G4-forming Sequences Research Articles

Targeting G-quadruplexes in an ageing epigenetic regulator promoter for rescuing mitochondrial dysfunction in Alzheimer's disease.

Here, we provide an out-of-the-box G-quadruplex (G4) targeting-based strategy for rescuing mitochondrial dysfunction in Alzheimer's disease. We predict and verify the presence of G4s within the promoter of an ageing epigenetic regulator BAZ2B. G4-specific ligands targeting BAZ2B G4s could significantly down-regulate the BAZ2B expression and relieve mitochondrial dysfunction. Therefore, this work may provide a new way of rescuing mitochondrial dysfunction in AD by targeting G4s in a specific ageing epigenetic regulator promoter.

Structural Polymorphism of Guanine Quadruplex-Containing Regions in Human Promoters.

Intramolecular guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by four guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4-forming DNA sequences are enriched in gene promoters and are implicated in the control of gene expression. Most G4-forming DNA contains more G residues than can simultaneously be incorporated into the core resulting in a variety of different possible G4 structures. Although this kind of structural polymorphism is well recognized in the literature, there remain unanswered questions regarding possible connections between G4 polymorphism and biological function. Here we report a detailed bioinformatic survey of G4 polymorphism in human gene promoter regions. Our analysis is based on identifying G4-containing regions (G4CRs), which we define as stretches of DNA in which every residue can form part of a G4. We found that G4CRs with higher degrees of polymorphism are more tightly clustered near transcription sites and tend to contain G4s with shorter loops and bulges. Furthermore, we found that G4CRs with well-characterized biological functions tended to be longer and more polymorphic than genome-wide averages. These results represent new evidence linking G4 polymorphism to biological function and provide new criteria for identifying biologically relevant G4-forming regions from genomic data.

Deciphering RNA G-quadruplex function during the early steps of HIV-1 infection.

G-quadruplexes (G4s) are four-stranded nucleic acid structures formed by the stacking of G-tetrads. Here we investigated their formation and function during HIV-1 infection. Using bioinformatics and biophysics analyses we first searched for evolutionary conserved G4-forming sequences in HIV-1 genome. We identified 10 G4s with conservation rates higher than those of HIV-1 regulatory sequences such as RRE and TAR. We then used porphyrin-based G4-binders to probe the formation of the G4s during infection of human cells by native HIV-1. The G4-binders efficiently inhibited HIV-1 infectivity, which is attributed to the formation of G4 structures during HIV-1 replication. Using a qRT-PCR approach, we showed that the formation of viral G4s occurs during the first 2 h post-infection and their stabilization by the G4-binders prevents initiation of reverse transcription. We also used a G4-RNA pull-down approach, based on a G4-specific biotinylated probe, to allow the direct detection and identification of viral G4-RNA in infected cells. Most of the detected G4-RNAs contain crucial regulatory elements such as the PPT and cPPT sequences as well as the U3 region. Hence, these G4s would function in the early stages of infection when the viral RNA genome is being processed for the reverse transcription step.

Potential G‐quadruplexes within the Promoter Nuclease Hypersensitive Sites of the Heat‐Responsive Genes in Rice

G-quadruplexes (G4s) have been shown to be involved in the regulation of multiple cellular processes. Exploring putative G4-forming sequences (PQSs) in heat-responsive genes of rice and their folding structures under different conditions will help to understand the mechanism in response to heat stress. In this work, we discovered a prevalence of PQSs in nuclease hypersensitive sites within the promoters of heat-responsive genes. Moreover, 50 % of the searched G3 PQSs ((G3+ L1-7 )3+ G3+ ) locate in heat shock transcription factors. Circular dichroism spectroscopy, thermal difference spectroscopy, and UV melting analysis demonstrated the representative PQSs could adopt stable G4s at physiological temperature and potassium concentration. These PQSs were able to stall Klenow fragment (KF) DNA polymerase by the formation of G4s. However, the G4s with Tm values around 50-60 °C could be increasingly unwound by KF with the increase of temperatures from 25 to 50 °C, implying that these G4s could sense the changes in temperature by structural switch. This work offers fresh clues to understanding the potential of G4-involved functions of PQSs and the molecular events in plants in response to heat stress.

