Four-stranded Structures Research Articles

Interaction of berberine with different forms of DNA in human telomeric region.

Guanine-rich oligonucleotide sequences have the potential to form four-stranded structure known as G-quadruplex. These structures are frequently observed in crucial regions of the human genome, including promoter and telomeric regions. Due to their involvement in regulating gene expression and cell division, G-quadruplexes have emerged as promising targets for anticancer drugs. This study investigated interaction of berberine with three distinct forms of DNA within human telomeric region. The results of absorption and fluorescence spectroscopy indicated that conformation of DNA plays an important role in the mode of binding. Circular dichroism suggested that berberine promotes compaction of the unstable quadruplex structure formed under non-saline conditions. Furthermore, interaction of berberine with the stable structures of G-quadruplex resulted in a change in their compactness without altering the type of DNA structure. 3D fluorescence spectra analysis by chemometrics methods showed formation of two distinct species probably attributed to the self-association and specific binding of berberin to the different forms of DNA. It can be also concluded that berberine forms a more stable complex with the human telomeric hybride type G-quadruplex structure compared with the basket type. In conclusion, the findings imply that the successful design of drugs targeting DNA within the human telomere region necessitates careful consideration of the diverse forms of DNA.

Coding relationship links RNA G-quadruplexes and protein RGG motifs in RNA-binding protein autoregulation

RNA G-quadruplexes (rG4s), the four-stranded structures formed by guanine-rich RNA sequences, are recognized by regions in RNA-binding proteins (RBPs) that are enriched in arginine-glycine repeats (RGG motifs). Importantly, arginine and glycine are encoded by guanine-rich codons, suggesting that some RGG motifs may both be encoded by and interact with rG4s in autogenous messenger RNAs (mRNAs). By analyzing transcriptome-wide rG4 datasets, we show that hundreds of RGG motifs in humans are at least partly encoded by rG4s, with an increased incidence for longer RGG motifs (~10 or more residues). Using randomized genetic codes, we demonstrate that the rG4/RGG coding relationship derives from the universal genetic code's structure. Moreover, we show that proteins, which contain RGG motifs encoded by experimentally detected rG4s, are significantly enriched in RNA binding relative to all RGG-containing proteins. Finally, using enhanced crosslinking and immunoprecipitation (eCLIP) data, we identify several prominent RBPs, including FUS, FMRP, and G3BP1, which interact with autogenous mRNAs in regions where RGG motifs are encoded by rG4s. Our results define a physically realistic mechanism behind autogenous mRNA/protein interactions that is hardwired in the genetic code structure and may contribute to the establishment of autoregulatory feedback loops in the cell.

Quencher-Free Fluorescence Monitoring of G-Quadruplex Folding.

Guanine-rich sequences exhibit a high degree of polymorphism and can form single-stranded, Watson-Crick duplex, and four-stranded G-quadruplex structures. These sequences have found a wide range of uses in synthetic biology applications, arising in part from their structural plasticity. High-throughput, low-cost tools for monitoring the folding and unfolding transitions of G-rich sequences would provide an enabling technology for accelerating the prototyping of synthetic biological systems and for accelerating design-build-test cycles. Here, we show that unfolding transitions of a range of G-quadruplex-forming DNA sequences can be monitored in a FRET-like format using DNA sequences that possess only a single dye label, with no quencher. These quencher-free assays can be performed at low cost, with both cost and lead times ca. 1 order of magnitude lower than FRET-labeled strands. Thus, quencher-free secondary structure monitoring promises to be a valuable tool for the testing and development of synthetic biology systems employing G-quadruplexes.

Efficient and Rapid Enrichment of Extracellular Vesicles Using DNA Nanotechnology-Enabled Synthetic Nano-Glue.

