G-Quadruplexes Regulate miRNA Biogenesis in Live Zebrafish Embryos.

RNA guanine quadruplexes (G4s) regulate RNA functions, metabolism, and processing. G4s formed within precursors of microRNAs (pre-miRNAs) may impair pre-miRNAs maturation by Dicer, thus repressing mature miRNA biogenesis. As miRNAs are essential for proper embryonic development, we studied the role of G4s on miRNA biogenesis in vivo during zebrafish embryogenesis. We performed a computational analysis on zebrafish pre-miRNAs to find putative G4 forming sequences (PQSs). The precursor of the miRNA 150 (pre-miR-150) was found to contain an evolutionarily conserved PQS formed by three G-tetrads and able to fold in vitro as G4. MiR-150 controls the expression of myb, which shows a well-defined knock-down phenotype in zebrafish developing embryos. We microinjected zebrafish embryos with in vitro transcribed pre-miR-150 synthesized using either GTP (G-pre-miR-150) or 7-Deaza-GTP, a GTP analogue unable to form G4s (7DG-pre-miR-150). Compared to embryos injected with G-pre-miR-150, embryos injected with 7DG-pre-miR-150 showed higher levels of miRNA 150 (miR-150) and lower levels of myb mRNA and stronger phenotypes associated with myb knock-down. The incubation of pre-miR-150 prior to the injection with the G4 stabilizing ligand pyridostatin (PDS) reverted gene expression variations and rescued the phenotypes related to myb knock-down. Overall, results suggest that the G4 formed in pre-miR-150 functions in vivo as a conserved regulatory structure competing with the stem-loop structure necessary for miRNA biogenesis.

Metal-Organic Compounds as Anticancer Agents: Versatile Building Blocks for Selective Action on G-quadruplexes.

BACKGROUND: Since the 1980s, cancer research has focused primarily on developing new therapeutic agents targeting DNA alterations rather than understanding cancer as an integrated system composed of several modules. In this sense, G-quadruplex (G4) nucleic acids are a promising target for drug development for cancer therapy since they exist in the chromosomal telomeric sequences and the promoter regions of numerous genes. The G4 structures within telomeric DNA can inhibit telomerase activity and prevent the proliferation and immortalization of cancer cells. Furthermore, such G4 systems within the promoter regions of oncogenes can inhibit the transcription and expression of the oncogene. OBJECTIVE: The rational design of small molecules such as organic ligands and their metal- organic derivative compounds can stabilize G4 structures through different binding modes on several G4 DNA topologies. Metal-based compounds have demonstrated their competitiveness compared to organic molecules to distinguish G4 over the DNA duplex owing to their convenient coordination features, positive charge, and electron density promoted by organic ligand. RESULTS: This article is a comprehensive review of metal compounds G4-binders and their structural features that confer them the ability to recognize G-quartets and stabilize several DNA G4s. CONCLUSION: This stabilization can be achieved through extended square aromatic surfaces, increased hydrophobicity, different auxiliary ligands, axially coordinated ligands, and the nature of the metal center.

What's new about CNBP? Divergent functions and activities for a conserved nucleic acid binding protein.

BACKGROUND: Cellular nucleic acid binding protein (CNBP) is a conserved single-stranded nucleic acid binding protein present in most eukaryotes, but not in plants. Expansions in the CNBP gene cause myotonic dystrophy type 2. Initially reported as a transcriptional regulator, CNBP was then also identified acting as a translational regulator. SCOPE OF REVIEW: The focus of this review was to link the CNBP structural features and newly reported biochemical activities with the recently described biological functions, in the context of its pathological significance. MAJOR CONCLUSIONS: Several post-translational modifications affect CNBP subcellular localization and activity. CNBP participates in the transcriptional and translational regulation of a wide range of genes by remodeling single-stranded nucleic acid secondary structures and/or by modulating the activity of trans-acting factors. CNBP is required for proper neural crest and heart development, and plays a role in cell proliferation control. Besides, CNBP has been linked with neurodegenerative, inflammatory, and congenital diseases, as well as with tumor processes. GENERAL SIGNIFICANCE: This review provides an insight into the growing functions of CNBP in cell biology. A unique and robust mechanistic or biochemical connection among these roles has yet not been elucidated. However, the ability of CNBP to dynamically integrate signaling pathways and to act as nucleic acid chaperone may explain most of the roles and functions identified so far.

CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands.

