46 results found
Liano D, Monti L, Chowdhury S, et al., 2022, Long-range DNA interactions: inter-molecular G-quadruplexes and their potential biological relevance, CHEMICAL COMMUNICATIONS, Vol: 58, Pages: 12753-12762, ISSN: 1359-7345
Rauchhaus J, Robinson J, Monti L, et al., 2022, G-quadruplexes mark sites of methylation instability associated with ageing and cancer, Genes, Vol: 13, Pages: 1-11, ISSN: 2073-4425
Regulation of the epigenome is critical for healthy cell function but can become disrupted with age, leading to aberrant epigenetic profiles including altered DNA methylation. Recent studies have indicated that DNA methylation homeostasis can be compromised by the formation of DNA secondary structures known as G-quadruplexes (G4s), which form in guanine-rich regions of the genome. G4s can be recognised and bound by certain methylation-regulating enzymes, and in turn perturb the surrounding methylation architecture. However, the effect G4 formation has on DNA 20methylation at critical epigenetic sites, remains elusive and poorly explored. In this work, we investigate the association between G4 sequences and prominent DNA methylation sites, termed ‘ageing clocks’, that act as bona fide dysregulated regions in aged and cancerous cells. Using a combination of in vitro (G4-seq) and in cellulo (BG4-ChIP) G4 distribution maps, we show that ageing clocks sites are significantly enriched with G4-forming sequences. The observed enrichment also varies across species and cell lines, being least significant in healthy cells and more pronounced in tumorigenic cells. Overall, our results suggest a biological significance of G4s in the realm of DNA methylation, which may be important for further deciphering the driving forces of diseases characterised by epigenetic abnormality, including ageing.
Chowdhury S, Wang J, Nuccio SP, et al., 2022, Short LNA-modified oligonucleotide probes as efficient disruptors of DNA G-quadruplexes, Nucleic Acids Research, Vol: 50, Pages: 7247-7259, ISSN: 0305-1048
G-quadruplexes (G4s) are well known non-canonical DNA secondary structures that can form in human cells. Most of the tools available to investigate G4-biology rely on small molecule ligands that stabilise these structures. However, the development of probes that disrupt G4s is equally important to study their biology. In this study, we investigated the disruption of G4s using Locked Nucleic Acids (LNA) as invader probes. We demonstrated that strategic positioning of LNA-modifications within short oligonucleotides (10 nts.) can significantly accelerate the rate of G4-disruption. Single-molecule experiments revealed that short LNA-probes can promote disruption of G4s with mechanical stability sufficient to stall polymerases. We corroborated this using a single-step extension assay, revealing that short LNA-probes can relieve replication dependent polymerase-stalling at G4 sites. We further demonstrated the potential of such LNA-based probes to study G4-biology in cells. By using a dual-luciferase assay, we found that short LNA probes can enhance the expression of c-KIT to levels similar to those observed when the c-KIT promoter is mutated to prevent the formation of the c-KIT1 G4. Collectively, our data suggest a potential use of rationally designed LNA-modified oligonucleotides as an accessible chemical-biology tool for disrupting individual G4s and interrogating their biological functions in cells.
Fabrini G, Minard A, Brady R, et al., 2022, Cation-responsive and photocleavable hydrogels from non-canonical amphiphilic DNA nanostructures, Nano Letters: a journal dedicated to nanoscience and nanotechnology, Vol: 22, Pages: 602-611, ISSN: 1530-6984
Thanks to its biocompatibility, versatility and programmable interactions, DNA has been proposed as a building block for functional, stimuli-responsive frameworks with applications in biosensing, tissue engineering and drug delivery. Of particular importance for in vivo applications is the possibility of making such nano-materials responsive to physiological stimuli. Here we demonstrate how combining non-canonical DNA G-quadruplex (G4) structures with amphiphilic DNA constructs yields nanos-tructures, which we termed “Quad-Stars”, capable of assembling into responsive hydrogel particles via a straightforward, enzyme-free, one-pot reaction. The embedded G4 structures allow one to trigger and control the assembly/disassembly in a reversible fashion by adding or removing K+ ions. Furthermore, the hydrogel aggregates can be photo disassembled upon near-UV irradiation in the presence of a porphyrin photosensitiser. The combinedreversibility of assembly, responsiveness and cargo-loading capabilities of the hydrophobic moieties make Quad-Stars a promising candidate for biosensors and responsive drug delivery carriers.