Swift and efficient enrichment and isolation of extracellular vesicles (EVs) are crucial for enhancing precise disease diagnostics and therapeutic strategies, as well as elucidating the complex biological roles of EVs. Conventional methods of isolating EVs are often marred by lengthy and laborious processes. In this study, we introduce an innovative approach to enrich and isolate EVs by leveraging the capabilities of DNA nanotechnology. We have developed a novel multivalent cholesterol-modified paranemic crossover DNA (PX-DNA-chol) construct, which is a four-stranded DNA structure containing adjacent double helices intertwined with their local helix axes parallel and serves as an effective synthetic nano-glue. This construct promotes the rapid coalescence of nanoscale EVs into clusters of micrometer scale, thereby streamlining their enrichment. Utilizing a conventional low-speed centrifuge, this intriguing methodology achieves a rapid concentration of EVs within minutes, bypassing the laborious and high-speed centrifugation steps typically required. The quality of EVs isolated by our technique is comparable to that obtained through ultracentrifugation methods. Given these advancements, our PX-DNA-chol-facilitated EVs enrichment protocol is poised to advance the field of EVs research, providing a robust and accessible tool for in-depth studies of EVs.

Loss of DHX36/G4R1, a G4 resolvase, drives genome instability and regulates innate immune gene expression in cancer cells.

G-quadruplexes (G4s) are four-stranded alternative secondary structures formed by guanine-rich nucleic acids and are prevalent across the human genome. G4s are enzymatically resolved using specialized helicases. Previous in vitro studies showed that DEAH-box Helicase 36 (DHX36/G4R1/RHAU), has the highest specificity and affinity for G4 structures. Here, by mapping genome-wide DNA double-strand breaks (DSBs), we demonstrate that knockout (KO) of DHX36 helicase increases DSB enrichment at G4 sites and that the presence of the G4 motif is a significant mediator of genome instability at regulatory regions. The loss of DHX36 corresponds with the significant upregulation of NF-κB transcriptional programs, culminating in the production and secretion of proinflammatory cytokines. Loss of DHX36 expression results in an increase in the innate immune signaling stimulator of interferon response cGAMP interactor 1 ( STING1 ) expression and activation of genes involved in immune response pathways. Importantly, higher levels of DHX36 mRNA expression in human B-cell acute lymphoblastic leukemia correlate with improved overall survival relative to lower expression of DHX36 , highlighting its critical role in preserving genome integrity at a cellular level and in the context of cancer.

Molecular Modelling and Investigation of Bioactive Stable DNA G-Quadruplex d(G4T4G4) under Different Physiological Conditions using Molecular Dynamics Simulation

We are reporting a molecular dynamics study on the structure and conformation of the DNA quadruplex. The single molecule quadruplex's coordinates were obtained and modeled using PDB ID 1d59. The simulation's sequence is d(GGGGTTTTGGGG). In the beginning, two hairpin structures were constructed with loops holding thymine residues at either end, forming a four-stranded helical structure. The cyclic hydrogen bonds created by the structure kept the guanine residues of all four strands in one plane. The molecule was subjected to molecular dynamics simulation for 100ns and at periodic intervals of 10ns, the dynamical pathway's trajectory was examined. The deviations and fluctuations viz. RMSD, RMSF and Rg plots were analyzed, the structure and shape of the time-evolved trajectory were examined. The outcome was compared with the crystal structure. Our findings reveal a few peculiar properties which we have discussed in this paper. The helix axis and torsion angle parameters were calculated. The results of this interpretation give the idea about quadruplex interaction with external agents and inspire to do further research on DNA quadruplex structures in telomeric regions of living chromosomes.

Kanamycin and G-Quadruplexes: An Exploration of Binding Interactions.

G-quadruplexes (G4s) are distinctive four-stranded nucleic acid structures formed by guanine-rich sequences, making them attractive targets for drug repurposing efforts. Modulating their stability and function holds promise for treating diseases like cancer. To identify potential drug candidates capable of interacting with these complex DNA formations, docking studies and molecular dynamics (MDs) simulations were conducted. Our analysis revealed kanamycin's ability to bind to various G4 structures, offering valuable insights into its potential as a modulator of G4 activity. Kanamycin exhibited favorable interactions with both parallel and hybrid G4 topologies in human structures, suggesting a broader mechanism of action for aminoglycosides. These findings may also shed light on aminoglycoside-associated toxicities, indicating that their effects might extend to binding non-ribosomal RNA structures. In summary, this research highlights kanamycin's potential as a promising tool for influencing G4 dynamics, paving the way for innovative therapeutic strategies targeting G4-related pathways.

