Chemistry and Biology of Noncanonical Nucleic Acid Structures: From Physicochemical Properties to Therapeutic Applications.

The aim of this Special Issue is to highlight significant and new aspects concerning the chemistry and biology of noncanonical nucleic acid structures, with emphasis on their structure, stability, and conformational equilibria, as well as on the biological relevance of their interactions with proteins and ligands [...].

Ag+-Mediated Folding of Long Polyguanine Strands to Double and Quadruple Helixes.

Metal-mediated base pairing of DNA has been a topic of extensive research spanning over more than four decades. Precise positioning of a single metal ion by predetermining the DNA sequence, as well as improved conductivity offered by the ions, make these structures interesting candidates in the context of using DNA in nanotechnology. Here, we report the formation and characterization of conjugates of long (kilo bases) homoguanine DNA strands with silver ions. We demonstrate using atomic force microscopy (AFM) and scanning tunneling microscope (STM) that binding of silver ions leads to folding of homoguanine DNA strands in a "hairpin" fashion to yield double-helical, left-handed molecules composed of G-G base pairs each stabilized by a silver ion. Further folding of the DNA-silver conjugate yields linear molecules in which the two halves of the double helix are twisted one against the other in a right-handed fashion. Quantum mechanical calculations on smaller molecular models support the helical twist directions obtained by the high resolution STM analysis. These long guanine-based nanostructures bearing a chain of silver ions have not been synthesized and studied before and are likely to possess conductive properties that will make them attractive candidates for nanoelectronics.

A reverse translational approach reveals the protective roles of Mangifera indica in inflammatory bowel disease.

Inflammatory bowel diseases (IBDs) are chronic intestinal disorders often characterized by a dysregulation of T cells, specifically T helper (Th) 1, 17 and T regulatory (Treg) repertoire. Increasing evidence demonstrates that dietary polyphenols from Mangifera indica L. extract (MIE, commonly known as mango) mitigate intestinal inflammation and splenic Th17/Treg ratio. In this study, we aimed to dissect the immunomodulatory and anti-inflammatory properties of MIE using a reverse translational approach, by initially using blood from an adult IBD inception cohort and then investigating the mechanism of action in a preclinical model of T cell-driven colitis. Of clinical relevance, MIE modulates TNF-α and IL-17 levels in LPS spiked sera from IBD patients as an ex vivo model of intestinal barrier breakdown. Preclinically, therapeutic administration of MIE significantly reduced colitis severity, pathogenic T-cell intestinal infiltrate and intestinal pro-inflammatory mediators (IL-6, IL-17A, TNF-α, IL-2, IL-22). Moreover, MIE reversed colitis-induced gut permeability and restored tight junction functionality and intestinal metabolites. Mechanistic insights revealed MIE had direct effects on blood vascular endothelial cells, blocking TNF-α/IFN-γ-induced up-regulation of COX-2 and the DP2 receptors. Collectively, we demonstrate the therapeutic potential of MIE to reverse the immunological perturbance during the onset of colitis and dampen the systemic inflammatory response, paving the way for its clinical use as nutraceutical and/or functional food.

In vivo biological validation of in silico analysis: A novel approach for predicting the effects of TLR4 exon 3 polymorphisms on brucellosis.

The role of the Toll-like receptor 4 (TLR4) is of recognising intracellular and extracellular pathogens and of activating the immune response. This process can be compromised by single nucleotide polymorphisms (SNPs) which might affect the activity of several TLRs. The aim of this study is of ascertaining whether SNPs in the TLR4 of Bubalus bubalis infected by Brucella abortus, compromise the protein functionality. For this purpose, a computational analysis was performed. Next, computational predictions were confirmed by performing genotyping analysis. Finally, NMR-based metabolomics analysis was performed to identify potential biomarkers for brucellosis. The results indicate two SNPs (c. 672 A > C and c. 902 G > C) as risk factor for brucellosis in Bubalus bubalis, and three metabolites (lactate, 3-hydroxybutyrate and acetate) as biological markers for predicting the risk of developing the disease. These metabolites, together with TLR4 structural modifications in the MD2 interaction domain, are a clear signature of the immune system alteration during diverse Gram-negative bacterial infections. This suggests the possibility to extend this study to other pathogens, including Mycobacterium tuberculosis. In conclusion, this study combines multidisciplinary approaches to evaluate the biological and structural effects of SNPs on protein function.