The Coronavirus Disease 2019 (COVID-19) pandemic has become a global health emergency with no effective medical treatment and with incipient vaccines. It is caused by a new positive-sense RNA virus called severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). G-quadruplexes (G4s) are nucleic acid secondary structures involved in the control of a variety of biological processes including viral replication. Using several G4 prediction tools, we identified highly putative G4 sequences (PQSs) within the positive-sense (+gRNA) and negative-sense (-gRNA) RNA strands of SARS-CoV-2 conserved in related betacoronaviruses. By using multiple biophysical techniques, we confirmed the formation of two G4s in the +gRNA and provide the first evidence of G4 formation by two PQSs in the -gRNA of SARS-CoV-2. Finally, biophysical and molecular approaches were used to demonstrate for the first time that CNBP, the main human cellular protein bound to SARS-CoV-2 RNA genome, binds and promotes the unfolding of G4s formed by both strands of SARS-CoV-2 RNA genome. Our results suggest that G4s found in SARS-CoV-2 RNA genome and its negative-sense replicative intermediates, as well as the cellular proteins that interact with them, are relevant factors for viral genes expression and replication cycle, and may constitute interesting targets for antiviral drugs development.

Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA.

Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

Folding Stability of Pax9 Intronic G-Quadruplex Correlates with Relative Molar Size in Eutherians.

Eutherian dentition has been the focus of a great deal of studies in the areas of evolution, development, and genomics. The development of molar teeth is regulated by an antero-to-posterior cascade mechanism of activators and inhibitors molecules, where the relative sizes of the second (M2) and third (M3) molars are dependent of the inhibitory influence of the first molar (M1). Higher activator/inhibitor ratios will result in higher M2/M1 or M3/M1. Pax9 has been shown to play a key role in tooth development. We have previously shown that a G-quadruplex in the first intron of Pax9 can modulate the splicing efficiency. Using a sliding window approach with we analyzed the association of the folding energy (Mfe) of the Pax9 first intron with the relative molar sizes in 42 mammalian species, representing 9 orders. The Mfe of two regions located in the first intron of Pax9 were shown to be significantly associated with the M2/M1 and M3/M1 areas and mesiodistal lengths. The first region is located at the intron beginning and can fold into a stable G4 structure, whereas the second is downstream the G4 and 265 bp from intron start. Across species, the first intron of Pax9 varied in G-quadruplex structural stability. The correlations were further increased when the Mfe of the two sequences were added. Our results indicate that this region has a role in the evolution of the mammalian dental pattern by influencing the relative size of the molars.

Novel complexes with ONNO tetradentate coumarin schiff-base donor ligands: x-ray structures, DFT calculations, molecular dynamics and potential anticarcinogenic activity.

The synthesis of eight novel Zn(II), Co(II), Cu(II), Ni(II) and Pt(II) complexes (2-9) derived from the ONNO tetradentate coumarin Schiff-Base donor ligands, L1 and the novel L2, was performed. All compounds were characterized by analytical, spectrometry and spectroscopy techniques. Complexes 2-4 were also characterized by DFT calculations and the structures of 5 and 6 were determined by single-crystal X-ray diffraction analysis. A cytotoxicity study was carried out through an MTT assay in the carcinogenic cell line HeLa and the noncarcinogenic cell lines HFF-1 and HaCaT. The results indicated that among all the evaluated compounds, 2 and 6 presented the best anticarcinogenic potential against HeLa cells with an IC50 of 3.5 and 4.1 µM, respectively. In addition, classical molecular dynamics simulations were performed on the synthesized coordination compounds bound to G4 DNA architectures in the scope of shedding light on their inhibition mode and the most conserved interactions that may lead to the biological activity of the compounds.

DNA G-quadruplex stability, position and chromatin accessibility are associated with CpG island methylation.

CpG islands (CGI) are genomic regions associated with gene promoters and involved in gene expression regulation. Despite their high CpG content and unlike bulk genomic DNA methylation pattern, these regions are usually hypomethylated. So far, the mechanisms controlling the CGI methylation patterning remain unclear. G-quadruplex (G4) structures can inhibit DNA methyltransferases 1 enzymatic activity, leading to CGI hypomethylation. Our aim was to analyse the association of G4 forming sequences (G4FS) and CGI methylation as well as to determine the intrinsic and extrinsic characteristics of G4FS that may modulate this phenomenon. Using methylation data from human embryonic stem cells (hESCs) and three hESC-derived populations, we showed that hypomethylated CpGs located inside CGI (CGI/CpG) tend to be associated with highly stable G4FS (Minimum free energy ≤ -30 kcal·mol-1 ). The association of highly stable G4FS and hypomethylation tend to be stronger when these structures are located at shorter distances (~ < 150 bp) from GCI/CpGs, when G4FS and CpGs are located within open chromatin and G4FS are inside CGI. Moreover, this association is not strongly influenced by the CpG content of CGI. Conversely, highly methylated CGI/CpG tend to be associated with low stability G4FS. Although CpGs inside CGIs without a G4FS tend to be more methylated, high stability G4FS within CGI neighbourhood were associated with decreased methylation. In summary, our data indicate that G4FS may act as protective cis elements against CGI methylation, and this effect seems to be influenced by the G4FS folding potential, its presence within CGI, CpG distance from G4FS and chromatin accessibility.

G-quadruplex in animal development: Contribution to gene expression and genomic heterogeneity.