Antiparallel G-Quadruplex Formation Hinders Conversion to a Parallel Topology.

G-quadruplexes (G4s) are four-stranded structures formed by guanine-rich sequences. While their structures, properties, and applications have been extensively studied, an understanding of their folding processes remains limited. In this study, we investigated the folding of the sequence d[(G3T2)3G3] in potassium solutions, focusing on the impact of a folding intermediate on the overall folding process. Our results indicate that this sequence eventually folds into a parallel G4 structure, either directly or through an antiparallel conformation intermediate, suggesting the existence of a specific competitive folding process. Detailed kinetic analysis using stopped-flow techniques reveals that the antiparallel conformation forms much faster than the parallel one. This antiparallel G4 slowly converts to the thermodynamically favored parallel topology, thus slowing the overall folding rate. As a result, the formation of the parallel quadruplex via an antiparallel G4 intermediate is slower than the direct process, indicating that this antiparallel conformation negatively impacts the overall folding process in a temperature-dependent manner. Interestingly, sodium was shown to facilitate the conversion from antiparallel to parallel. These analyses highlight the complexity of the G4 folding process, which is crucial for most biological applications.

Small molecule-based regulation of gene expression in human astrocytes switching on and off the G-quadruplex control systems

A great deal of attention is being paid to strategies seeking to uncover the biology of the four-stranded nucleic acid structure G-quadruplex (G4) via their stabilization in cells with G4-specific ligands. The conventional definition of chemical biology implies that a complete assessment of G4 biology can only be achieved by implementing a complementary approach involving destabilization of cellular G4s by ad hoc molecular effectors. We report here on an unprecedented comparison of the cellular consequences of G4 chemical stabilization by pyridostatin (PDS) and destabilization by phenylpyrrolocytosine (PhpC) at both transcriptome- and proteome-wide scales in patient-derived primary human astrocytes. Our results show that the stabilization of G4s by PDS triggers the dysregulation of many cellular circuitries, the most drastic effects originating in downregulation of 354 transcripts and 158 proteins primarily involved in RNA transactions. In contrast, destabilization of G4s by PhpC modulates the G4 landscapes in a far more focused manner with upregulation of 295 proteins, mostly involved in RNA transactions as well, thus mirroring the effects of PDS. Our study is the first of its kind to report the extent of G4-associated cellular circuitries in human cells by systematically pitting the effect of G4 stabilization against destabilization in a direct and unbiased manner.

Machine learning-based prediction of DNA G-quadruplex folding topology with G4ShapePredictor

Deoxyribonucleic acid (DNA) is able to form non-canonical four-stranded helical structures with diverse folding patterns known as G-quadruplexes (G4s). G4 topologies are classified based on their relative strand orientation following the 5’ to 3’ phosphate backbone polarity. Broadly, G4 topologies are either parallel (4+0), antiparallel (2+2), or hybrid (3+1). G4s play crucial roles in biological processes such as DNA repair, DNA replication, transcription and have thus emerged as biological targets in drug design. While computational models have been developed to predict G4 formation, there is currently no existing model capable of predicting G4 folding topology based on its nucleic acid sequence. Therefore, we introduce G4ShapePredictor (G4SP), an application featuring a collection of multi-classification machine learning models that are trained on a custom G4 dataset combining entries from existing literature and in-house circular dichroism experiments. G4ShapePredictor is designed to accurately predict G4 folding topologies in potassium (K+\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\hbox {K}^+$$\\end{document}) buffer based on its primary sequence and is able to incorporate a threshold optimization strategy allowing users to maximise precision. Furthermore, we have identified three topological sequence motifs that suggest specific G4 folding topologies of (4+0), (2+2) or (3+1) when utilising the decision-making mechanisms of G4ShapePredictor.