G-quadruplexes Mark Sites of Methylation Instability Associated with Ageing and Cancer.

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 methylation 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.

Identification and functional characterization of G-quadruplexes in plants.

DNA (G4-DNA) or RNA (G4-RNA) G-quadruplexes, which are formed within guanine-rich nucleic acid context harboring putative G4-forming sequences (PQSs), are prevalent in mammalian and plant genomes. The presence of G4-DNA/RNA in mammals has been extensively investigated using high throughput sequencing methodologies. Recently, BG4-IP-seq and (r)G4-seq have been successfully applied for characterization of G4-DNA/RNA in Arabidopsis thaliana and rice (Oryza sativum), thereby establishing a key step towards understanding the biological relevance of G4s in plants.

A Machine Learning Perspective on DNA and RNA G-quadruplexes

Abstract: G-quadruplexes (G4s) are particular structures found in guanine-rich DNA and RNA sequences that exhibit a wide diversity of three-dimensional conformations and exert key functions in the control of gene expression. G4s are able to interact with numerous small molecules and endogenous proteins, and their dysregulation can lead to a variety of disorders and diseases. Characterization and prediction of G4-forming sequences could elucidate their mechanism of action and could thus represent an important step in the discovery of potential therapeutic drugs. In this perspective, we propose an overview of G4s, discussing the state of the art of methodologies and tools developed to characterize and predict the presence of these structures in genomic sequences. In particular, we report on machine learning (ML) approaches and artificial neural networks (ANNs) that could open new avenues for the accurate analysis of quadruplexes, given their potential to derive informative features by learning from large, high-density datasets.

Affinity Chromatography-Based Assays for the Screening of Potential Ligands Selective for G-Quadruplex Structures.

DNA G‐quadruplexes (G4s) are key structures for the development of targeted anticancer therapies. In this context, ligands selectively interacting with G4s can represent valuable anticancer drugs. Aiming at speeding up the identification of G4‐targeting synthetic or natural compounds, we developed an affinity chromatography‐based assay, named G‐quadruplex on Oligo Affinity Support (G4‐OAS), by synthesizing G4‐forming sequences on commercially available polystyrene OAS. Then, due to unspecific binding of several hydrophobic ligands on nude OAS, we moved to Controlled Pore Glass (CPG). We thus conceived an ad hoc functionalized, universal support on which both the on‐support elongation and deprotection of the G4‐forming oligonucleotides can be performed, along with the successive affinity chromatography‐based assay, renamed as G‐quadruplex on Controlled Pore Glass (G4‐CPG) assay. Here we describe these assays and their applications to the screening of several libraries of chemically different putative G4 ligands. Finally, ongoing studies and outlook of our G4‐CPG assay are reported.

Targeting KRAS Regulation with PolyPurine Reverse Hoogsteen Oligonucleotides.

KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4mid structure downregulates transcription of KRAS; the function and formation of other G4s within the gene are unknown. Herein we identify three putative G4-forming sequences (G4FS) within the KRAS gene, explore their G4 formation, and develop oligonucleotides targeting these three regions and the G4mid forming sequence. We tested Polypurine Reverse Hoogsteen hairpins (PPRHs) for their effects on KRAS regulation via enhancing G4 formation or displacing G-rich DNA strands, downregulating KRAS transcription and mediating an anti-proliferative effect. Five PPRH were designed, two against the KRAS promoter G4mid and three others against putative G4FS in the distal promoter, intron 1 and exon 5. PPRH binding was confirmed by gel electrophoresis. The effect on KRAS transcription was examined by luciferase, FRET Melt2, qRT-PCR. Cytotoxicity was evaluated in pancreatic and ovarian cancer cells. PPRHs decreased activity of a luciferase construct driven by the KRAS promoter. PPRH selectively suppressed proliferation in KRAS dependent cancer cells. PPRH demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound, NSC 317605, in KRAS-dependent pancreatic cells. PPRHs selectively stabilize G4 formation within the KRAS mid promoter region and represent an innovative approach to both G4-stabilization and to KRAS modulation with potential for development into novel therapeutics.

Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands.

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.

G-Quadruplexes Formation at the Upstream Region of Replication Origin (OriL) of the Pseudorabies Virus: Implications for Antiviral Targets

Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease, which still causes large economic losses for the swine industry. Therefore, it is urgent to find a new strategy to prevent and control PRV infection. Previous studies have proven that guanine (G)-rich DNA or RNA sequences in some other viruses’ genomes have the potential to form G-quadruplex (G4), which serve as promising antivirus targets. In this study, we identified two novel G4-forming sequences, OriL-A and OriL-S, which are located at the upstream origin of replication (OriL) in the PRV genome and conserved across 32 PRV strains. Circular dichroism (CD) spectroscopy and a gel electrophoresis assay showed that the two G-rich sequences can fold into parallel G4 structures in vitro. Moreover, fluorescence resonance energy transfer (FRET) melting and a Taq polymerase stop assay indicated that the G4 ligand PhenDC3 has the capacity to bind and stabilize the G4. Notably, the treatment of PRV-infected cells with G4-stabilizer PhenDC3 significantly inhibited PRV DNA replication in host cells but did not affect PRV’s attachment and entry. These results not only expand our knowledge about the G4 characteristics in the PRV genome but also suggest that G4 may serve as an innovative therapeutic target against PRV.

Direct genome-wide identification of G-quadruplex structures by whole-genome resequencing

We present a user-friendly and transferable genome-wide DNA G-quadruplex (G4) profiling method that identifies G4 structures from ordinary whole-genome resequencing data by seizing the slight fluctuation of sequencing quality. In the human genome, 736,689 G4 structures were identified, of which 45.9% of all predicted canonical G4-forming sequences were characterized. Over 89% of the detected canonical G4s were also identified by combining polymerase stop assays with next-generation sequencing. Testing using public datasets of 6 species demonstrated that the present method is widely applicable. The detection rates of predicted canonical quadruplexes ranged from 32% to 58%. Because single nucleotide variations (SNVs) influence the formation of G4 structures and have individual differences, the given method is available to identify and characterize G4s genome-wide for specific individuals.

The role of G-Quadruplex DNA in Paraspeckle formation in cancer

Paraspeckles are RNA–protein structures within the nucleus of mammalian cells, capable of orchestrating various biochemical processes. An overexpression of the architectural component of paraspeckles, a long non-coding RNA called NEAT1 (Nuclear Enriched Abundant Transcript 1), has been linked to a variety of cancers and is often associated with poor patient prognosis. Thus, there is an accumulating interest in the role of paraspeckles in carcinogenesis, however there is a limited understanding of how NEAT1 expression is regulated. Here, we demonstrate that both nuclear G-quadruplex (G4) and paraspeckle formation are significantly increased in a human breast cancer cell line compared to non-tumorigenic breast cells. Moreover, we identified and characterized G4-forming sequences within the NEAT1 promoter and demonstrate stabilization of G4 DNA with a G4-stabilizing small molecule results in a significant alteration in both paraspeckle formation and NEAT1 expression. This G4-mediated alteration of NEAT1 at both the transcriptional and post-transcriptional levels was evident in U2OS osteosarcoma cells, MCF-7 breast adenocarcinoma and MDA-MB-231 triple negative breast cancer cells.

FeII Metallohelices Stabilize DNA G-Quadruplexes and Downregulate the Expression of G-Quadruplex-Regulated Oncogenes.