Liano D, Chowdhury S, Di Antonio M, 2021, Cockayne Syndrome B protein selectively interacts and resolves intermolecular DNA G-quadruplex structures., Journal of the American Chemical Society, Vol: 143, Pages: 20988-21002, ISSN: 0002-7863
Guanine-rich DNA can fold into secondary structures known as G-quadruplexes (G4s). G4s can form from a single DNA strand (intramolecular) or from multiple DNA strands (intermolecular), but studies on their biological functions have been often limited to intramolecular G4s, owing to the low probability of intermolecular G4s to form within genomic DNA. Herein, we report the first example of an endogenous protein, Cockayne Syndrome B (CSB), that can bind selectively with picomolar affinity toward intermolecular G4s formed within rDNA while displaying negligible binding toward intramolecular structures. We observed that CSB can selectively resolve intermolecular over intramolecular G4s, demonstrating that its selectivity toward intermolecular structures is also reflected at the resolvase level. Immunostaining of G4s with the antibody BG4 in CSB-impaired cells (CS1AN) revealed that G4-staining in the nucleolus of these cells can be abrogated by transfection of viable CSB, suggesting that intermolecular G4s can be formed within rDNA and act as binding substrate for CSB. Given that loss of function of CSB elicits premature aging phenotypes, our findings indicate that the interaction between CSB and intermolecular G4s in rDNA could be of relevance to maintain cellular homeostasis.
Di Antonio M, Robinson J, Raguseo F, et al., 2021, DNA G-quadruplex structures: more than simple roadblocks to transcription?, Nucleic Acids Research, Vol: 49, Pages: 8419-8431, ISSN: 0305-1048
It has been >20 years since the formation of G-quadruplex (G4) secondary structures in gene promoters was first linked to the regulation of gene expression. Since then, the development of small molecules to selectively target G4s and their cellular application have contributed to an improved understanding of how G4s regulate transcription. One model that arose from this work placed these non-canonical DNA structures as repressors of transcription by preventing polymerase processivity. Although a considerable number of studies have recently provided sufficient evidence to reconsider this simplistic model, there is still a misrepresentation of G4s as transcriptional roadblocks. In this review, we will challenge this model depicting G4s as simple ‘off switches’ for gene expression by articulating how their formation has the potential to alter gene expression at many different levels, acting as a key regulatory element perturbing the nature of epigenetic marks and chromatin architecture.
Di Antonio M, Overkleeft H, Wang C, et al., 2021, Outstanding Reviewers for RSC Chemical Biology in 2020, RSC CHEMICAL BIOLOGY, Vol: 2, Pages: 684-684, ISSN: 2633-0679
Balcerowicz M, Di Antonio M, Chung BYW, 2021, Monitoring real-time temperature dynamics of a short RNA hairpin using Förster resonance energy transfer and Circular Dichroism, Bio-protocol, Vol: 11
RNA secondary structures are highly dynamic and subject to prompt changes in response to the environment. Temperature in particular has a strong impact on RNA structural conformation, and temperature-sensitive RNA hairpin structures have been exploited by multiple organisms to modify the rate of translation in response to temperature changes. Observing RNA structural changes in real-time over a range of temperatures is therefore highly desirable. A variety of approaches exists that probe RNA secondary structures, but many of these either require large amount and/or extensive processing of the RNA or cannot be applied under physiological conditions, rendering the observation of structural dynamics over a range of temperatures difficult. Here, we describe the use of a dually fluorescently labelled RNA oligonucleotide (containing the predicted hairpin structure) that can be used to monitor subtle RNA-structural dynamics by Förster Resonance Energy Transfer (FRET) at different temperatures with RNA concentration as low as 200 nM. FRET efficiency varies as a function of the fluorophores' distance; high efficiency can thus be correlated to a stable hairpin structure, whilst a reduction in FRET efficiency reflects a partial opening of the hairpin or a destabilisation of this structure. The same RNA sequence can also be used for Circular Dichroism spectroscopy to observe global changes of RNA secondary structure at a given temperature. The combination of these approaches allowed us to monitor RNA structural dynamics over a range of temperatures in real-time and correlate structural changes to plant biology phenotypes.
Di Antonio M, Ponjavic A, Radzevičius A, et al., 2020, Single-molecule visualization of DNA G-quadruplex formation in live cells., Nature Chemistry, Vol: 12, Pages: 832-837, ISSN: 1755-4330
Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.