G-quadruplex DNA and RNA in cellular senescence.

Normal cells divide, are damaged, and are repaired across their lifetime. As cells age, they enter cellular senescence, characterized by a permanent state of cell-cycle arrest triggered by various stressors. The molecular mechanisms that regulate senescent phenotypes have been actively investigated over the last several decades; however, one area that has been neglected is how G-quadruplex (G4) DNA and RNA (G4-DNA and G4-RNA) mediate senescence. These non-canonical four-stranded DNA and RNA structures regulate most normative DNA and RNA-dependent processes, such as transcription, replication, and translation, as well as pathogenic mechanisms, including genomic instability and abnormal stress granule function. This review also highlights the contribution of G4s to sex differences in age-associated diseases and emphasizes potential translational approaches to target senescence and anti-aging mechanisms through G4 manipulation.

Insights into the Molecular Structure, Stability, and Biological Significance of Non-Canonical DNA Forms, with a Focus on G-Quadruplexes and i-Motifs

This article provides a comprehensive examination of non-canonical DNA structures, particularly focusing on G-quadruplexes (G4s) and i-motifs. G-quadruplexes, four-stranded structures formed by guanine-rich sequences, are stabilized by Hoogsteen hydrogen bonds and monovalent cations like potassium. These structures exhibit diverse topologies and are implicated in critical genomic regions such as telomeres and promoter regions of oncogenes, playing significant roles in gene expression regulation, genome stability, and cellular aging. I-motifs, formed by cytosine-rich sequences under acidic conditions and stabilized by hemiprotonated cytosine–cytosine (C:C+) base pairs, also contribute to gene regulation despite being less prevalent than G4s. This review highlights the factors influencing the stability and dynamics of these structures, including sequence composition, ionic conditions, and environmental pH. Molecular dynamics simulations and high-resolution structural techniques have been pivotal in advancing our understanding of their folding and unfolding mechanisms. Additionally, the article discusses the therapeutic potential of small molecules designed to selectively bind and stabilize G4s and i-motifs, with promising implications for cancer treatment. Furthermore, the structural properties of these DNA forms are explored for applications in nanotechnology and molecular devices. Despite significant progress, challenges remain in observing these structures in vivo and fully elucidating their biological functions. The review underscores the importance of continued research to uncover new insights into the genomic roles of G4s and i-motifs and their potential applications in medicine and technology. This ongoing research promises exciting developments in both basic science and applied fields, emphasizing the relevance and future prospects of these intriguing DNA structures.

Strategy for modeling higher-order G-quadruplex structures recalcitrant to NMR determination

Guanine-rich nucleic acids can form intramolecularly folded four-stranded structures known as G-quadruplexes (G4s). Traditionally, G4 research has focused on short, highly modified DNA or RNA sequences that form well-defined homogeneous compact structures. However, the existence of longer sequences with multiple G4 repeats, from proto-oncogene promoters to telomeres, suggests the potential for more complex higher-order structures with multiple G4 units that might offer selective drug-targeting sites for therapeutic development. These larger structures present significant challenges for structural characterization by traditional high-resolution methods like multi-dimensional NMR and X-ray crystallography due to their molecular complexity. To address this current challenge, we have developed an integrated structural biology (ISB) platform, combining experimental and computational methods to determine self-consistent molecular models of higher-order G4s (xG4s). Here we outline our ISB method using two recent examples from our lab, an extended c-Myc promoter and long human telomere G4 repeats, that highlights the utility and generality of our approach to characterizing biologically relevant xG4s.

