Mg2+ concentration in the binding buffer, 100 mM/0.5 mM. The binding examination by fluorescence microscopy showed that MA4 had a stronger binding ability to P. minimum than the original aptamer. Taken together, this study not only obtained an aptamer with higher affinity than the original aptamer, which laid a good foundation for subsequent application, but also may provide a feasible reference method for aptamer optimization.

Bifunctional G-Quadruplex Aptamer Targeting Nucleolin and Topoisomerase 1: Antiproliferative Activity and Synergistic Effect of Conjugated Drugs.

As a counterpart to antibody-drug conjugates (ADCs), aptamer-drug conjugates (ApDCs) have been considered a promising strategy for targeted therapy due to the various benefits of aptamers. However, an aptamer merely serves as a targeting ligand in ApDCs, whereas the antibody enables the unexpected therapeutic efficacy of ADCs through antibody-dependent cellular cytotoxicity (ADCC). In this study, we developed a tumor-specific aptamer with an effector function and used it to confirm the feasibility of more potent ApDCs. First, we designed a nucleolin (NCL)-binding G-quadruplex (GQ) library based on the ability of NCL to bind to telomeric sequences. We then identified a bifunctional GQ aptamer (BGA) inhibiting the catalytic activity of topoisomerase 1 (TOP1) by forming an irreversible cleavage complex. Our BGA specifically targeted NCL-positive MCF-7 cells, exhibiting antiproliferative activity, and this suggested that tumor-specific therapeutic aptamers can be developed by using a biased library to screen aptamer candidates for functional targets. Finally, we utilized DM1, which has a synergistic interaction with TOP1 inhibitors, as a conjugated drug. BGA-DM1 exerted an anticancer effect 20-fold stronger than free DM1 and even 10-fold stronger than AS1411 (NCL aptamer)-DM1, highlighting our approach to develop synergistic ApDCs. Therefore, we anticipate that our library might be utilized for the identification of aptamers with effector functions. Furthermore, by employing such aptamers and appropriate drugs, synergistic ApDCs can be developed for targeted cancer therapy in a manner distinct from how ADCs exhibit additional therapeutic efficacy.

Homopurine guanine-rich sequences in complex with N-methyl mesoporphyrin IX form parallel G-quadruplex dimers and display a unique symmetry tetrad

DNA can fold into G-quadruplexes (GQs), non-canonical secondary structures formed by π-π stacking of G-tetrads. GQs are important in many biological processes, which makes them promising therapeutic targets. We identified a 42-nucleotide long, purine-only G-rich sequence from human genome, which contains eight G-stretches connected by A and AAAA loops. We divided this sequence into five unique segments, four guanine stretches each, named GA1-5. In order to investigate the role of adenines in GQ structure formation, we performed biophysical and X-ray crystallographic studies of GA1-5 and their complexes with a highly selective GQ ligand, N-methyl mesoporphyrin IX (NMM). Our data indicate that all variants form parallel GQs whose stability depends on the number of flexible AAAA loops. GA1-3 bind NMM with 1:1 stoichiometry. The Ka for GA1 and GA3 is modest, ∼0.3 μM −1, and that for GA2 is significantly higher, ∼1.2 μM −1. NMM stabilizes GA1-3 by 14.6, 13.1, and 7.0 °C, respectively, at 2 equivalents. We determined X-ray crystal structures of GA1-NMM (1.98 Å resolution) and GA3-NMM (2.01 Å). The structures confirm the parallel topology of GQs with all adenines forming loops and display NMM binding at the 3′ G-tetrad. Both complexes dimerize through the 5′ interface. We observe two novel structural features: 1) a ‘symmetry tetrad’ at the dimer interface, which is formed by two guanines from each GQ monomer and 2) a NMM dimer in GA1-NMM. Our structural work confirms great flexibility of adenines as structural elements in GQ formation and contributes greatly to our understanding of the structural diversity of GQs and their modes of interaction with small molecule ligands.

Shedding light on the base-pair opening dynamics of nucleic acids in living human cells

Base-pair opening is a fundamental property of nucleic acids that plays important roles in biological functions. However, studying the base-pair opening dynamics inside living cells has remained challenging. Here, to determine the base-pair opening kinetics inside living human cells, the exchange rate constant ({k}_{{{{{{rm{ex}}}}}}}) of the imino proton with the proton of solvent water involved in hairpin and G-quadruplex (GQ) structures is determined by the in-cell NMR technique. It is deduced on determination of {k}_{{{{{{rm{ex}}}}}}} values that at least some G-C base pairs of the hairpin structure and all G-G base-pairs of the GQ structure open more frequently in living human cells than in vitro. It is suggested that interactions with endogenous proteins could be responsible for the increase in frequency of base-pair opening. Our studies demonstrate a difference in dynamics of nucleic acids between in-cell and in vitro conditions.

G-quadruplexes in the monkeypox virus are potential antiviral targets.