Volatile organic compounds (VOCs) from diffuse degassing areas: Interstitial soil gases as message bearers from deep hydrothermal reservoirs.

The chemical composition of volatile organic compounds (VOCs) in interstitial soil gases from hydrothermal areas is commonly shaped by both deep hydrothermal conditions (e.g., temperature, redox, sulfur fugacity) and shallow secondary processes occurring near the soil-atmosphere interface. Caldara di Manziana and Solfatara di Nepi, i.e., two hydrothermal systems characterized by diverse physicochemical conditions located in the Sabatini Volcanic District and Vicano-Cimino Volcanic District, respectively (Central Italy), were investigated to evaluate the capability of VOCs in soil gases to preserve information from the respective feeding deep fluid reservoirs. Hierarchical cluster analyses and robust principal component analyses allowed recognition of distinct groups of chemical parameters of soil gases collected from the two study areas. The compositional dissimilarities from the free-gas discharges were indeed reflected by the chemical features of soil gases collected from each site, despite the occurrence of shallow processes, e.g., air mixing and microbial degradation processes, affecting VOCs. Four distinct groups of VOCs were recognized suggesting similar sources and/or geochemical behaviors, as follows: (i) S-bearing compounds, whose abundance (in particular that of thiophenes) was strictly dependent on the sulfur fugacity in the feeding system; (ii) C4,5,7+ alkanes, n-hexane, cyclics and alkylated aromatics, related to relatively low-temperature conditions at the gas source; (iii) C2,3 alkanes, benzene, benzaldehyde and phenol, i.e., stable compounds and thermal degradation products; and (iv) aliphatic O-bearing compounds, largely influenced by shallow processes within the soil. However, they maintain a chemical speciation that preserves a signature derived from the supplying deep-fluids, with aldehydes and ketones becoming more enriched after intense interaction of the hypogenic fluids with shallow aquifers. Accordingly, the empirical results of this study suggest that the chemical composition of VOCs in soil gases from hydrothermal areas provides insights into both deep source conditions and fluid circulation dynamics, identifying VOCs as promising geochemical tracers for geothermal exploration.

G-quadruplexes in cancer-related gene promoters: from identification to therapeutic targeting.

INTRODUCTION: Guanine-rich DNA sequences can fold into four-stranded noncanonical secondary structures called G-quadruplexes (G4s) which are widely distributed in functional regions of the human genome, such as telomeres and gene promoter regions. Compelling evidence suggests their involvement in key genome functions such as gene expression and genome stability. Notably, the abundance of G4-forming sequences near transcription start sites suggests their potential involvement in regulating oncogenes. AREAS COVERED: This review provides an overview of current knowledge on G4s in human oncogene promoters. The most representative G4-binding ligands have also been documented. The objective of this work is to present a comprehensive overview of the most promising targets for the development of novel and highly specific anticancer drugs capable of selectively impacting the expression of individual or a limited number of genes. EXPERT OPINION: Modulation of G4 formation by specific ligands has been proposed as a powerful new tool to treat cancer through the control of oncogene expression. Actually, most of G4-binding small molecules seem to simultaneously target a range of gene promoter G4s, potentially influencing several critical driver genes in cancer, thus producing significant therapeutic benefits.

Unveiling the interaction between DNA G-quadruplexes and RG-rich peptides.