Research progress in targeting DNA G-quadruplexes using natural products

DNA G-quadruplex(G4) is a guanine-rich single-stranded DNA sequence that spontaneously folds into a spherical four-stranded DNA secondary structure in oncogene promoter sequences and telomeres. G4s are highly associated with the occurrence and development of cancer and have emerged as promising anticancer targets. Natural products have long been important sources of anticancer drug development. In recent years, significant progress has been made in the discovery of natural drugs targeting DNA G4s, with many DNA G4s have been confirmed as promising targets of natural products, including MYC-G4, KRAS-G4, PDGFR-β-G4, BCL-2-G4, VEGF-G4, and telomeric G4. This review summarizes the research progress in discovering natural small molecules that target DNA G4s and their binding mechanisms. It also discusses the opportunities of and challenges in developing drugs targeting DNA G4s. This review will serve as a valuable reference for the research on natural products, particularly in the development of novel antitumor medications.

Balancing act: BRCA2's elaborate management of telomere replication through control of G-quadruplex dynamicity.

In billion years of evolution, eukaryotes preserved the chromosome ends with arrays of guanine repeats surrounded by thymines and adenines, which can form stacks of four-stranded planar structure known as G-quadruplex (G4). The rationale behind the evolutionary conservation of the G4 structure at the telomere remained elusive. Our recent study has shed light on this matter by revealing that telomere G4 undergoes oscillation between at least two distinct folded conformations. Additionally, tumor suppressor BRCA2 exhibits a unique mode of interaction with telomere G4. To elaborate, BRCA2 directly interacts with G-triplex (G3)-derived intermediates that form during the interconversion of the two different G4 states. In doing so, BRCA2 remodels the G4, facilitating the restart of stalled replication forks. In this review, we succinctly summarize the findings regarding the dynamicity of telomeric G4, emphasize its importance in maintaining telomere replication homeostasis, and the physiological consequences of losing G4 dynamicity at the telomere.

IMab antibody binds single-stranded cytosine-rich sequences and unfolds DNA i-motifs.

i-Motifs (iMs) are non-canonical, four-stranded secondary structures formed by stacking of hemi-protonated CH+·C base pairs in cytosine-rich DNA sequences, predominantly at pH<7. The presence of iM structures in cells was a matter of debate until the recent development of iM-specific antibody, iMab, which was instrumental for several studies that suggested the existence of iMs in live cells and their putative biological roles. We assessed the interaction of iMab with cytosine-rich oligonucleotides by biolayer interferometry (BLI), pull-down assay and bulk-FRET experiments. Our results suggest that binding of iMab to DNA oligonucleotides is governed by the presence of runs of at least two consecutive cytosines and is generally increased in acidic conditions, irrespectively of the capacity of the sequence to adopt, or not, an iM structure. Moreover, the results of the bulk-FRET assay indicate that interaction with iMab results in unfolding of iM structures even in acidic conditions, similarly to what has been observed with hnRNP K, well-studied single-stranded DNA binding protein. Taken together, our results strongly suggest that iMab actually binds to blocks of 2-3 cytosines in single-stranded DNA, and call for more careful interpretation of results obtained with this antibody.

Near-infrared fluorescent probe for sensitive detection and imaging of DNA G4s in living cells

G-quadruplexs (G4s), four-stranded nucleic acid secondary structures, which formed by guanine-rich sequences play a vital role in human biological systems. Studies have shown that the formation of G4s is closely related to tumor development and apoptosis, which is considered as a new target for the development of anti-tumor drugs. Therefore, it is important to develop novel probes for G4s imaging. In this article, we engineered a near-infrared fluorescent probe (TOH) which can be activated by DNA G4s in living cells and tumor. TOH exhibits high selectivity to the structure of DNA G4s with the limit of detection for DNA G4s (Mito-0.5–2) is calculated to be 0.43 nM. Imaging studies of different cell lines revealed that the brighter fluorescence in cancer cell lines than in normal, indicating that DNA G4s maybe highly express in tumor cell lines. Simultaneously, TOH is also introduced into live tumor tissue imaging and found that the fluorescence intensity of tumor is the brightest relative to normal tissue, further validating the high expression of DNA G4s structures in tumor tissue. These features demonstrate TOH not only have the ability to image DNA G4 structures in real time, but also may have tumor diagnostic capabilities.