DNA G-quadruplexes (G4s) have been identified within the promoter regions of many proto-oncogenes. Thus, G4s represent attractive targets for cancer therapy, and the design and development of new drugs as G4 binders is a very active field of medicinal chemistry. Here, molecular biophysics and biology methods were employed to investigate the interaction of chiral metallohelices with a series of four DNA G4s (hTelo, c-myc, c-kit1, c-kit2) that are formed by the human telomeric sequence (hTelo) and in the promoter regions of c-MYC and c-KIT proto-oncogenes. We show that the investigated water-compatible, optically pure metallohelices, which are made by self-assembly of simple nonpeptidic organic components around FeII ions and exhibit bioactivity emulating the natural systems, bind with high affinity to G4 DNA and much lower affinity to duplex DNA. Notably, both enantiomers of a metallohelix containing a m-xylenyl bridge (5 b) were found to effectively inhibit primer elongation catalyzed by Taq DNA polymerase by stabilizing G4 structures formed in the template strands containing c-myc and c-kit2 G4-forming sequences. Moreover, both enantiomers of 5 b downregulated the expression of c-MYC and c-KIT oncogenes in human embryonic kidney cells at mRNA and protein levels. As metallohelices also bind alternative nucleic acid structures, they hold promise as potential multitargeted drugs.

Mechanical diversity and folding intermediates of parallel-stranded G-quadruplexes with a bulge.

A significant number of sequences in the human genome form noncanonical G-quadruplexes (G4s) with bulges or a guanine vacancy. Here, we systematically characterized the mechanical stability of parallel-stranded G4s with a one to seven nucleotides bulge at various positions. Our results show that G4-forming sequences with a bulge form multiple conformations, including fully-folded G4 with high mechanical stability (unfolding forces > 40 pN), partially-folded intermediates (unfolding forces < 40 pN). The folding probability and folded populations strongly depend on the positions and lengths of the bulge. By combining a single-molecule unfolding assay, dimethyl sulfate (DMS) footprinting, and a guanine-peptide conjugate that selectively stabilizes guanine-vacancy-bearing G-quadruplexes (GVBQs), we identified that GVBQs are the major intermediates of G4s with a bulge near the 5′ or 3′ ends. The existence of multiple structures may induce different regulatory functions in many biological processes. This study also demonstrates a new strategy for selectively stabilizing the intermediates of bulged G4s to modulate their functions.

G-Quadruplexes as pathogenic drivers in neurodegenerative disorders.

G-quadruplexes (G4s), higher-order DNA and RNA secondary structures featuring guanine-rich nucleic acid sequences with various conformations, are widely distributed in the human genome. These structural motifs are known to participate in basic cellular processes, including transcription, splicing, and translation, and their functions related to health and disease are becoming increasingly recognized. In this review, we summarize the landscape of G4s involved in major neurodegenerative disorders, describing the genes that contain G4-forming sequences and proteins that have high affinity for G4-containing elements. The functions of G4s are diverse, with potentially protective or deleterious effects in the pathogenic cascades of various neurological diseases. While the studies of the functions of G4s in vivo, including those involved in pathophysiology, are still in their early stages, we will nevertheless discuss the evidence pointing to their biological relevance. A better understanding of this unique structural element in the biological context is important for unveiling its potential roles in the pathogenesis of diseases such as neurodegeneration and for designing new diagnostic and therapeutic strategies.

PfGBP2is a novel G‐quadruplex binding protein inPlasmodium falciparum

Guanine-quadruplexes (G4s) are non-canonical DNA structures that can regulate key biological processes such as transcription, replication and telomere maintenance in several organisms including eukaryotes, prokaryotes and viruses. Recent reports have identified the presence of G4s within the AT-rich genome of Plasmodium falciparum, the protozoan parasite causing malaria. In Plasmodium, potential G4-forming sequences (G4FS) are enriched in the telomeric and sub-telomeric regions of the genome where they are associated with telomere maintenance and recombination events within virulence genes. However, there is a little understanding about the biological role of G4s and G4-binding proteins. Here, we provide the first snapshot of G4-interactome in P. falciparum using DNA pull-down assay followed by LC-MS/MS. Interestingly, we identified ~24 potential G4-binding proteins (G4-BP) that bind to a stable G4FS (AP2_G4). Furthermore, we characterised the role of G-strand binding protein 2 (PfGBP2), a putative telomere-binding protein in P. falciparum. We validated the interaction of PfGBP2 with G4 in vitro as well as in vivo. PfGBP2 is expressed throughout the intra-erythrocytic developmental cycle and is essential for the parasites in the presence of G4-stabilising ligand, pyridostatin. Gene knockout studies showed the role of PfGBP2 in the expression of var genes. Taken together, this study suggests that PfGBP2 is a bona fide G4-binding protein, which is likely to be involved in the regulation of G4-related functions in these malarial parasites. In addition, this study sheds light on this understudied G4 biology in P. falciparum.

