Maintaining the health of the proteome is a critical cellular task. Recently, we found G-quadruplex (G4) nucleic acids are especially potent at preventing protein aggregation in vitro and could at least indirectly improve the protein folding environment of Escherichia coli. However, the roles of G4s in protein folding were not yet explored. Here, through in vitro protein folding experiments, we discover that G4s can accelerate protein folding by rescuing kinetically trapped intermediates to both native and near-native folded states. Time-course folding experiments in E. coli further demonstrate that these G4s primarily improve protein folding quality in E. coli as opposed to preventing protein aggregation. The ability of a short nucleic acid to rescue protein folding opens up the possibility of nucleic acids and ATP-independent chaperones to play considerable roles in dictating the ultimate folding fate of proteins.

Parasitic helminths infecting humans are highly prevalent infecting ∼2 billion people worldwide, causing inflammatory responses, malnutrition and anemia that are the primary cause of morbidity. In addition, helminth infections of cattle have a significant economic impact on livestock production, milk yield and fertility. The etiological agents of helminth infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms). G-quadruplexes (G4) are unusual nucleic acid structures formed by G-rich sequences that can be recognized by specific G4 ligands. Here we used the G4Hunter Web Tool to identify and compare potential G4 sequences (PQS) in the nuclear and mitochondrial genomes of various helminths to identify G4 ligand targets. PQS are nonrandomly distributed in these genomes and often located in the proximity of genes. Unexpectedly, a Nematode, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This species can tolerate high-stability G4 structures, which are not counter selected at all, in stark contrast to most other species. We experimentally confirmed G4 formation for sequences found in four different parasitic helminths. Small molecules able to selectively recognize G4 were found to bind to Schistosoma mansoni G4 motifs. Two of these ligands demonstrated potent activity both against larval and adult stages of this parasite.

Influenza viruses are dangerous pathogens. Seventy-Seven genomes of recently emerged genotype 4 reassortant Eurasian avian-like H1N1 virus (G4-EA-H1N1) are currently available. We investigated the presence and variation of potential G-quadruplex forming sequences (PQS), which can serve as targets for antiviral treatment.

Alternative splicing is central to metazoan gene regulation, but the regulatory mechanisms are incompletely understood. Here, we show that G-quadruplex (G4) motifs are enriched ~3-fold near splice junctions. The importance of G4s in RNA is emphasised by a higher enrichment for the non-template strand. RNA-seq data from mouse and human neurons reveals an enrichment of G4s at exons that were skipped following depolarisation induced by potassium chloride. We validate the formation of stable RNA G4s for three candidate splice sites by circular dichroism spectroscopy, UV-melting and fluorescence measurements. Moreover, we find that sQTLs are enriched at G4s, and a minigene experiment provides further support for their role in promoting exon inclusion. Analysis of >1,800 high-throughput experiments reveals multiple RNA binding proteins associated with G4s. Finally, exploration of G4 motifs across eleven species shows strong enrichment at splice sites in mammals and birds, suggesting an evolutionary conserved splice regulatory mechanism.

Environments surrounding G-rich sequences remarkably affect the conformations of these structures. A proper evaluation system mimicking the crowded environment in a cell with macromolecules should be developed to perform structural and functional studies on G-quadruplexes. In this study, the topology and stability of a G-quadruplex formed by human telomeric repeat sequences were investigated in a macromolecule-crowded environment created by polyethylene glycol 200 (PEG200), tumor cell extract, and Xenopus laevis egg extract. The interactions between small molecules and telomeric G-quadruplexes were also evaluated in the different systems. The results suggested that the actual behavior of G-quadruplex structures in cells extract is quite different from that in the PEG crowding system, and proteins or other factors in extracts might play a very important role in G-quadruplex structures.