G-Quadruplex Forming DNA Sequence Context Is Enriched around Points of Somatic Mutations in a Subset of Multiple Myeloma Patients.

Multiple myeloma (MM) is the second most common hematological malignancy, which remains incurable despite recent advances in treatment strategies. Like other forms of cancer, MM is characterized by genomic instability, caused by defects in DNA repair. Along with mutations in DNA repair genes and genotoxic drugs used to treat MM, non-canonical secondary DNA structures (four-stranded G-quadruplex structures) can affect accumulation of somatic mutations and chromosomal abnormalities in the tumor cells of MM patients. Here, we tested the hypothesis that G-quadruplex structures may influence the distribution of somatic mutations in the tumor cells of MM patients. We sequenced exomes of normal and tumor cells of 11 MM patients and analyzed the data for the presence of G4 context around points of somatic mutations. To identify molecular mechanisms that could affect mutational profile of tumors, we also analyzed mutational signatures in tumor cells as well as germline mutations for the presence of specific SNPs in DNA repair genes or in genes regulating G-quadruplex unwinding. In several patients, we found that sites of somatic mutations are frequently located in regions with G4 context. This pattern correlated with specific germline variants found in these patients. We discuss the possible implications of these variants for mutation accumulation and specificity in MM and propose that the extent of G4 context enrichment around somatic mutation sites may be a novel metric characterizing mutational processes in tumors.

Targeting G4 motifs of various stem cell makers with designed peptide for therapeutic applications

Noncanonical secondary structures formed by Guanine-rich DNA sequences fold into four-stranded structures called the G-quadruplexes (G4s). Targeting G-quadruplexes is considered an attractive approach toward drug intervention. Here, we have studied the targeting of G4s of stem cell markers with designed short peptide (named as QW10) using biophysical and biochemical techniques. Our CD studies showed that G4 sequences of stem cell markers formed mixed G-quadruplexes in 100 mM Na+, 100 mM K+ and 100 mM K+ +40 wt% PEG 200. On titrating these structures with an increasing concentration of QW10 peptide, we observed a significant decrease in CD intensity followed by the complete disappearance of G4 CD signatures confirming their destabilization not only in dilute conditions but also under cell-mimicking molecular crowding conditions. Our electrophoretic mobility shift assay and significant decrease in the Tm values confirmed the significant destabilization of G4 structures Fluorescence results showed the formation of high-affinity G4 complex-peptide complex with binding affinities in the micromolar (µM) range of 2–8 µM in different ionic conditions. First time, this study may give insight into the use of peptides as leads for the development of more potent and selective ligands to regulate the potential therapeutic applications of cancer stem cell markers.

Design, Synthesis, and Investigation of the Pharmacokinetics and Anticancer Activities of Indenoisoquinoline Derivatives That Stabilize the G-Quadruplex in the MYC Promoter and Inhibit Topoisomerase I.

G-quadruplexes are noncanonical four-stranded DNA secondary structures. MYC is a master oncogene and the G-quadruplex formed in the MYC promoter functions as a transcriptional silencer and can be stabilized by small molecules. We have previously revealed a novel mechanism of action for indenoisoquinoline anticancer drugs, dual-downregulation of MYC and inhibition of topoisomerase I. Herein, we report the design and synthesis of novel 7-aza-8,9-methylenedioxyindenoisoquinolines based on desirable substituents and π-π stacking interactions. These compounds stabilize the MYC promoter G-quadruplex, significantly lower MYC levels in cancer cells, and inhibit topoisomerase I. MYC targeting was demonstrated by differential activities in Raji vs CA-46 cells and cytotoxicity in MYC-dependent cell lines. Cytotoxicities in the NCI-60 panel of human cancer cell lines were investigated. Favorable pharmacokinetics were established, and in vivo anticancer activities were demonstrated in xenograft mouse models. Furthermore, favorable brain penetration, brain pharmacokinetics, and anticancer activity in an orthotopic glioblastoma mouse model were demonstrated.