G-quadruplexes are non-canonical DNA secondary structures formed within guanine-rich strands that play important roles in various biological processes, including gene regulation, telomere maintenance and DNA replication. The biological functions and formation of these DNA structures are strictly controlled by several proteins that bind and stabilize or resolve them. Many G-quadruplex-binding proteins feature an arginine and glycine-rich motif known as the RGG or RG-rich motif. Although this motif plays a crucial role in the recognition of such non-canonical structures, their interaction is still poorly understood. Here, we employed a combination of several biophysical techniques to provide valuable insights into the interaction between a peptide containing an RGG motif shared by numerous human G-quadruplex-binding proteins (NIQI) and various biologically relevant G-quadruplex DNA structures with different topologies. We also shed light on the key amino acids involved in the binding process. Our findings contribute to lay the basis for the development of a new class of peptide-based G-quadruplex ligands as an alternative to small molecules. These ligands may serve as valid tools for interfering in DNA-protein interactions, with potential therapeutic applications.

Hit identification of novel small molecules interfering with MALAT1 triplex by a structure-based virtual screening.

Nowadays, RNA is an attractive target for the design of new small molecules with different pharmacological activities. Among several RNA molecules, long noncoding RNAs (lncRNAs) are extensively reported to be involved in cancer pathogenesis. In particular, the overexpression of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in the development of multiple myeloma (MM). Starting from the crystallographic structure of the triple-helical stability element at the 3'-end of MALAT1, we performed a structure-based virtual screening of a large commercial database, previously filtered according to the drug-like properties. After a thermodynamic analysis, we selected five compounds for the in vitro assays. Compound M5, characterized by a diazaindene scaffold, emerged as the most promising molecule enabling the destabilization of the MALAT1 triplex structure and antiproliferative activity on in vitro models of MM. M5 is proposed as a lead compound to be further optimized for improving its affinity toward MALAT1.

Comparison of the biological effects of gadodiamide (Omniscan) and gadoteridol (ProHance) by means of multi-organ and plasma metabolomics.

Gadolinium-based contrast agents (GBCAs) are massively employed in radiology to increase the diagnostic power of MRI. However, investigations aiming at detecting possible metabolic perturbations or adverse health effects due to gadolinium deposition are still lacking. In this work, aqueous organs extract and plasma samples were analyzed by GC-MS and 1H-NMR, respectively, to investigate the effects of multiple administrations of one linear (Omniscan) and one macrocyclic (ProHance) GBCA, on the main metabolic pathways in healthy mice. Multivariate analysis revealed that plasma metabolome was not differently perturbed by the two GBCAs, while, the multiorgan analysis displayed a clear separation of the Omniscan-treated from the control and the ProHance-treated groups. Interestingly, the most affected organs were the brain, cerebellum and liver. Thus, this work paves the way to both the safest use of the commercially available GBCAs and the development of new GBCAs characterized by lower general toxicity.

Targeting G-quadruplex motifs interferes with differentiation of adipose-derived mesenchymal stem cells.

BACKGROUND: G-quadruplex (G4) motifs are nucleic acid secondary structures observed in mammalian genomes and transcriptomes able to regulate various cellular processes. Several small molecules have been developed so far to modulate G4 stability, frequently associated with anticancer activity. However, how G4 structures are regulated over homeostatic conditions is mostly unexplored. Here, we used human adipose-derived mesenchymal stem cells (ASCs) to address the role of G4 motifs during adipogenic differentiation. METHODS: Adipocyte differentiation of ASCs was investigated in the presence or absence of a well-known G4 ligand, Braco-19. Cell viability was determined by sulforhodamine B assay. Cell dimension and granularity, DNA G4 motifs and cell cycle were detected by flow cytometry. Lipid droplet accumulation was assessed by Oil Red O staining. Cell senescence was evaluated by ß-galactosidase staining. Gene expression was measured by qPCR. Protein release in the extracellular medium was quantified by ELISA. RESULTS: Braco-19 used at non-cytotoxic concentrations induced morphological changes in mature adipocytes partially restoring an undifferentiated-like status. Braco-19 reduced lipid vacuolization and PPARG, AP2, LEP and TNFA mRNA levels in terminally differentiated cells. No effect was observed in cell senescence, fibrotic markers, IL-6 and IL-8 production, while the secretion of VEGF was dose-dependently reduced. Interestingly, G4 structures were increased in differentiated adipocytes compared to their precursors. Braco-19 treatment reduced G4 content in mature adipocytes. CONCLUSIONS: Our data highlight a new role of G4 motifs as genomic structural elements related to human ASC differentiation into mature adipocytes, with potential implications in physio-pathological processes.

