G-quadruplex Formation Research Articles

Single-molecule Fluorescence Technique to Monitor the Co-transcriptional Formation of G-quadruplex and R-loop Structures.

G-quadruplexes (GQ) and R-loops are non-canonical nucleic acid structures related to gene regulation and genome instability that can be formed during transcription; however, their formation mechanisms remain elusive. To address this question, we developed a single-molecule fluorescence technique to monitor the formation of G-quadruplex and R-loop structures during transcription. Using this technique, we found that R-loop formation precedes GQ formation and that there exists a positive feedback loop between G-quadruplex and R-loop formation.

Reversible photo-regulation on the folding/unfolding of telomere G-quadruplexes with solid-state nanopores.

The formation of G-quadruplexes (G4) in human telomere and other important biological regions inhibits the replication and transcription of DNA, thereby influencing further cell proliferation. The investigation of G4 formation and unfolding is vital for understanding their modulation in biological processes and life science. Photo regulation is a facile and sensitive approach for monitoring the structures of biomacromolecules and material surface properties. The nanopore-based technique is also prevalent for label-free single-molecule characterization with high accuracy. This study provides a combination of solid-state nanopore technology with light-switch as a platform for the modulation of human telomere G4 formation and splitting under switchable light exposure. The introduction of molecular switch, namely azobenzene moiety at different positions of the DNA sequence influences the formation and stability of G4. Three azobenzenes immobilized on each of the G-quartet plane (hTelo-3azo-p) or four azobenzenes on the same plane (hTelo-4azo-4p) of the human telomere G4 sequence realized the reversible control of G4 folding/unfolding at the temporal scale upon photo regulation, and the formation and splitting of G4 with hTelo-4azo-4p is slower and not thorough compared to that with hTelo-3azo-p due to the coplanar steric hindrance. Moreover, the G4 formation recorded with the combined nanopore and photo-responsive approach was also characterized with fluorescence, and the variation in the fluorescence intensity of the NMM and G4 complex exhibited a different tendency under reverse light irradiation due to the distinct interactions of NMM with the azobenzene-modified G4. Our study demonstrated a controllable and sensitive way for the manipulation of G4 structures, which will be inspiring for the intervention of G4-related cell senescence, cancer diagnosis and drug exploration.

A novel minimal motif for left-handed G-quadruplex formation.

A recent study on the left-handed G-quadruplex (LHG4) DNA revealed a 12-nt minimal motif GTGGTGGTGGTG with the ability to independently form an LHG4 and to drive an adjacent sequence to LHG4 formation. Here we have identified a second LHG4-forming motif, GGTGGTGGTGTG, and determined the X-ray crystal structure of an LHG4 involving this motif. Our structural analysis indicated the role of split guanines and single thymine loops in promoting LHG4 formation.

A Reusable Electrochemical Aptasensor for the Sensitive Detection of Pb (II) with an Electrodeposited AuNP-Modified Electrode based on the Formation of a Target-Induced G-Quadruplex

A Reusable Electrochemical Aptasensor for the Sensitive Detection of Pb (II) with an Electrodeposited AuNP-Modified Electrode based on the Formation of a Target-Induced G-Quadruplex

GCG repeat expansion in <i>ARX</i> gene inhibits ARX protein translation through the ectopic formation of RNA G-quadruplex

GCG repeat expansion in <i>ARX</i> gene inhibits ARX protein translation through the ectopic formation of RNA G-quadruplex

The combined action of cations and anions of ionic liquids modulates the formation and stability of G-quadruplex DNA.