Monkeypox virus (MPXV) is a member of Orthopoxvirus in the Poxviridae family, causing a Public Health Emergency of International Concern. The number of cases and geographic range has increased significantly in 2022. Identification of MPXV-specific therapeutic targets is urgent. G-quadruplex (GQ) secondary structures attract great attention as potential targets for antiviral strategy. Whether GQs are present in theMPXV genome remains inconclusive. In this study, we aim to characterize the GQs encoded by MPXV. Through a series of biophysical experiments, we characterized the formation potential of MPXV-encoded GQs and evaluated the binding and stabilization abilities of GQ ligands including BRACO-19, pyridostatin, and TMPyP4 to GQs encoded by MPXV. Moreover, GQ ligands suppressed the gene transcription of MPXV sequences containing GQ. BRACO-19 and TMPyP4 were able to inhibit vaccinia virus replication. We demonstrated the existence of MPXV GQ and reinforced the idea that GQs could be novel antiviral targets. Targeting these GQ sequences with GQ-binding molecules may represent a new approach for MPXV therapy.

A Spectroscopic Approach to Unravel the Local Conformations of a G-Quadruplex Using CD-Active Fluorescent Base Analogues.

The formation of a stable G-quadruplex (GQ) can inhibit the increased telomerase activity that is common in most cancers. The global structure and the thermal stability of the GQs are usually evaluated by spectroscopic methods and thermal denaturation properties. However, most biochemical processes involving GQs might require local conformational changes at the guanine tetrad (G4) level. These local conformational changes of individual G4 layers during protein and drug interactions have not yet been explored in detail. In this study, we monitored the local conformations of individual G4 layers in GQs using 6-methylisoxanthopterine (6MI) chromophores, which are circular dichroism (CD)-active fluorescent base analogues of guanine, as local conformational probes. A synthetic, tetramolecular, parallel GQ with site-specifically positioned 6MI monomers or dimers was used as the experimental construct. Analytical ultracentrifugation studies and gel electrophoretic studies showed that properly positioned 6MI monomers and dimers could form stable GQs with CD-active fluorescent G4 layers. The local conformation of individual fluorescent G4 layers in the GQ structure was then tracked by monitoring the absorbance, fluorescence intensity, thermal melting, fluorescence quenching, and CD changes of the incorporated probes. Overall, these studies showed that site-specifically incorporated fluorescent base analogues could be used as probes to monitor the local conformational changes of individual G4 layers of a GQ structure. This method can be applied to explore the details of small molecule-GQ interaction at the level of the individual G4 layers, which may prove to be useful in designing drugs to treat GQ-related genetic disorders, cancer, and aging.

Small Heterocyclic Ligands as Anticancer Agents: QSAR with a Model G-Quadruplex.

G-quadruplexes (GQs) have become valid targets for anticancer studies in recent decades due to their multifaceted biological function. Herewith, we aim to quantify interactions of potential heterocyclic ligands (Ls) with model GQs. For seven 4-aminoquinazolines and three 2-heteroaryl perimidines, seven of this ten-membered group so far unknown, we use routine quantum chemical modeling. As shown in the literature, a preferred mode of interaction of heterocycles with cellular structures is stacking to exposable faces of G-quadruplexes. To exploit the energy of this interaction as a molecular descriptor and achieve the necessary chemical precision, we use state of the art large-scale density functional theory (DFT) calculations of stacked heterocycles to a GQ. Actually, the GQ has been simplified for the computation by stripping it off all pentose phosphate residues into a naked model of stacked guanine quartets. The described model thus becomes computable. The obtained heterocyclic ligand GQ.L stacking energies, that is, their GQ affinities, are the necessary ligand descriptors. Using the ligand biological inhibitory activities (IC50) on a human malignant melanoma A375 cell line, we obtain a good linear relationship between computed ligand stacking affinities to GQ, and experimental log (IC50) values. Based on the latter relationship, we discuss a putative mechanism of anticancer activity of heterocyclic ligands via stacking interactions with GQs and thereby controlling cell regulatory activity. This mechanism may tentatively be applied to other condensed five- and six-membered small heterocycles as well.

Zeptoliter DNA Origami Reactor to Reveal Cosolute Effects on Nanoconfined G-Quadruplexes.

Cellular environments such as nanoconfinement and molecular crowding can change biomolecular properties. However, in nanoconfinement, it is extremely challenging to investigate effects of crowding cosolutes on macromolecules. By using optical tweezers, here, we elucidated the effects of hexaethylene glycol (HEG) on the mechanical stability of a telomeric G-quadruplex (GQ) in a zeptoliter DNA origami reactor (zepto-reactor). When HEG molecules were introduced in the GQ-containing zepto-reactor at different positions, we found that the GQ species split into two equilibrated populations, reflecting diverse effects of the oligoethylene glycol on the GQ via either a long-range dehydration effect or direct interactions. When the number of HEG molecules was increased, the stability of the GQ unexpectedly decreased, suggesting that the direct destabilizing interaction between the GQ and HEG is dominating over the long-range stabilizing dehydration effects of the HEG in hydrophilic nanocavities. These findings indicate that a nanoconfined environment can alter regular effects of cosolutes on biomacromolecules.