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The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications. G-quadruplexes are four-stranded DNA secondary structures (G4s) that have been implicated in transcriptional regulation and cancer. Here, we show that G4s are key genomic structural features linked to cellular differentiation. We find that G4s are highly abundant in human embryonic stem cells and are lost during lineage specification. G4s are prevalent in enhancers and promoters. G4s that are found in common between embryonic and downstream lineages are tightly linked to transcriptional stabilisation of genes involved in essential cellular functions as well as transitions in the histone post-translational modification landscape. Furthermore, the application of small molecules that stabilise G4s causes a delay in stem cell differentiation, keeping cells in a more pluripotent-like state. Collectively, our data highlight G4s as important epigenetic features that are coupled to stem cell pluripotency and differentiation. Whether G-quadruplexes (G4s) regulate stem cell self-renewal and fate determination during embryonic development is not well understood. Here, the authors reveal that the embryonic stem cell state is defined by very high G4 abundance. G4s are progressively lost during differentiation as cells transit to lower lineage potential while artificial G4 stabilisation leads to delayed differentiation. [ABSTRACT FROM AUTHOR]

G-quadruplexes (G4s) are four-stranded DNA secondary structures that form in guanine-rich regions of the genome. G4s have important roles in transcription and replication and have been implicated in genome instability and cancer. Thus far most work has profiled the G4 landscape in an ensemble of cell populations, therefore it is critical to explore the structure–function relationship of G4s in individual cells to enable detailed mechanistic insights into G4 function. With standard ChIP-seq methods it has not been possible to determine if G4 formation at a given genomic locus is variable between individual cells across a population. For the first time, we demonstrate the mapping of a DNA secondary structure at single-cell resolution. We have adapted single-nuclei (sn) CUT&Tag to allow the detection of G4s in single cells of human cancer cell lines. With snG4-CUT&Tag, we can distinguish cellular identity from a mixed cell-type population solely based on G4 features within individual cells. Our methodology now enables genomic investigations on cell-to-cell variation of a DNA secondary structure that were previously not possible. [ABSTRACT FROM AUTHOR]

Four-stranded G-quadruplex (G4) structures form from guanine-rich tracts, but the extent of their formation in cellular RNA and details of their role in RNA biology remain poorly defined. Herein, we first delineate the presence of endogenous RNA G4s in the human cytoplasmic transcriptome via the binding sites of G4-interacting proteins, DDX3X (previously published), DHX36 and GRSF1. We demonstrate that a sub-population of these RNA G4s are reliably detected as folded structures in cross-linked cellular lysates using the G4 structure-specific antibody BG4. The 5′ UTRs of protein coding mRNAs show significant enrichment in folded RNA G4s, particularly those for ribosomal proteins. Mutational disruption of G4s in ribosomal protein UTRs alleviates translation in vitro, whereas in cells, depletion of G4-resolving helicases or treatment with G4-stabilising small molecules inhibit the translation of ribosomal protein mRNAs. Our findings point to a common mode for translational co-regulation mediated by G4 structures. The results reveal a potential avenue for therapeutic intervention in diseases with dysregulated translation, such as cancer. [ABSTRACT FROM AUTHOR]

The G-quadruplex (G4) is a non-canonical nucleic acid structure which regulates important cellular processes. RNA G4s have recently been shown to exist in human cells and be biologically significant. Described herein is a new approach to detect and map RNA G4s in cellular transcripts. This method exploits the specific control of RNA G4-cation and RNA G4-ligand interactions during reverse transcription, by using a selective reverse transcriptase to monitor RNA G4-mediated reverse transcriptase stalling (RTS) events. Importantly, a ligation-amplification strategy is coupled with RTS, and enables detection and mapping of G4s in important, low-abundance cellular RNAs. Strong evidence is provided for G4 formation in full-length cellular human telomerase RNA, offering important insights into its cellular function. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Four-stranded G-quadruplex DNA secondary structures have recently been visualized in the nuclei of human cultured cells. Here, we show that BG4, a G-quadruplex-specific antibody, can be used to stain DNA G-quadruplex structures in patient-derived tissues using immunohistochemistry. We observe a significantly elevated number of G-quadruplex-positive nuclei in human cancers of the liver and stomach as compared to background non-neoplastic tissue. Our results suggest that G-quadruplex formation can be detected and measured in patient-derived material and that elevated G-quadruplex formation may be a characteristic of some cancers. [ABSTRACT FROM AUTHOR]

Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs. [ABSTRACT FROM AUTHOR]

While the discovery of B-form DNA 60 years ago has defined our molecular view of the genetic code, other postulated DNA secondary structures, such as A-DNA, Z-DNA, H-DNA, cruciform and slipped structures have provoked consideration of DNA as a more dynamic structure. Four-stranded G-quadruplex DNA does not use Watson-Crick base pairing and has been subject of considerable speculation and investigation during the past decade, particularly with regard to its potential relevance to genome integrity and gene expression. Here, we discuss recent data that collectively support the formation of G-quadruplexes in genomic DNA and the consequences of formation of this structural motif in biological processes. [ABSTRACT FROM AUTHOR]

We report here on the screening of a fragment library against a G-quadruplex element in the human c-MYC promoter. The ten fragment hits had significant concordance between a biophysical assay, in silico modelling and c-MYC expression inhibition, highlighting the feasibility of applying a fragment-based approach to the targeting of a quadruplex nucleic acid. [ABSTRACT FROM AUTHOR]

Following extensive evidence for the formation of four-stranded DNA G-quadruplex structures in vitro, DNA G-quadruplexes have been observed within human cells. Although chemically distinct, RNA can also fold in vitro into G-quadruplex structures that are highly stable because of the 2′-hydroxyl group. However, RNA G-quadruplexes have not yet been reported in cells. Here, we demonstrate the visualization of RNA G-quadruplex structures within the cytoplasm of human cells using a G-quadruplex structure-specific antibody. We also demonstrate that small molecules that bind to G-quadruplexes in vitro can trap endogenous RNA G-quadruplexes when applied to cells. Furthermore, a small molecule that exhibits a preference for RNA G-quadruplexes rather than DNA G-quadruplexes in biophysical experiments also shows the same selectivity within a cellular context. Our findings provide substantive evidence for RNA G-quadruplex formation in the human transcriptome, and corroborate the selectivity and application of stabilizing ligands that target G-quadruplexes within a cellular context. [ABSTRACT FROM AUTHOR]

Four-stranded G-quadruplex nucleic acid structures are of great interest as their high thermodynamic stability under near-physiological conditions suggests that they could form in cells. Here we report the generation and application of an engineered, structure-specific antibody employed to quantitatively visualize DNA G-quadruplex structures in human cells. We show explicitly that G-quadruplex formation in DNA is modulated during cell-cycle progression and that endogenous G-quadruplex DNA structures can be stabilized by a small-molecule ligand. Together these findings provide substantive evidence for the formation of G-quadruplex structures in the genome of mammalian cells and corroborate the application of stabilizing ligands in a cellular context to target G-quadruplexes and intervene with their function. [ABSTRACT FROM AUTHOR]

The folding kinetics of G-quadruplex forming sequences is critical to their capacity to influence biological function. While G-quadruplex structure and stability have been relatively well studied, little is known about the kinetics of their folding. We employed a stopped-flow mixing technique to systematically investigate the potassium-dependent folding kinetics of telomeric RNA and DNA G-quadruplexes and RNA G-quadruplexes containing only two G-quartets formed from sequences r[(GGA)3GG] and r[(GGUUA)3GG]. Our findings suggest a folding mechanism that involves two kinetic steps with initial binding of a single K+, irrespective of the number of G-quartets involved or whether the G-quadruplex is formed from RNA or DNA. The folding rates for telomeric RNA and DNA G-quadruplexes are comparable at near physiological [K+] (90 mM) (τ = ∼60 ms). The folding of a 2-quartet RNA G-quadruplex with single nucleotide A loops is considerably slower (τ = ∼700 ms), and we found that the time required to fold a UUA looped variant (τ > 100 s, 500 mM K+) exceeds the lifetimes of some regulatory RNAs. We discuss the implications of these findings with respect to the fundamental properties of G-quadruplexes and their potential functions in biology. [ABSTRACT FROM AUTHOR]

