RESUMO
Single-stranded guanine-rich DNA sequences can fold into four-stranded DNA structures called G-quadruplexes (G4s) that arise from the self-stacking of two or more guanine quartets. There has been considerable recent progress in the detection and mapping of G4 structures in the human genome and in biologically relevant contexts. These advancements, many of which align with predictions made previously in computational studies, provide important new insights into the functions of G4 structures in, for example, the regulation of transcription and genome stability, and uncover their potential relevance for cancer therapy.
Assuntos
Quadruplex G , Genoma Humano , Animais , Replicação do DNA , DNA de Cadeia Simples/química , Regulação da Expressão Gênica , Instabilidade Genômica , Humanos , Neoplasias/tratamento farmacológico , Oligonucleotídeos , Transcrição GênicaRESUMO
Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress.
Assuntos
DNA , Quadruplex G , Humanos , Genoma Humano , Nucleotidiltransferases , Replicação do DNARESUMO
Over the past decade, appreciation of the roles of G-quadruplex (G4) structures in cellular regulation and maintenance has rapidly grown, making the establishment of robust methods to visualize G4s increasingly important. Fluorescent probes are commonly used for G4 detection in vitro; however, achieving sufficient selectivity to detect G4s in a dense and structurally diverse cellular environment is challenging. The use of fluorescent probes for G4 detection is further complicated by variations of probe uptake into cells, which may affect fluorescence intensity independently of G4 abundance. In this work, we report an alternative small-molecule approach to visualize G4s that does not rely on fluorescence intensity switch-on and, thus, does not require the use of molecules with exclusive G4 binding selectivity. Specifically, we have developed a novel thiazole orange derivative, TOR-G4, that exhibits a unique fluorescence lifetime when bound to G4s compared to other structures, allowing G4 binding to be sensitively distinguished from non-G4 binding, independent of the local probe concentration. Furthermore, TOR-G4 primarily colocalizes with RNA in the cytoplasm and nucleoli of cells, making it the first lifetime-based probe validated for exploring the emerging roles of RNA G4s in cellulo.
Assuntos
Corantes Fluorescentes , Quadruplex G , Corantes Fluorescentes/química , RNA , Microscopia de Fluorescência , Citoplasma/metabolismoRESUMO
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.
Assuntos
Quadruplex G , Sondas de Oligonucleotídeos/química , Oligonucleotídeos/química , DNA/química , Humanos , Regiões Promotoras GenéticasRESUMO
G-quadruplexes (G4s) are nucleic acid secondary structures that have been linked to the functional regulation of eukaryotic organisms. G4s have been extensively characterised in humans and emerging evidence suggests that they might also be biologically relevant for human pathogens. This indicates that G4s might represent a novel class of therapeutic targets for tackling infectious diseases. Bioinformatic studies revealed a high prevalence of putative quadruplex-forming sequences (PQSs) in the genome of protozoans, which highlights their potential roles in regulating vital processes of these parasites, including DNA transcription and replication. In this work, we focus on the neglected trypanosomatid parasites, Trypanosoma and Leishmania spp., which cause debilitating and deadly diseases across the poorest populations worldwide. We review three examples where G4-formation might be key to modulate transcriptional activity in trypanosomatids, providing an overview of experimental approaches that can be used to exploit the regulatory roles and relevance of these structures to fight parasitic infections.
Assuntos
Quadruplex G , Parasitos , Trypanosoma , Animais , Humanos , Parasitos/genética , Trypanosoma/genética , DNA/química , GenomaRESUMO
This symposium is the third PSL (Paris Sciences & Lettres) Chemical Biology meeting (2016, 2019, 2023) held at Institut Curie. This initiative originally started at Institut de Chimie des Substances Naturelles (ICSN) in Gif-sur-Yvette (2013, 2014), under the directorship of Professor Max Malacria, with a strong focus on chemistry. It was then continued at the Institut Curie (2015) covering a larger scope, before becoming the official PSL Chemical Biology meeting. This latest edition was postponed twice for the reasons that we know. This has given us the opportunity to invite additional speakers of great standing. This year, Institut Curie hosted around 300 participants, including 220 on site and over 80 online. The pandemic has had, at least, the virtue of promoting online meetings, which we came to realize is not perfect but has its own merits. In particular, it enables those with restricted time and resources to take part in events and meetings, which can now accommodate unlimited participants. We apologize to all those who could not attend in person this time due to space limitation at Institut Curie.