G-Quadruplex (Gq) formation and stabilization by any molecule is an essential requirement for its application in therapy, especially in oncology. Metal cations have shown higher propensity of the formation of the Gq structure and its stabilization. In this study, the role of both cations and anions of ionic liquids (ILs) on the Gq formation of human telomere (hTeloG) and its stability was investigated using spectroscopic and molecular dynamics simulation techniques. Irrespective of the nature of anions of ILs, tetramethylguanidinium (TMG) cations associated with different anions can form an antiparallel Gq structure in hTeloG. However, the propensity of the formation of an antiparallel Gq structure and its stability depend on the chain length of anions of ILs. Gq is significantly less stable in ILs having longer hydrocarbon chain anions compared to the short chain anions suggesting that the hydrophobicity of the anion plays a critical role in the stability and formation of the Gq structure by ILs. The data indicate that longer hydrocarbon chain anions of ILs preferably interact in the loop region of Gq through hydrophobic interaction which enhances the overall binding of the cation of ILs with Gq causing a decrease in the stacking energy between the G-quartets as well as Hoogsteen hydrogen bonds between the guanine bases leading to the destabilization of the antiparallel Gq structure.

Inhibition of protein synthesis through RNA-based tandem G-quadruplex formation.

We demonstrate that an RNA template containing eight GGG repeat sequences exhibits a unique tandem G-quadruplex structure in which two individual G-quadruplexes are aligned in close proximity. Because of their unexpected stability, tandem G-quadruplexes formed in the coding region of mRNA strands effectively inhibited in vitro protein synthesis.

G-Quadruplex Modulation of SP1 Functional Binding Sites at the KIT Proximal Promoter.

The regulation of conformational arrangements of gene promoters is a physiological mechanism that has been associated with the fine control of gene expression. Indeed, it can drive the time and the location for the selective recruitment of proteins of the transcriptional machinery. Here, we address this issue at the KIT proximal promoter where three G-quadruplex forming sites are present (kit1, kit2 and kit*). On this model, we focused on the interplay between G-quadruplex (G4) formation and SP1 recruitment. By site directed mutagenesis, we prepared a library of plasmids containing mutated sequences of the WT KIT promoter that systematically exploited different G4 formation attitudes and SP1 binding properties. Our transfection data showed that the three different G4 sites of the KIT promoter impact on SP1 binding and protein expression at different levels. Notably, kit2 and kit* structural features represent an on-off system for KIT expression through the recruitment of transcription factors. The use of two G4 binders further helps to address kit2-kit* as a reliable target for pharmacological intervention.

Superiority of an Asymmetric Perylene Diimide in Terms of Hydrosolubility, G-Quadruplex Binding, Cellular Uptake, and Telomerase Inhibition in Prostate Cancer Cells.

Perylene diimide (PDI) derivatives have been studied as G-quadruplex ligands that suppress telomerase activity by facilitating G-quadruplex formation of telomeric DNA and the hTERT promoter. PIPER, the prototypical PDI, reduces telomerase activity in lung and prostate cancer cells, leading to telomere shortening and cellular senescence of these cells. However, PIPER suffers from poor hydrosolubility and the propensity to aggregate at neutral pH. In this report, we synthesized a new asymmetric PDI, aPDI-PHis, which maintains one N-ethyl piperidine side chain of PIPER and has histidine as another side chain. The results show that aPDI-PHis is superior to its symmetric counterparts, PIPER and PDI-His, in terms of hydrosolubility, G-quadruplex binding, cellular uptake, and telomerase inhibition in prostate cancer cells. These results suggest that one N-ethyl piperidine side chain of PDI is sufficient for G-quadruplex binding, while another side chain can be tuned to elicit desirable properties. These findings might lead to better PDIs for use as anticancer drugs.

Structure of a DNA G-Quadruplex Related to Osteoporosis with a G-A Bulge Forming a Pseudo-loop.

Bone remodeling is a fine-tuned process principally regulated by a cascade triggered by interaction of receptor activator of NF-κB (RANK) and RANK ligand (RANKL). Excessive activity of the RANKL gene leads to increased bone resorption and can influence the incidence of osteoporosis. Although much has been learned about the intracellular signals activated by RANKL/RANK complex, significantly less is known about the molecular mechanisms of regulation of RANKL expression. Here, we report on the structure of an unprecedented DNA G-quadruplex, well-known secondary structure-mediated gene expression regulator, formed by a G-rich sequence found in the regulatory region of a RANKL gene. Solution-state NMR structural study reveals the formation of a three-layered parallel-type G-quadruplex characterized by an unique features, including a G-A bulge. Although a guanine within a G-tract occupies syn glycosidic conformation, bulge-forming residues arrange in a pseudo-loop conformation to facilitate partial 5/6-ring stacking, typical of G-quadruplex structures with parallel G-tracts orientation. Such distinctive structural features protruding from the core of the structure can represent a novel platform for design of highly specific ligands with anti-osteoporotic function. Additionally, our study suggests that the expression of RANKL gene may be regulated by putative folding of its G-rich region into non-B-DNA structure(s).