Over-Expressed GATA-1S, the Short Isoform of the Hematopoietic Transcriptional Factor GATA-1, Inhibits Ferroptosis in K562 Myeloid Leukemia Cells by Preventing Lipid Peroxidation.

Ferroptosis is a recently recognized form of regulated cell death involving lipid peroxidation. Glutathione peroxidase 4 (GPX4) plays a central role in the regulation of ferroptosis through the suppression of lipid peroxidation generation. Connections have been reported between ferroptosis, lipid metabolism, cancer onset, and drug resistance. Recently, interest has grown in ferroptosis induction as a potential strategy to overcome drug resistance in hematological malignancies. GATA-1 is a key transcriptional factor controlling hematopoiesis-related gene expression. Two GATA-1 isoforms, the full-length protein (GATA-1FL) and a shorter isoform (GATA-1S), are described. A balanced GATA-1FL/GATA-1S ratio helps to control hematopoiesis, with GATA-1S overexpression being associated with hematological malignancies by promoting proliferation and survival pathways in hematopoietic precursors. Recently, optical techniques allowed us to highlight different lipid profiles associated with the expression of GATA-1 isoforms, thus raising the hypothesis that ferroptosis-regulated processes could be involved. Lipidomic and functional analysis were conducted to elucidate these mechanisms. Studies on lipid peroxidation production, cell viability, cell death, and gene expression were used to evaluate the impact of GPX4 inhibition. Here, we provide the first evidence that over-expressed GATA-1S prevents K562 myeloid leukemia cells from lipid peroxidation-induced ferroptosis. Targeting ferroptosis is a promising strategy to overcome chemoresistance. Therefore, our results could provide novel potential therapeutic approaches and targets to overcome drug resistance in hematological malignancies.

Degradation of biogas in a simulated landfill cover soil at laboratory scale: Compositional changes of main components and volatile organic compounds.

A laboratory experiment lasting 28 days was run to simulate a typical landfill system and to investigate the compositional changes affecting the main components (CH4, CO2, and H2) and nonmethane volatile organic compounds from biogas generated by anaerobic digestion of food waste and passing through a soil column. Gas samples were periodically collected from both the digester headspace and the soil column at increasing distances from the biogas source. CH4 and H2 were efficiently degraded along the soil column. The isotopic values of δ13C measured in CH4 and CO2 from the soil column were relatively enriched in 13C compared to the biogas. Aromatics and alkanes were the most abundant groups in the biogas samples. Among these compounds, alkylated benzenes and long-chain C3+ alkanes were significantly degraded within the soil column, whereas benzene and short-chain alkanes were recalcitrant. Terpene and O-substituted compounds were relatively stable under oxidising conditions. Cyclic, alkene, S-substituted, and halogenated compounds, which exhibited minor amounts in the digester headspace, were virtually absent in the soil column. These results pointed out how many recalcitrant potentially toxic and polluting compounds tend to be relatively enriched along the soil column, claiming action to minimise diffuse landfill gas (LFG) emissions. The proposed experimental approach represents a reliable tool for investigating the attenuation capacities of landfill cover soils for LFG components and developing optimised covers by adopting proper soil treatments and operating conditions to improve their degradation efficiencies.

Ligand-based drug repurposing strategy identified SARS-CoV-2 RNA G-quadruplex binders.

The single-stranded RNA genome of SARS-CoV-2 contains some G-quadruplex-forming G-rich elements which are putative drug targets. Here, we performed a ligand-based pharmacophore virtual screening of FDA approved drugs to find candidates targeting such RNA structures. Further in silico and in vitro assays identified three drugs as emerging SARS-CoV-2 RNA G-quadruplex binders.

Balancing Affinity, Selectivity, and Cytotoxicity of Hydrazone-Based G-Quadruplex Ligands for Activation of Interferon ß Genes in Cancer Cells.