Assuntos
Biologia , Humanos , ParisRESUMO
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.
Assuntos
Epigênese Genética , Quadruplex G , Regulação da Expressão Gênica/genética , Transcrição Gênica , Cromatina/genética , DNA Polimerase Dirigida por DNA/genética , Humanos , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/genéticaRESUMO
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 nanomaterials responsive to physiological stimuli. Here, we demonstrate how combining noncanonical DNA G-quadruplex (G4) structures with amphiphilic DNA constructs yields nanostructures, 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 photosensitizer. The combined reversibility of assembly, responsiveness, and cargo-loading capabilities of the hydrophobic moieties make Quad-Stars a promising candidate for biosensors and responsive drug delivery carriers.
Assuntos
Quadruplex G , Nanoestruturas , Cátions , DNA/química , Hidrogéis/química , Nanoestruturas/químicaRESUMO
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.
Assuntos
DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA/metabolismo , Quadruplex G , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Recombinases/metabolismo , Aminoquinolinas/farmacologia , Animais , Benzotiazóis/farmacologia , Nucléolo Celular/metabolismo , DNA/genética , Células HeLa , Humanos , Naftiridinas/farmacologia , Ácidos Picolínicos/farmacologia , Ligação Proteica/efeitos dos fármacos , Células Sf9 , SpodopteraRESUMO
Poly (ADP-ribose) polymerase 1 (PARP1) has emerged as an attractive target for cancer therapy due to its key role in DNA repair processes. Inhibition of PARP1 in BRCA-mutated cancers has been observed to be clinically beneficial. Recent genome-mapping experiments have identified a non-canonical G-quadruplex-forming sequence containing bulges within the PARP1 promoter. Structural features, like bulges, provide opportunities for selective chemical targeting of the non-canonical G-quadruplex structure within the PARP1 promoter, which could serve as an alternative therapeutic approach for the regulation of PARP1 expression. Here we report the G-quadruplex structure formed by a 23-nucleotide G-rich sequence in the PARP1 promoter. Our study revealed a three-layered intramolecular (3+1) hybrid G-quadruplex scaffold, in which three strands are oriented in one direction and the fourth in the opposite direction. This structure exhibits unique structural features such as an adenine bulge and a G·G·T base triple capping structure formed between the central edgewise loop, propeller loop and 5' flanking terminal. Given the highly important role of PARP1 in DNA repair and cancer intervention, this structure presents an attractive opportunity to explore the therapeutic potential of PARP1 inhibition via G-quadruplex DNA targeting.
Assuntos
DNA/química , Quadruplex G , Conformação de Ácido Nucleico , Poli(ADP-Ribose) Polimerase-1/química , Adenina/química , DNA/genética , Reparo do DNA/genética , Guanina/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Poli(ADP-Ribose) Polimerase-1/genética , Regiões Promotoras GenéticasRESUMO
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.
Assuntos
Indolquinonas/química , Alquilação , Química Click , Reação de Cicloadição , DNA/química , DNA/metabolismo , Humanos , Indolquinonas/metabolismo , Microscopia de Fluorescência , Proteínas/química , Proteínas/metabolismo , Interferência de RNA , Raios UltravioletaRESUMO
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.
RESUMO
A wide variety of organic dyes form, under certain conditions, clusters know as J- and H-aggregates. Cyanine dyes are such a class of molecules where the spatial proximity of several dyes leads to overlapping electron orbitals and thus to the creation of a new energy landscape compared to that of the individual units. In this work, we create artificial H-aggregates of exactly two Cyanine 3 (Cy3) dyes by covalently linking them to a DNA molecule with controlled subnanometer distances. The absorption spectra of these coupled systems exhibit a blue-shifted peak, whose intensity varies depending on the distance between the dyes and the rigidity of the DNA template. Simulated vibrational resolved spectra, based on molecular orbital theory, excellently reproduce the experimentally observed features. Circular dichroism spectroscopy additionally reveals distinct signals, which indicates a chiral arrangement of the dye molecules. Molecular dynamic simulations of a Cy3-Cy3 construct including a 14-base pair DNA sequence verified chiral stacking of the dye molecules.