Guanine-rich DNA sequences that can adopt non-Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here we show that the G-quadruplex-interacting drug pyridostatin promotes growth arrest in human cancer cells by inducing replication- and transcription-dependent DNA damage. A chromatin immunoprecipitation sequencing analysis of the DNA damage marker ?H2AX provided the genome-wide distribution of pyridostatin-induced sites of damage and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein abundance and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target of this drug. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions. [ABSTRACT FROM AUTHOR]

Ligands that stabilize the formation of telomeric DNA G-quadruplexes have potential as cancer treatments, because the G-quadruplex structure cannot be extended by telomerase, an enzyme over-expressed in many cancer cells. Understanding the kinetic, thermodynamic and mechanical properties of small-molecule binding to these structures is therefore important, but classical ensemble assays are unable to measure these simultaneously. Here, we have used a laser tweezers method to investigate such interactions. With a force jump approach, we observe that pyridostatin promotes the folding of telomeric G-quadruplexes. The increased mechanical stability of pyridostatin-bound G-quadruplex permits the determination of a dissociation constant Kd of 490 ± 80 nM. The free-energy change of binding obtained from a Hess-like process provides an identical Kd for pyridostatin and a Kd of 42 ± 3 µM for a weaker ligand RR110. We anticipate that this single-molecule platform can provide detailed insights into the mechanical, kinetic and thermodynamic properties of liganded bio-macromolecules, which have biological relevance. [ABSTRACT FROM AUTHOR]

G-quadruplexes are four-stranded DNA structures that are over-represented in gene promoter regions and are viewed as emerging therapeutic targets in oncology, as transcriptional repression of oncogenes through stabilization of these structures could be a novel anticancer strategy. Many gene promoter G-quadruplexes have physicochemical properties and structural characteristics that might make them druggable, and their structural diversity suggests that a high degree of selectivity might be possible. Here, we describe the evidence for G-quadruplexes in gene promoters and discuss their potential as therapeutic targets, as well as progress in the development of strategies to harness this potential through intervention with small-molecule ligands. [ABSTRACT FROM AUTHOR]

Nucleic acids containing stretches of tandem guanines can fold into four-stranded structures called G-quadruplexes. The existence of such sequences in genomic DNA suggests the occurrence of these motifs in cells, with potential implications in a number of biological processes relevant to cancer. Small molecules have proven to be valuable tools to dissect cell circuitry. Here, we describe a synthetic small molecule derived from an N,N′-bis(2-quinolinyl)pyridine-2,6-dicarboxamide, which is designed to mediate the selective isolation of G-quadruplex nucleic acids. The methodology was successfully applied to a range of DNA and RNA G-quadruplexes in vitro. We demonstrate the general applicability of the method by isolating telomeric DNA-containing G-quadruplex motifs from cells. We show that telomeres are targets for the probe, providing further evidence of the formation of G-quadruplexes in human cells. [ABSTRACT FROM AUTHOR]

Nucleic acid sequences containing several short runs of guanine nucleotides can form complex higher order structures, termed quadruplexes. Their occurrence has been most extensively characterised at the telomeric ends of eukaryotic chromosomes, whose DNA comprises such sequences, and where the extreme 3′ ends are single-stranded. This enables relatively facile formation of quadruplex arrangements under the influence of a quadruplex-selective small molecule to compete effectively with telomeric protein–DNA interactions. Occurrences of quadruplexes within the human and other genomes have been mapped by bioinformatics surveys, which have revealed over-representations in promoter regions, especially of genes involved in replication, such as oncogenes, as well as in 5′UTR regions. The highly distinctive nature of quadruplex topologies suggests that they can act as novel therapeutic targets, for example in the selective inhibition of transcription of a given oncogene, using designed small molecules to stabilise a particular quadruplex. This offers the prospect of an alternative to, for example, direct kinase targeting with small molecules, without the attendant issues of active-site resistance. We survey here the basis of these approaches, together with current progress, and discuss the mechanistic issues posed by quadruplex targeting. [Copyright &y& Elsevier]