Crucial Role of the C-Terminal Domain of Hfq Protein in Genomic Instability.

G-rich DNA repeats that can form G-quadruplex structures are prevalent in bacterial genomes and are frequently associated with regulatory regions of genes involved in virulence, antigenic variation, and antibiotic resistance. These sequences are also inherently mutagenic and can lead to changes affecting cell survival and adaptation. Transcription of the G-quadruplex-forming repeat (G3T)n in E. coli, when mRNA comprised the G-rich strand, promotes G-quadruplex formation in DNA and increases rates of deletion of G-quadruplex-forming sequences. The genomic instability of G-quadruplex repeats may be a source of genetic variability that can influence alterations and evolution of bacteria. The DNA chaperone Hfq is involved in the genetic instability of these G-quadruplex sequences. Inactivation of the hfq gene decreases the genetic instability of G-quadruplex, demonstrating that the genomic instability of this regulatory element can be influenced by the E. coli highly pleiotropic Hfq protein, which is involved in small noncoding RNA regulation pathways, and DNA organization and packaging. We have shown previously that the protein binds to and stabilizes these sequences, increasing rates of their genomic instability. Here, we extend this analysis to characterize the role of the C-terminal domain of Hfq protein in interaction with G-quadruplex structures. This allows to better understand the function of this specific region of the Hfq protein in genomic instability.

Widespread intron retention impairs protein homeostasis in C9orf72 ALS brains.

The GGGGCC hexanucleotide expansion in C9orf72 (C9) is the most frequent known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet a clear understanding of how C9 fits into the broader context of ALS/FTD pathology has remained lacking. The repetitive RNA derived from the C9 repeat is known to sequester hnRNPH, a splicing regulator, into insoluble aggregates, resulting in aberrant alternative splicing. Furthermore, hnRNPH insolubility and altered splicing of a robust set of targets have been observed to correlate in C9 and sporadic ALS/FTD patients alike, suggesting that changes along this axis are a core feature of disease pathogenesis. Here, we characterize previously uncategorized RNA splicing defects involving widespread intron retention affecting almost 2000 transcripts in C9ALS/FTD brains exhibiting a high amount of sequestered, insoluble hnRNPH. These intron retention events appear not to alter overall expression levels of the affected transcripts but rather the protein-coding regions. These retained introns affect transcripts in multiple cellular pathways predicted to be involved in C9 as well as sporadic ALS/FTD etiology, including the proteasomal and autophagy systems. The retained intron pre-mRNAs display a number of characteristics, including enrichment of hnRNPH-bound splicing enhancer motifs and a propensity for G-quadruplex (G-Q) formation, linking the defective splicing directly to high amounts of sequestered hnRNPH. Together, our results reveal previously undetected splicing defects in high insoluble hnRNPH-associated C9ALS brains, suggesting a feedback between effective RNA-binding protein dosage and protein quality control in C9, and perhaps all, ALS/FTD.

Development of Specific Inhibitors for Oncogenic Phosphatase PPM1D by Using Ion-Responsive DNA Aptamer Library

(1) Background: Ser/Thr protein phosphatase PPM1D is an oncogenic protein. In normal cells, however, PPM1D plays essential roles in spermatogenesis and immune response. Hence, it is necessary to develop novel PPM1D inhibitors without side effects on normal cells. Stimuli-responsive molecules are suitable for the spatiotemporal regulation of inhibitory activity. (2) Methods: In this study, we designed an ion-responsive DNA aptamer library based on G-quadruplex DNA that can change its conformation and function in response to monovalent cations. (3) Results: Using this library, we identified the PPM1D specific inhibitor M1D-Q5F aptamer. The M1D-Q5F aptamer showed anti-cancer activity against breast cancer MCF7 cells. Interestingly, the induction of the structural change resulting in the formation of G-quadruplex upon stimulation by monovalent cations led to the enhancement of the inhibitory activity and binding affinity of M1D-Q5F. (4) Conclusions: These data suggest that the M1D-Q5F aptamer may act as a novel stimuli-responsive anti-cancer agent.