rRNA is the single most abundant polymer in most cells. Mammalian rRNAs are nearly twice as large as those of prokaryotes. Differences in rRNA size are due to expansion segments, which contain extended tentacles in metazoans. Here we show that the terminus of an rRNA tentacle of Homo sapiens contains 10 tandem G-tracts that form highly stable G-quadruplexes in vitro. We characterized rRNA of the H. sapiens large ribosomal subunit by computation, circular dichroism, UV melting, fluorescent probes, nuclease accessibility, electrophoretic mobility shifts, and blotting. We investigated Expansion Segment 7 (ES7), oligomers derived from ES7, intact 28S rRNA, 80S ribosomes, and polysomes. We used mass spectrometry to identify proteins that bind to rRNA G-quadruplexes in cell lysates. These proteins include helicases (DDX3, CNBP, DDX21, DDX17) and heterogeneous nuclear ribonucleoproteins. Finally, by multiple sequence alignments, we observe that G-quadruplex-forming sequences are a general feature of LSU rRNA of Chordata but not, as far as we can tell, of other species. Chordata ribosomes present polymorphic tentacles with the potential to switch between inter- and intramolecular G-quadruplexes. To our knowledge, G-quadruplexes have not been reported previously in ribosomes.

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.

G-quadruplex (G4) DNA structures with a left-handed backbone progression have unique and conserved structural features. Studies on sequence dependency of the structures revealed the prerequisites and some minimal motifs required for left-handed G4 formation. To extend the boundaries, we explore the adaptability of left-handed G4s towards the existence of bulges. Here we present two X-ray crystal structures and an NMR solution structure of left-handed G4s accommodating one, two and three bulges. Bulges in left-handed G4s show distinct characteristics as compared to those in right-handed G4s. The elucidation of intricate structural details will help in understanding the possible roles and limitations of these unique structures.

G-quadruplex (G4) sites in the human genome frequently colocalize with CCCTC-binding factor (CTCF)-bound sites in CpG islands (CGIs). We aimed to clarify the role of G4s in CTCF positioning. Molecular modeling data suggested direct interactions, so we performed in vitro binding assays with quadruplex-forming sequences from CGIs in the human genome. G4s bound CTCF with Kd values similar to that of the control duplex, while respective i-motifs exhibited no affinity for CTCF. Using ChIP-qPCR assays, we showed that G4-stabilizing ligands enhance CTCF occupancy at a G4-prone site in STAT3 gene. In view of the reportedly increased CTCF affinity for hypomethylated DNA, we next questioned whether G4s also facilitate CTCF recruitment to CGIs via protecting CpG sites from methylation. Bioinformatics analysis of previously published data argued against such a possibility. Finally, we questioned whether G4s facilitate CTCF recruitment by affecting chromatin structure. We showed that three architectural chromatin proteins of the high mobility group colocalize with G4s in the genome and recognize parallel-stranded or mixed-topology G4s in vitro. One of such proteins, HMGN3, contributes to the association between G4s and CTCF according to our bioinformatics analysis. These findings support both direct and indirect roles of G4s in CTCF recruitment.

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.

About 10-15% of all human cancer cells employ a telomerase-independent recombination-based telomere maintenance method, known as alternative lengthening of telomere (ALT), of which the full mechanism remains incompletely understood. While implicated in previous studies as the initiating signals for ALT telomere repair, the prevalence of non-canonical nucleic acid structures in ALT cancers remains unclear. Extending earlier reports, we observe higher levels of DNA/RNA hybrids (R-loops) in ALT-positive (ALT+) compared to telomerase-positive (TERT+) cells. Strikingly, we observe even more pronounced differences for an associated four-stranded nucleic acid structure, G-quadruplex (G4). G4 signals are found at the telomere and are broadly associated with telomere length and accompanied by DNA damage markers. We establish an interdependent relationship between ALT-associated G4s and R-loops and confirm that these two structures can be spatially linked into unique structures, G-loops, at the telomere. Additionally, stabilization of G4s and R-loops cooperatively enhances ALT-activity. However, co-stabilization at higher doses resulted in cytotoxicity in a synergistic manner. Nuclear G4 signals are significantly and reproducibly different between ALT+ and TERT+ low-grade glioma tumours. Together, we present G4 as a novel hallmark of ALT cancers with potential future applications as a convenient biomarker for identifying ALT+ tumours and as therapeutic targets.