Minard A, Morgan D, Raguseo F, et al., 2020, A short peptide that preferentially binds c-MYC G-quadruplex DNA, CHEMICAL COMMUNICATIONS, Vol: 56, Pages: 8940-8943, ISSN: 1359-7345
Chung BYW, Balcerowicz M, Di Antonio M, et al., 2020, An RNA thermoswitch regulates daytime growth in Arabidopsis, NATURE PLANTS, Vol: 6, Pages: 522-+, ISSN: 2055-026X
Raguseo F, Chowdhury S, Minard A, et al., 2020, Chemical-biology approaches to probe DNA and RNA G-quadruplex structures in the genome, CHEMICAL COMMUNICATIONS, Vol: 56, Pages: 1317-1324, ISSN: 1359-7345
Weber J, Bollepalli L, Belenguer AM, et al., 2019, An Activatable Cancer-Targeted Hydrogen Peroxide Probe for Photoacoustic and Fluorescence Imaging, CANCER RESEARCH, Vol: 79, Pages: 5407-5417, ISSN: 0008-5472
Di Antonio M, Minard A, Liano D, et al., 2019, The unexplored potential of quinone methides in chemical biology, Bioorganic and Medicinal Chemistry, Vol: 27, Pages: 2298-2305, ISSN: 0968-0896
Quinone methides (QMs) are transient reactive species that can be efficiently generated from stable precursors under a variety of biocompatible conditions. Due to their electrophilic nature, QMs have been widely explored as cross-linking agents of DNA and proteins under physiological conditions. However, QMs also have a diene character and can irreversibly react via Diels-Alder reaction with electron-rich dienophiles. This particular reactivity has been recently exploited to label biomolecules with fluorophores in living cells.QMs are characterised by two unique properties that make them ideal candidates for chemical biology applications: i) they can be efficiently generated in situ from very stable precursors by means of bio-orthogonal protocols ii) they are reversible cross-linking agents, making them suitable for “catch and release” target-enrichment experiments. Nevertheless, there are only few examples reported to date that truly take advantage of QMs unique chemistry in the context of chemical-biology assay development. In this review, we will examine the most relevant examples that illustrate the benefit of using QMs for chemical biology purposes and we will anticipate novel approaches to further their applications in biologically relevant contexts.
Marsico G, Chambers VS, Sahakyan AB, et al., 2019, Whole genome experimental maps of DNA G-quadruplexes in multiple species, Nucleic Acids Research, Vol: 47, Pages: 3862-3874, ISSN: 0305-1048
Genomic maps of DNA G-quadruplexes (G4s) can help elucidate the roles that these secondary structures play in various organisms. Herein, we employ an improved version of a G-quadruplex sequencing method (G4-seq) to generate whole genome G4 maps for 12 species that include widely studied model organisms and also pathogens of clinical relevance. We identify G4 structures that form under physiological K+ conditions and also G4s that are stabilized by the G4-targeting small molecule pyridostatin (PDS). We discuss the various structural features of the experimentally observed G-quadruplexes (OQs), highlighting differences in their prevalence and enrichment across species. Our study describes diversity in sequence composition and genomic location for the OQs in the different species and reveals that the enrichment of OQs in gene promoters is particular to mammals such as mouse and human, among the species studied. The multi-species maps have been made publicly available as a resource to the research community. The maps can serve as blueprints for biological experiments in those model organisms, where G4 structures may play a role.
Sengar A, Vandana JJ, Chambers VS, et al., 2019, Structure of a (3+1) hybrid G-quadruplex in the PARP1 promoter, NUCLEIC ACIDS RESEARCH, Vol: 47, Pages: 1564-1572, ISSN: 0305-1048
Mao S-Q, Ghanbarian AT, Spiegel J, et al., 2018, DNA G-quadruplex structures mold the DNA methylome, Nature Structural and Molecular Biology, Vol: 25, Pages: 951-957, ISSN: 1545-9985
Control of DNA methylation level is critical for gene regulation, and the factors that govern hypomethylation at CpG islands (CGIs) are still being uncovered. Here, we provide evidence that G-quadruplex (G4) DNA secondary structures are genomic features that influence methylation at CGIs. We show that the presence of G4 structure is tightly associated with CGI hypomethylation in the human genome. Surprisingly, we find that these G4 sites are enriched for DNA methyltransferase 1 (DNMT1) occupancy, which is consistent with our biophysical observations that DNMT1 exhibits higher binding affinity for G4s as compared to duplex, hemi-methylated, or single-stranded DNA. The biochemical assays also show that the G4 structure itself, rather than sequence, inhibits DNMT1 enzymatic activity. Based on these data, we propose that G4 formation sequesters DNMT1 thereby protecting certain CGIs from methylation and inhibiting local methylation.
Greenfield JL, Evans EW, Di Nuzzo D, et al., 2018, Unraveling Mechanisms of Chiral Induction in Double-Helical Metallopolymers, Journal of the American Chemical Society, Vol: 140, Pages: 10344-10353, ISSN: 0002-7863
© 2018 American Chemical Society. Self-assembled helical polymers hold great promise as new functional materials, where helical handedness controls useful properties such as circularly polarized light emission or electron spin. The technique of subcomponent self-assembly can generate helical polymers from readily prepared monomers. Here we present three distinct strategies for chiral induction in double-helical metallopolymers prepared via subcomponent self-assembly: (1) employing an enantiopure monomer, (2) polymerization in a chiral solvent, (3) using an enantiopure initiating group. Kinetic and thermodynamic models were developed to describe the polymer growth mechanisms and quantify the strength of chiral induction, respectively. We found the degree of chiral induction to vary as a function of polymer length. Ordered, rod-like aggregates more than 70 nm long were also observed in the solid state. Our findings provide a basis to choose the most suitable method of chiral induction based on length, regiochemical, and stereochemical requirements, allowing stereochemical control to be established in easily accessible ways.