Human ribosomal G-quadruplexes regulate heme bioavailability

The in vitro formation of stable G-quadruplexes (G4s) in human rRNA was recently reported. However, their formation in cells and their cellular roles were not resolved. Here, by taking a chemical biology approach that integrates results from immunofluorescence, G4 ligands, heme-affinity reagents, and a genetically encoded fluorescent heme sensor, we report that human ribosomes can form G4s in vivo that regulate heme bioavailability. Immunofluorescence experiments indicate that the vast majority of extra-nuclear G4s are associated with rRNA. Moreover, titrating human cells with a G4 ligand alters the ability of ribosomes to bind heme and disrupts cellular heme bioavailability as measured by a genetically encoded fluorescent heme sensor. Overall, these results suggest that ribosomes play a role in regulating heme homeostasis.

Novel electrochemical biosensing platform for microRNA detection based on G-quadruplex formation in nanochannels

Ultrasensitive detection of microRNA is crucial important in the diagnosis of related diseases. By immobilizing the capture probes in nanochannels, we developed a label-free electrochemical biosensing platform based on guanine-quadruplex (G-quadruplex) formation in nanochannels. The designed capture probe contains both the microRNA-21 (MiR-21) recognition sequence and guanine-rich sequence. The presence of the target MiR-21 would trigger the enzymatic cleaving of the RNA/DNA heteroduplexes which led some of the guanine-rich sequences escape from the nanochannels and thus reduced the formation of the G-quadruplex/hemin complex. Therefore, the quantity of methylene blue fluxed through the nanochannels changed because of the variation of the steric hindrance. A optimized carbon nanofibers modified electrode was applied to monitor the quantity difference of methylene blue. In this way, the biosensing platform realized the label-free electrochemical detection of MiR-21 with a ultralow detection limit of 0.5 aM. Meanwhile, this biosensing platform was assessed for MiR-21 detection in serum samples. The biosensing platform can be readily extended to the assay of other scarcetumor-related genes.

Highly Sensitive MicroRNA Detection by Coupling Nicking-Enhanced Rolling Circle Amplification with MoS2 Quantum Dots.

In this work, a label-free and highly sensitive fluorescence assay was constructed for microRNA detection. Nicking-enhanced rolling circle amplification (RCA) induced by G-quadruplex formation is coupled with inner filter effect (IFE)-based quenching effects of MoS2 quantum dots (MoS2 QDs). The padlock probe contains a recognition sequence to target microRNA and an accessible nicking site. The padlock probe is cyclized upon hybridization with target microRNA. Sequentially, amplification initiates a production of a long-concatenated sequence of circular probes. Abundant G-quadruplex sequences are produced via the nicking process and then used as the trigger to initiate the next RCA. In the presence of hemin, numerous hemin/G-quadruplex DNAzymes are formed, which catalyze the oxidation of o-phenylenediamine (OPD) into the colored product 2,3-diaminophenazine, resulting in quenching of the fluorescence of MoS2 QDs. This sensing strategy enables detection of microRNA let-7a with high selectivity and a detection limit of 4.6 fM. The as-prepared sensor was applied for detecting microRNA let-7a in dilute human serum samples and achieved a satisfactory recovery rate, demonstrating its potential in clinic diagnosis of microRNA-associated disease and biochemical research.