MicroRNAs (miRNAs) are small noncoding RNAs that repress the translation of their target genes. It has previously been shown that a target's availability to miRNA can be affected by its structure. G-quadruplexes (G4) are noncanonical structures adopted by G-rich nucleic acids that have been shown to have multiple biological functions. In this study, whether or not G4 structures' presence in the 3' UTRs of mRNAs can hinder miRNA binding was investigated. Putative G4 overlapping with predicted miRNAs' binding sites was searched for, and 44,294 hits were found in humans. The FADS2 mRNA/mir331-3p pair was selected as a model example. In-line probing and G4-specific fluorescent ligand experiments binding were performed and confirmed the presence of a G4 near the predicted miRNA binding site. Subsequent luciferase assays showed that the presence of the G4 prevents the binding of mir331-3p in cellulo. Together, these results served as proof of concept that a G4 structure present in a 3' UTR sequence should be taken into consideration when predicting miRNA binding sites.

G-Quadruplex (G4), as a non-canonical nucleic acid secondary structure, has been proved to be prevalent in genomes and plays important roles in many biological processes. Ligands targeting G4, especially small-molecular fluorescent light-up probes with selectivity for special conformations, are essential for studying the relationship between G4 folding and the cellular response. However, their development still remains challenging but is attracting massive attention. Here, we synthesized a new tetraphenylethene derivative, namely TPE-B, as a parallel G4 probe. Fluorescence experiments showed that TPE-B could give out a strong fluorescence response to the G4 structure. Moreover, it gave a much higher fluorescence intensity response to parallel G4s than anti-parallel ones, which indicated that TPE-B could serve as a special tool for probing parallel G4s. The circular dichroism (CD) spectra and melting curves showed that TPE-B could selectively bind and stabilize parallel G4s without changing their topology. ESI-MS studies showed that TPE-B could bind to parallel G4 with a 1 : 1 stoichiometry. The gel staining results showed that TPE-B was a good candidate for probing parallel G4s. Altogether, the TPE-B molecule may serve as a promising new probe that can discriminate parallel G4s.

G-quadruplexes (G4) are alternative nucleic acid structures involved in transcription, translation and replication. Aberrant G4 formation and stabilisation is linked to genome instability and cancer. G4 ligand treatment disrupts key biological processes leading to cell death. To discover genes and pathways involved with G4s and gain mechanistic insights into G4 biology, we present the first unbiased genome-wide study to systematically identify human genes that promote cell death when silenced by shRNA in the presence of G4-stabilising small molecules. Many novel genetic vulnerabilities were revealed opening up new therapeutic possibilities in cancer, which we exemplified by an orthogonal pharmacological inhibition approach that phenocopies gene silencing. We find that targeting the WEE1 cell cycle kinase or USP1 deubiquitinase in combination with G4 ligand treatment enhances cell killing. We also identify new genes and pathways regulating or interacting with G4s and demonstrate that the DDX42 DEAD-box helicase is a newly discovered G4-binding protein.

Independent surveys of human gene promoter regions have demonstrated an overrepresentation of G(3)X(n1)G3X(n2)G(3)X(n3)G(3) motifs which are known to be capable of forming intrastrand quadruple helix structures. In spite of the widely recognized importance of G-quadruplex structures in gene regulation and growing interest around this unusual DNA structure, there are at present only few such structures available in the Nucleic Acid Database. In the present work we generate by molecular modeling feasible G-quadruplex structures which may be useful for interpretation of experimental data.