Sahakyan AB, Chambers VS, Marsico G, et al., 2017, Machine learning model for sequence-driven DNA G-quadruplex formation, SCIENTIFIC REPORTS, Vol: 7, ISSN: 2045-2322
Hansel-Hertsch R, Di Antonio M, Balasubramanian S, 2017, DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential, NATURE REVIEWS MOLECULAR CELL BIOLOGY, Vol: 18, Pages: 279-284, ISSN: 1471-0072
Nieto-Orellana A, Di Antonio M, Conte C, et al., 2017, Effect of polymer topology on non-covalent polymer-protein complexation: miktoarm versus linear mPEG-poly(glutamic acid) copolymers, POLYMER CHEMISTRY, Vol: 8, Pages: 2210-2220, ISSN: 1759-9954
Xu H, Di Antonio M, McKinney S, et al., 2017, CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours, Nature Communications, Vol: 8, Pages: 1-18, ISSN: 2041-1723
G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016).
Nicoli F, Roos MK, Hemmig EA, et al., 2016, Proximity-Induced H-Aggregation of Cyanine Dyes on DNA-Duplexes, JOURNAL OF PHYSICAL CHEMISTRY A, Vol: 120, Pages: 9941-9947, ISSN: 1089-5639
Hansel-Hertsch R, Beraldi D, Lensing SV, et al., 2016, G-quadruplex structures mark human regulatory chromatin, Nature Genetics, Vol: 48, Pages: 1267-1272, ISSN: 1061-4036
G-quadruplex (G4) structural motifs have been linked to transcription1,2, replication3 and genome instability4,5 and are implicated in cancer and other diseases6,7,8. However, it is crucial to demonstrate the bona fide formation of G4 structures within an endogenous chromatin context9,10. Herein we address this through the development of G4 ChIP–seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We find ∼10,000 G4 structures in human chromatin, predominantly in regulatory, nucleosome-depleted regions. G4 structures are enriched in the promoters and 5′ UTRs of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as MYC. Strikingly, de novo and enhanced G4 formation are associated with increased transcriptional activity, as shown by HDAC inhibitor–induced chromatin relaxation and observed in immortalized as compared to normal cellular states. Our findings show that regulatory, nucleosome-depleted chromatin and elevated transcription shape the endogenous human G4 DNA landscape.
Chambers VS, Marsico G, Boutell JM, et al., 2015, High-throughput sequencing of DNA G-quadruplex structures in the human genome, Nature Biotechnology, Vol: 33, Pages: 877-881, ISSN: 1087-0156
G-quadruplexes (G4s) are nucleic acid secondary structures that form within guanine-rich DNA or RNA sequences. G4 formation can affect chromatin architecture and gene regulation and has been associated with genomic instability, genetic diseases and cancer progression1,2,3,4. Here we present a high-resolution sequencing–based method to detect G4s in the human genome. We identified 716,310 distinct G4 structures, 451,646 of which were not predicted by computational methods5,6,7. These included previously uncharacterized noncanonical long loop and bulged structures8,9. We observed a high G4 density in functional regions, such as 5′ untranslated regions and splicing sites, as well as in genes previously not predicted to contain these structures (such as BRCA2). G4 formation was significantly associated with oncogenes, tumor suppressors and somatic copy number alterations related to cancer development10. The G4s identified in this study may therefore represent promising targets for cancer intervention.
Yangyuoru PM, DiAntonio M, Ghimire C, et al., 2015, Dual Binding of an Antibody and a Small Molecule Increases the Stability of TERRA G-Quadruplex, Angewandte Chemie, Vol: 127, Pages: 924-927, ISSN: 0044-8249
Yangyuoru PM, Di Antonio M, Ghimire C, et al., 2015, Dual Binding of an Antibody and a Small Molecule Increases the Stability of TERRA G-Quadruplex, ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, Vol: 54, Pages: 910-913, ISSN: 1433-7851
Le DD, Di Antonio M, Chan LKM, et al., 2015, G-quadruplex ligands exhibit differential G-tetrad selectivity, CHEMICAL COMMUNICATIONS, Vol: 51, Pages: 8048-8050, ISSN: 1359-7345
Di Antonio M, McLuckie KIE, Balasubramanian S, 2014, Reprogramming the Mechanism of Action of Chlorambucil by Coupling to a G-Quadruplex Ligand, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 136, Pages: 5860-5863, ISSN: 0002-7863
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