Stabilization of human telomeric RNA G-quadruplex by the water-compatible optically pure and biologically-active metallohelices

RNA G-quadruplexes have been suggested to play key roles in fundamental biological processes and are linked to human diseases. Thus, they also represent good potential therapeutic targets. Here, we describe, using the methods of molecular biophysics, interactions of a series of biologically-active supramolecular cationic metallohelices with human telomeric RNA G-quadruplex. We demonstrate that the investigated metallohelices bind with a high affinity to human telomeric RNA G-quadruplex and that their binding selectivity considerably differs depending on the dimensions and overall shape of the metallohelices. Additionally, the investigated metallohelices inhibit DNA synthesis on the RNA template containing four repeats of the human telomeric sequence by stabilizing the RNA G-quadruplex structure. Collectively, the results of this study suggest that stabilization of RNA sequences capable of G-quadruplex formation by metallohelices investigated in this work might contribute to the mechanism of their biological activity.

A 5' UTR GGN repeat controls localisation and translation of a potassium leak channel mRNA through G-quadruplex formation.

RNA G-quadruplexes (G4s) are secondary structures proposed to function as regulators of post-transcriptional mRNA localisation and translation. G4s within some neuronal mRNAs are known to control distal localisation and local translation, contributing to distinct local proteomes that facilitate the synaptic remodelling attributed to normal cellular function. In this study, we characterise the G4 formation of a (GGN)13 repeat found within the 5′ UTR of the potassium 2-pore domain leak channel Task3 mRNA. Biophysical analyses show that this (GGN)13 repeat forms a parallel G4 in vitro exhibiting the stereotypical potassium specificity of G4s, remaining thermostable under physiological ionic conditions. Through mouse brain tissue G4-RNA immunoprecipitation, we further confirm that Task3 mRNA forms a G4 structure in vivo. The G4 is inhibitory to translation of Task3 in vitro and is overcome through activity of a G4-specific helicase DHX36, increasing K+ leak currents and membrane hyperpolarisation in HEK293 cells. Further, we observe that this G4 is fundamental to ensuring delivery of Task3 mRNA to distal primary cortical neurites. It has been shown that aberrant Task3 expression correlates with neuronal dysfunction, we therefore posit that this G4 is important in regulated local expression of Task3 leak channels that maintain K+ leak within neurons.

RNA G-quadruplex structures exist and function in vivo in plants

BackgroundGuanine-rich sequences are able to form complex RNA structures termed RNA G-quadruplexes in vitro. Because of their high stability, RNA G-quadruplexes are proposed to exist in vivo and are suggested to be associated with important biological relevance. However, there is a lack of direct evidence for RNA G-quadruplex formation in living eukaryotic cells. Therefore, it is unclear whether any purported functions are associated with the specific sequence content or the formation of an RNA G-quadruplex structure.ResultsUsing rG4-seq, we profile the landscape of those guanine-rich regions with the in vitro folding potential in the Arabidopsis transcriptome. We find a global enrichment of RNA G-quadruplexes with two G-quartets whereby the folding potential is strongly influenced by RNA secondary structures. Using in vitro and in vivo RNA chemical structure profiling, we determine that hundreds of RNA G-quadruplex structures are strongly folded in both Arabidopsis and rice, providing direct evidence of RNA G-quadruplex formation in living eukaryotic cells. Subsequent genetic and biochemical analyses show that RNA G-quadruplex folding is able to regulate translation and modulate plant growth.ConclusionsOur study reveals the existence of RNA G-quadruplex in vivo and indicates that RNA G-quadruplex structures act as important regulators of plant development and growth.

Recent advances in the development of ligands specifically targeting telomeric multimeric G-quadruplexes

Long human telomeric DNA sequence could form higher-order G-quadruplex structures, namely telomeric multimeric G-quadruplexes. The formation of telomeric multimeric G-quadruplexes has been demonstrated. Several efforts have been devoted to the development of ligands targeting telomeric multimeric G-quadruplexes in recent years. The reported ligands specifically targeting telomeric multimeric G-quadruplexes exhibited either high anticancer activity with effective stabilization ability or distinct fluorescence responses to telomeric multimeric G-quadruplexes. In this review, the ligands including three types of small molecules are discussed which focus on their structural features and binding modes. Furthermore, we put forward the promising prospects and current challenges. This presented review might provide new strategies to exploit more sophisticated ligands targeting telomeric multimeric G-quadruplexes.