Assuntos
Carbocianinas/química , DNA/química , Dicroísmo Circular , Simulação de Dinâmica MolecularRESUMO
In investigating the binding interactions between the human telomeric RNA (TERRA) G-quadruplex (GQ) and its ligands, it was found that the small molecule carboxypyridostatin (cPDS) and the GQ-selective antibody BG4 simultaneously bind the TERRA GQ. We previously showed that the overall binding affinity of BG4 for RNA GQs is not significantly affected in the presence of cPDS. However, single-molecule mechanical unfolding experiments revealed a population (48%) with substantially increased mechanical and thermodynamic stability. Force-jump kinetic investigations suggested competitive binding of cPDS and BG4 to the TERRA GQ. Following this, the two bound ligands slowly rearrange, thereby leading to the minor population with increased stability. Given the relevance of G-quadruplexes in the regulation of biological processes, we anticipate that the unprecedented conformational rearrangement observed in the TERRA-GQ-ligand complex may inspire new strategies for the selective stabilization of G-quadruplexes in cells.
Assuntos
Aminoquinolinas/metabolismo , Anticorpos/imunologia , Quadruplex G , Ácidos Picolínicos/metabolismo , Telômero/metabolismo , Aminoquinolinas/química , Humanos , Ligantes , Conformação de Ácido Nucleico , Pinças Ópticas , Ácidos Picolínicos/química , RNA/química , TermodinâmicaRESUMO
The nitrogen mustard Chlorambucil (Chl) generates covalent adducts with double-helical DNA and inhibits cell proliferation. Among these adducts, interstrand cross-links (ICLs) are the most toxic, as they stall replication by generating DNA double strand breaks (DSBs). Conversely, intrastrand cross-links generated by Chl are efficiently repaired by a dedicated Nucleotide Excision Repair (NER) enzyme. We synthesized a novel cross-linking agent that combines Chl with the G-quadruplex (G4) ligand PDS (PDS-Chl). We demonstrated that PDS-Chl alkylates G4 structures at low µM concentrations, without reactivity toward double- or single-stranded DNA. Since intramolecular G4s arise from a single DNA strand, we reasoned that preferential alkylation of such structures might prevent the generation of ICLs, while favoring intrastrand cross-links. We observed that PDS-Chl selectively impairs growth in cells genetically deficient in NER, but did not show any sensitivity to the repair gene BRCA2, involved in double-stranded break repair. Our findings suggest that G4 targeting of this clinically important alkylating agent alters the overall mechanism of action. These insights may inspire new opportunities for intervention in diseases specifically characterized by genetic impairment of NER, such as skin and testicular cancers.
Assuntos
Clorambucila/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Quadruplex G/efeitos dos fármacos , Aminoquinolinas/metabolismo , Linhagem Celular , Adutos de DNA/química , Adutos de DNA/metabolismo , Humanos , Ligantes , Ácidos Picolínicos/metabolismoRESUMO
The development of methods that enabled genome-wide mapping of DNA G-quadruplex structures in chromatin has played a critical role in providing evidence to support the formation of these structures in living cells. Over the past decade, a variety of methods aimed at mapping G-quadruplexes have been reported in the literature. In this critical review, we have sought to provide a technical overview on the relative strengths and weaknesses of the genomics approaches currently available, offering step-by-step guidance to assessing experimental needs and selecting the most appropriate method to achieve effective genome-wide mapping of DNA G-quadruplexes.
RESUMO
RNA molecules play crucial roles in gene expression regulation and cellular signaling, and these functions are governed by the formation of RNA secondary and tertiary structures. These structures are highly dynamic and subject to rapid changes in response to environmental cues, temperature in particular. Thermosensitive RNA secondary structures have been harnessed by multiple organisms to survey their temperature environment and to adjust gene expression accordingly. It is thus highly desirable to observe RNA structural changes in real time over a range of temperatures. Multiple approaches have been developed to study structural dynamics, but many of these require extensive processing of the RNA, large amounts of RNA input, and/or cannot be applied under physiological conditions. Here, we describe the use of a dually fluorescently labeled RNA oligonucleotide (containing a predicted hairpin structure) to monitor subtle RNA structural dynamics in vitro by Förster resonance energy transfer (FRET) and circular dichroism (CD) spectroscopy. These approaches can be employed under physiologically relevant conditions over a range of temperatures and with RNA concentrations as low as 200 nM; they enable us to observe RNA structural dynamics in real time and to correlate these dynamics with changes in biological processes such as translation.