During animal development, gene expression is orchestrated by specific and highly evolutionarily conserved mechanisms that take place accurately, both at spatial and temporal levels. The last decades have provided compelling evidence showing that chromatin state plays essential roles in orchestrating most of the stages of development. The DNA molecule can adopt alternative structures different from the helical duplex architecture. G-rich DNA sequences can fold as intrastrand quadruple helix structures called G-quadruplexes or G4-DNA. G4 can also be formed in RNA molecules, such as mRNA, lncRNA and pre-miRNA. Emerging evidences suggest that G4s have crucial roles in a variety of biological processes, including transcription, recombination, replication, translation and chromosome stability. In this review, we have collected recent information gathered by various laboratories showing the important role of G4 DNA and RNA structures in several steps of animal development.

Human-specific features of the G-quadruplex in the androgen receptor gene promoter: A comparative structural and dynamics study.

The androgen receptor (AR) promoter contains guanine-rich regions that are able to fold into polymorphic G-quadruplex (GQ) structures, and whose deletion decreases AR gene transcription. Our attention was focused on this region because of the frequent termination of sequencing reactions during promoter methylation studies. UV and circular dichroism (CD) spectroscopy of synthetic oligonucleotides encompassing these guanine-rich regions suggested a parallel quadruplex topology with three guanine quartets and three side loops in the three cases. Melting curves revealed a lower thermostability of the human GQ compared to the rat/mouse QG structures, which is attributed to the presence of a longer central loop in the former. One molecular model is proposed for the highly similar sequences in the rat/mouse. Due to the polymorphism resulting from possible arrangements of the guanine tracts, two models were derived for the human GQ. Molecular dynamics (MD) simulations determined that both models for the human GQ had higher flexibility and lower stability than the rodent GQ models. These properties result from the presence of a longer central loop in the human GQ models, which contains 11 and 13 nucleotides, in comparison to the 2-nucleotide long loop in the rat/mouse GQ. Overall, the unveiled structural and dynamics features provide sufficient detail for the intelligent design of drugs targeting the human AR promoter.

"In-House" Production of Single Stranded Oligodeoxyribonucleotides.

The most widely used technique for the production of DNA aptamers/oligonucleotides is chemical synthesis. Despite its effectiveness, this technique cannot be performed "in house", making the user fully dependent on a supplier. In this work, we present a simplified method by which it is possible to enzymatically produce DNA aptamers "in house". This new method uses the rolling circle replication followed by a unique cleavage step using the SchI endonuclease. Potentially, any oligonucleotide can be produced by the enzymatic method proposed in this study. To illustrate, we present the production of three variations of the 31-TBA aptamer, a single stranded DNA which has anticoagulant action.

A simple G-quadruplex molecular beacon-based biosensor for highly selective detection of microRNA.

MicroRNAs (miRNAs) family members are usually different from each other in one-base variation. The high sequence homology poses a challenge for miRNA analysis with single-base selectivity. On the basis of G-quadruplex molecular beacons (G4MB) and duplex-specific nuclease (DSN), we developed a simple and highly selective amplification biosensor for miRNA detection. G4MB with a G4 motif stem is used as recognition probe. In the present of target miRNAs, G4MB hybridizes with target miRNA perfectly and forms a G4MB-miRNA duplex. Then, DSN subsequently cleaves the G4MB of the G4MB-miRNA duplex to recycle the target miRNA, which leads to fluorescence signal amplification. In the absence of target miRNAs, DSN can not digest the stem of G4MB because of the protection of G4 motif, which eliminates the false positive signal, and produces low fluorescence background. Importantly, the powerful discriminating abilities of both G4MB and DSN make the novel sensor suitable for miRNAs detection with high single-base selectivity. Comparing with traditional linear ssDNA probe-DSN-based method, the signal response of similar miRNA sequences with one-base difference has been reduced from 24% to 6% by using this G4MB-DSN-based method. Moreover, this simple sensor also exhibits a good applicability in cancer cell samples and a multiplex capability in one sample with different miRNA targets, making it a promising strategy for clinical diagnostics.

Tuning supramolecular G-quadruplexes with mono- and divalent cations.

Supramolecular G-quadruplexes (SGQs) are formed via the cation promoted self-assembly of guanine derivatives into stacks of planar hydrogen-bonded tetramers. Here, we present results on the formation of SGQs made from the 8-(m-acetylphenyl)-2'-deoxyguanosine (mAGi) derivative in the presence of various mono- and divalent cations. NMR and HR ESI-MS data indicate that varying the cation can efficiently tune the molecularity, the fidelity and stability (thermal and kinetic) of the resulting SGQs. The results show that, parallel to the previously reported potassium-templated hexadecamer (mAGi16·3K+), Na+, Rb+ and [Formula: see text] also promote the formation of similar supramolecules with high fidelity and molecularity. In contrast, the divalent cations Pb2+, Sr2+ and Ba2+ template the formation of octamers (mAGi8), with the latter two inducing higher thermal stabilities. Molecular dynamics simulations for the hexadecamers containing monovalent cations enabled critical insights that help explain the experimental observations.