Abstract: Single molecule fluorescence resonance energy transfer (FRET) can be employed to study conformational heterogeneity and real-time dynamics of biological macromolecules. Here we present single molecule studies on human genomic DNA G-quadruplex sequences that occur in the telomeres and in the promoter of a proto-oncogene. The findings are discussed with respect to the proposed biological function(s) of such motifs in living cells. [Copyright &y& Elsevier]

G-Rich sequences found within biologically important regions of the genome have been shown to form intramolecular G-quadruplexes with varied loop lengths and sequences. Many of these quadruplexes will be distinguishable from each other on the basis of their thermodynamic stabilities and folded conformations. It has been proposed that loop lengths can strongly influence the topology and stability of intramolecular G-quadruplexes. Previous studies have been limited to the analysis of quadruplex sequences with particular loop sequences, making it difficult to make generalizations. Here, we describe an original study that aimed to elucidate the effect of loop length on the biophysical properties of G-quadruplexes in a sequence-independent context. We employed UV melting and circular dichroism spectroscopy to examine and compare the properties of 21 DNA quadruplex libraries, each comprising partially randomized loop sequences with lengths ranging from one to three nucleotides. Our work supports a number of general predictions that can be made solely on the basis of loop lengths. In particular, the results emphasize the strong influence of single-nucleotide loops on quadruplex properties. This study provides a predictive framework that may help identify or classify biologically relevant G-quadruplex-forming sequences. [ABSTRACT FROM AUTHOR]

The use of a caged G-quadruplex ligand allows for transcriptional control of quadruplex-containing genes using UV light as an external trigger. An important oncogene, SRC, involved in the initiation and proliferation of epithelial tumours is shown to be significantly downregulated in cells treated by the caged ligand in synergy with UV light treatment. [ABSTRACT FROM AUTHOR]

The nitrogen mustard Chlorambucil (Chi) generates covalent adducts with double-helical DNA and inhibits cell proliferation. Among these adducts, inter-strand cross-links (ICLs) are the most toxic, as they stall replication by generating DNA double strand breaks (DSBs). Conversely, intrastrand cross-links generated by Chi are efficiently repaired by a dedicated Nucleotide Excision Repair (NER) enzyme. We synthesized a novel cross-linking agent that combines Chi with the G-quadruplex (G4) ligand PDS (PDS-Chl). We demonstrated that PDS-Chl alkylates G4 structures at low fiM 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. [ABSTRACT FROM AUTHOR]

RNA molecules can fold into noncanonical structures such as the four-stranded structures known as G-quadruplexes. G-quadruplexes in the transcriptome have recently emerged as relevant regulatory elements of gene expression. Conformational transitions in RNA molecules offer an important way to regulate their biological functions. Here we report on the competition between a canonical hairpin structure and a G-quadruplex structure within an RNA molecule. We show that the conformational preference strongly depends on the relative amounts of mono- and divalent metal ions present in solution. In our system, the G-quadruplex, whose formation is not predicted by available predictive RNA folding programs, is the major conformer at physiologically relevant K+ and Mg2+ concentrations. Furthermore, we show that a synthetic small molecule can displace the structural dynamic equilibrium in favor of the hairpin conformer. This work highlights a new and important level of complexity in RNA folding that could be relevant to the biological functions and targeting of RNAs comprising G-quadruplex motifs. [ABSTRACT FROM AUTHOR]