Assuntos
Transferência Ressonante de Energia de Fluorescência , RNA , RNA/química , Temperatura , Dicroísmo Circular , OligonucleotídeosRESUMO
Condensation of RNA and proteins is central to cellular functions, and the ability to program it would be valuable in synthetic biology and synthetic cell science. Here we introduce a modular platform for engineering synthetic RNA condensates from tailor-made, branched RNA nanostructures that fold and assemble co-transcriptionally. Up to three orthogonal condensates can form simultaneously and selectively accumulate fluorophores through embedded fluorescent light-up aptamers. The RNA condensates can be expressed within synthetic cells to produce membrane-less organelles with a controlled number and relative size, and showing the ability to capture proteins using selective protein-binding aptamers. The affinity between otherwise orthogonal nanostructures can be modulated by introducing dedicated linker constructs, enabling the production of bi-phasic RNA condensates with a prescribed degree of interphase mixing and diverse morphologies. The in situ expression of programmable RNA condensates could underpin the spatial organization of functionalities in both biological and synthetic cells.
RESUMO
Androgen receptor (AR) signaling remains an important regulatory pathway in castrate-resistant prostate cancer, and its transcriptional downregulation could provide a new line of therapy. A number of small-molecule ligands have previously demonstrated the ability to stabilize G-quadruplex structures and affect gene transcription for those genes whose promoters contain a quadruplex-forming sequence. Herein, we report the probable formation of new G-quadruplex structure present in the AR promoter in a transcriptionally important location. NMR spectroscopy, circular dichroism, UV spectroscopy, and UV thermal melting experiments for this sequence are consistent with G-quadruplex formation. Fluorescence resonance energy transfer (FRET) melting studies have identified a novel compound, MM45, which appears to stabilize this G-quadruplex at submicromolar concentrations. The effects of MM45 have been investigated in prostate cancer cell lines where it has been shown to inhibit cell growth. A reporter assay intended to isolate the effect of MM45 on the G-quadruplex sequence showed dose-dependent transcriptional repression only when the AR promoter G-quadruplex sequence is present. Dose-dependent transcriptional repression of the AR by MM45 has been demonstrated at both a protein and mRNA level. This proof of concept study paves the route toward a potential alternative treatment pathway in castrate-resistant prostate cancer.
Assuntos
Antineoplásicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Imidas/farmacologia , Naftalenos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Transferência Ressonante de Energia de Fluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Ressonância Magnética Nuclear Biomolecular , Regiões Promotoras Genéticas/efeitos dos fármacos , Próstata/efeitos dos fármacos , Próstata/metabolismo , Neoplasias da Próstata/genética , Espectrofotometria Ultravioleta , Ativação Transcricional/efeitos dos fármacosRESUMO
Synthetic lethality is a genetic concept in which cell death is induced by the combination of mutations in two sensitive genes, while mutation of either gene alone is not sufficient to affect cell survival. Synthetic lethality can also be achieved "chemically" by combination of drug-like molecules targeting distinct but cooperative pathways. Previously, we reported that the small molecule pyridostatin (PDS) stabilizes G-quadruplexes (G4s) in cells and elicits a DNA damage response by causing the formation of DNA double strand breaks (DSB). Cell death mediated by ligand-induced G4 stabilization can be potentiated in cells deficient in DNA damage repair genes. Here, we demonstrate that PDS acts synergistically both with NU7441, an inhibitor of the DNA-PK kinase crucial for nonhomologous end joining repair of DNA DSBs, and BRCA2-deficient cells that are genetically impaired in homologous recombination-mediated DSB repair. G4 targeting ligands have potential as cancer therapeutic agents, acting synergistically with inhibition or mutation of the DNA damage repair machinery.