Photosensitizers Based on G-Quadruplex Ligand for Cancer Photodynamic Therapy.

G-quadruplex (G4) is the non-canonical secondary structure of DNA and RNA formed by guanine-rich sequences. G4-forming sequences are abundantly located in telomeric regions and in the promoter and untranslated regions (UTR) of cancer-related genes, such as RAS and MYC. Extensive research has suggested that G4 is a potential molecular target for cancer therapy. Here, we reviewed G4 ligands as photosensitizers for cancer photodynamic therapy (PDT), which is a minimally invasive therapeutic approach. The photosensitizers, such as porphyrins, were found to be highly toxic against cancer cells via the generation of reactive oxidative species (ROS) upon photo-irradiation. Several porphyrin derivatives and analogs, such as phthalocyanines, which can generate ROS upon photo-irradiation, have been reported to act as G4 ligands. Therefore, they have been implicated as promising photosensitizers that can selectively break down cancer-related DNA and RNA forming G4. In this review, we majorly focused on the potential application of G4 ligands as photosensitizers, which would provide a novel strategy for PDT, especially molecularly targeted PDT (mtPDT).

Targeting RNA G-Quadruplex in SARS-CoV-2: A Promising Therapeutic Target for COVID-19?

The COVID-19 pandemic caused by SARS-CoV-2 has become a global threat. Understanding the underlying mechanisms and developing innovative treatments are extremely urgent. G-quadruplexes (G4s) are important noncanonical nucleic acid structures with distinct biofunctions. Four putative G4-forming sequences (PQSs) in the SARS-CoV-2 genome were studied. One of them (RG-1), which locates in the coding sequence region of SARS-CoV-2 nucleocapsid phosphoprotein (N), has been verified to form a stable RNA G4 structure in live cells. G4-specific compounds, such as PDP (pyridostatin derivative), can stabilize RG-1 G4 and significantly reduce the protein levels of SARS-CoV-2 N by inhibiting its translation both in vitro and in vivo. This result is the first evidence that PQSs in SARS-CoV-2 can form G4 structures in live cells, and that their biofunctions can be regulated by a G4-specific stabilizer. This finding will provide new insights into developing novel antiviral drugs against COVID-19.

G-Quadruplexes in the Archaea Domain.

The importance of unusual DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, G-quadruplexes (G4s) have gained in popularity during the last decade, and their presence and functional relevance at the DNA and RNA level has been demonstrated in a number of viral, bacterial, and eukaryotic genomes, including humans. Here, we performed the first systematic search of G4-forming sequences in all archaeal genomes available in the NCBI database. In this article, we investigate the presence and locations of G-quadruplex forming sequences using the G4Hunter algorithm. G-quadruplex-prone sequences were identified in all archaeal species, with highly significant differences in frequency, from 0.037 to 15.31 potential quadruplex sequences per kb. While G4 forming sequences were extremely abundant in Hadesarchaea archeon (strikingly, more than 50% of the Hadesarchaea archaeon isolate WYZ-LMO6 genome is a potential part of a G4-motif), they were very rare in the Parvarchaeota phylum. The presence of G-quadruplex forming sequences does not follow a random distribution with an over-representation in non-coding RNA, suggesting possible roles for ncRNA regulation. These data illustrate the unique and non-random localization of G-quadruplexes in Archaea.