Abstract: Guanine-rich nucleic acids can fold into non-canonical DNA secondary structures called G-quadruplexes. The formation of these structures can interfere with the biology that is crucial to sustain cellular homeostases and metabolism via mechanisms that include transcription, translation, splicing, telomere maintenance and DNA recombination. Thus, due to their implication in several biological processes and possible role promoting genomic instability, G-quadruplex forming sequences have emerged as potential therapeutic targets. There has been a growing interest in the development of synthetic molecules and biomolecules for sensing G-quadruplex structures in cellular DNA. In this review, we summarise and discuss recent methods developed for cellular imaging of G-quadruplexes, and the application of experimental genomic approaches to detect G-quadruplexes throughout genomic DNA. In particular, we will discuss the use of engineered small molecules and natural proteins to enable pull-down, ChIP-Seq, ChIP-chip and fluorescence imaging of G-quadruplex structures in cellular DNA. [Copyright &y& Elsevier]

G-Quadruplexes are noncanonical nucleic acid secondary structures based on guanine association that are readily adopted by G-rich RNA and DNA sequences. Naturally occurring genomic G-quadruplex-forming sequences have functional roles in biology that are mediated through structure. To appreciate how this is achieved, an understanding of the likelihood of G-quadruplex formation and the structural features of the folded species under a defined set of conditions is informative. We previously systematically investigated the thermodynamic stability and folding topology of DNA G-quadruplexes and found a strong dependence of these properties on loop length and loop arrangement [Bugaut, A., and Balasubramanian, S. (2008) Biochemistry 47, 689-697]. Here we report on a complementary analysis of RNA G-quadruplexes using UV melting and circular dichroism spectroscopy that also serves as a comparison to the equivalent DNA G-quadruplex-forming sequences. We found that the thermodynamic stability of G-quadruplex RNA can be modulated by loop length while the overall structure is largely unaffected. The systematic design of our study also revealed subtle loop length dependencies in RNA G-quadruplex structure. [ABSTRACT FROM AUTHOR]

Telomeric repeat-containing RNA (TERRA) is important for telomere regulation, but the structural basis for how TERRA localizes to chromosome ends is unknown. Here we report on studies exploring whether the TERRA G-quadruplex structure is critical for binding to telomeres. We demonstrate that the telomeric protein TRF2 binds TERRA via interactions that necessitate the formation of a G-quadruplex structure rather than the TERRA sequence per Se. We also show that TRF2 simultaneously binds TERRA and telomeric duplex or G-quadruplex DNA. These observations suggest that the TERRA G-quadruplex is a key feature of telomere organization. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

G-Quadruplexes are nudeic acid secondary stnlctures consisting of a planar arrangement of four guanine residues. Potential G.quadruplex.forming sequences are widely distributed throughout the genome. Significantly, they are present in telomeres and are enriched in gene promoters and first introns, raising the possibility that perturbation of Gquadruplex stability might have therapeutic potential, for example in the treatment of cancer. Ligands that interact selectively with G-quadruplexes indude both proteins and small molecules, although the interactions between ligands and their G-quadruplex targets have-been monitored using indirect methods. li~ addition, the G-quadruplex targets have often been short DNA fragments. Here, we have used atomic force microscopy imaging to examine directly at the single-molecule level the interaction of ligands with G-quadruplexes generated during transcription of a plasmid containing a G-rich insert. We show that the structures produced during transcription are decorated specifically by the single-chain antibody HFI and by the nuclear protein PARP-l, both of which are known to recognize G-quadruplexes. Our results provide clear structural evidence of G-quadruplex formation in a transcription-dependent case and demonstrate directly how small-molecule stabilizers and destabilizers can manipulate these structures in a biochemically functional system. [ABSTRACT FROM AUTHOR]

The article reports the tight and selective binding of cationic trimetric macrocycle and helically folded tetramer to quadruplex DNA. According to the authors, the new ligands showed significant potential for potent G-quadruplex stabilization without any evidence of duplex stabilization. It is also stated that the relative ease of synthesis of these molecules makes them amenable to chemical modification to improve affinity and tailor quadruplex selectivity.