G-quadruplex structures inhibit telomerase activity and must be disrupted for telomere elongation during S phase. It has been suggested that the replication protein A (RPA) could unwind and maintain single-stranded DNA in a state amenable to the binding of telomeric components. We show here that under near-physiological in vitro conditions, human RPA is able to bind and unfold G-quadruplex structures formed from a 21mer human telomeric sequence. Analyses by native gel electrophoresis, cross-linking and fluorescence resonance energy transfer indicate the formation of both 1:1 and 2:1 complexes in which G-quadruplexes are unfolded. In addition, quadruplex opening by hRPA is much faster than observed with the complementary DNA, demonstrating that this protein efficiently unfolds G-quartets. A two-step mechanism accounting for the binding of hRPA to G-quadruplexes is proposed. These data point to the involvement of hRPA in regulation of telomere maintenance.

Heme is an essential cofactor for many enzymes, but free heme is toxic and its levels are tightly regulated. G-quadruplexes bind heme avidly in vitro, raising the possibility that they may sequester heme in vivo. If so, then treatment that displaces heme from quadruplexes is predicted to induce expression of genes involved in iron and heme homeostasis. Here we show that PhenDC3, a G-quadruplex ligand structurally unrelated to heme, displaces quadruplex-bound heme in vitro and alters transcription in cultured human cells, upregulating genes that support heme degradation and iron homeostasis, and most strikingly causing a 30-fold induction of heme oxidase 1, the key enzyme in heme degradation. We propose that G-quadruplexes sequester heme to protect cells from the pathophysiological consequences of free heme.

The reaction mechanism by which the shelterin protein POT1 (Protection of Telomeres 1) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. Stopped-flow kinetic and spectroscopic titration studies, along with isothermal calorimetry, were used to show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is both fast (80 ms) and strong (-10.1 ± 0.3 kcal mol-1). In sharp contrast, kinetic studies showed the binding of POT1 to an initially folded 24 nt G-quadruplex structure is four orders of magnitude slower. Fluorescence, circular dichroism and analytical ultracentrifugation studies showed that POT1 binding is coupled to quadruplex unfolding, with a final complex with a stoichiometry of 2 POT1 per 24 nt DNA. The binding isotherm for the POT1-quadruplex interaction was sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provided an excellent quantitative fit to the experimental binding data. POT1 unfolded and bound to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid, parallel monomers or a 48 nt sequence with two contiguous quadruplexes), but did not avidly interact with duplex DNA or with other G-quadruplex structures. Finally, molecular dynamics simulations provided a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.

Maintaining proteome health is important for cell survival. Nucleic acids possess the ability to prevent protein aggregation more efficiently than traditional chaperone proteins. In this study, we explore the sequence specificity of the chaperone activity of nucleic acids. Evaluating over 500 nucleic acid sequences' effects on protein aggregation, we show that the holdase chaperone effect of nucleic acids is sequence-dependent. G-Quadruplexes prevent protein aggregation via quadruplex:protein oligomerization. They also increase the folded protein level of a biosensor in E. coli. These observations contextualize recent reports of quadruplexes playing important roles in aggregation-related diseases, such as fragile X and amyotrophic lateral sclerosis (ALS), and provide evidence that nucleic acids have the ability to modulate the folding environment of E. coli.

We previously identified quinoline-based oligoamide helical foldamers and a trimeric macrocycle as selective ligands of DNA quadruplexes. Their helical structures might permit targeting of the backbone loops and grooves of G-quadruplexes instead of the G-tetrads. Given the vast array of morphologies G-quadruplex structures can adopt, this might be a way to achieve sequence selective binding. Here, we describe the design and synthesis of molecules based on macrocyclic and helically folded oligoamides. We tested their ability to interact with the human telomeric G-quadruplex and an array of promoter G-quadruplexes by using FRET melting assay and single-molecule FRET. Our results show that they constitute very potent ligands--comparable to the best so far reported. Their modes of interaction differ from those of traditional tetrad binders, thus opening avenues for the development of molecules specific for certain G-quadruplex conformations.