G-quadruplex (G4) ligands are investigated to discover new anticancer drugs with increased cell-killing potency. These ligands can induce genome instability and activate innate immune genes at non-cytotoxic doses, opening the discovery of cytostatic immune-stimulating ligands. However, the interplay of G4 affinity/selectivity with cytotoxicity and immune gene activation is not well-understood. We investigated a series of closely related hydrazone derivatives to define the molecular bases of immune-stimulation activity. Although they are closely related to each other, such derivatives differ in G4 affinity, cytotoxicity, genome instability, and immune gene activation. Our findings show that G4 affinity of ligands is a critical feature for immune gene activation, whereas a high cytotoxic potency interferes with it. The balance of G4 stabilization versus cytotoxicity can determine the level of immune gene activation in cancer cells. Thus, we propose a new rationale based on low cell-killing potency and high immune stimulation to discover effective anticancer G4 ligands.

Combination of dl922-947 Oncolytic Adenovirus and G-Quadruplex Binders Uncovers Improved Antitumor Activity in Breast Cancer.

G-quadruplexes (G4s) are nucleic secondary structures characterized by G-tetrads. G4 motif stabilization induces DNA damage and cancer cell death; therefore, G4-targeting small molecules are the focus of clinical investigation. DNA destabilization induced by G4 ligands might potentiate the anticancer activity of agents targeting DNA or inhibiting its repair such as oncolytic viruses. This study represents the first approach combining G4 ligands, BRACO-19 (B19), pyridostatin (PDS), and the adenovirus dl922-947 in breast cancer cells. We demonstrated that G4 binders and dl922-947 induce cytotoxicity in breast cancer cells (MDA-MB-231 and MCF-7) and at higher doses in other neoplastic cell lines of thyroid (BHT-101 cells) and prostate (PC3 cells). G4 binders induce G4 motifs distributed in the S and G2/M phases in MCF-7 cells. G4 binder/dl922-947 combination increases cell cytotoxicity and the accumulation in subG0/G1. Indeed, G4 binders favor viral entry and replication with no effect on coxsackie and adenovirus receptor. Notably, dl922-947 induces G4 motifs and its combination with PDS potentiates this effect in MCF-7 cells. The agents alone or in combination similarly enhanced cell senescence. Additionally, PDS/dl922-947 combination inactivates STING signaling in MDA-MB-231 cells. Our results suggest that G4 binder/virotherapy combination may represent a novel therapeutic anticancer approach.

Histone lysine demethylase inhibition reprograms prostate cancer metabolism and mechanics.

OBJECTIVE: Aberrant activity of androgen receptor (AR) is the primary cause underlying development and progression of prostate cancer (PCa) and castration-resistant PCa (CRPC). Androgen signaling regulates gene transcription and lipid metabolism, facilitating tumor growth and therapy resistance in early and advanced PCa. Although direct AR signaling inhibitors exist, AR expression and function can also be epigenetically regulated. Specifically, lysine (K)-specific demethylases (KDMs), which are often overexpressed in PCa and CRPC phenotypes, regulate the AR transcriptional program. METHODS: We investigated LSD1/UTX inhibition, two KDMs, in PCa and CRPC using a multi-omics approach. We first performed a mitochondrial stress test to evaluate respiratory capacity after treatment with MC3324, a dual KDM-inhibitor, and then carried out lipidomic, proteomic, and metabolic analyses. We also investigated mechanical cellular properties with acoustic force spectroscopy. RESULTS: MC3324 induced a global increase in H3K4me2 and H3K27me3 accompanied by significant growth arrest and apoptosis in androgen-responsive and -unresponsive PCa systems. LSD1/UTX inhibition downregulated AR at both transcriptional and non-transcriptional level, showing cancer selectivity, indicating its potential use in resistance to androgen deprivation therapy. Since MC3324 impaired metabolic activity, by modifying the protein and lipid content in PCa and CRPC cell lines. Epigenetic inhibition of LSD1/UTX disrupted mitochondrial ATP production and mediated lipid plasticity, which affected the phosphocholine class, an important structural element for the cell membrane in PCa and CRPC associated with changes in physical and mechanical properties of cancer cells. CONCLUSIONS: Our data suggest a network in which epigenetics, hormone signaling, metabolite availability, lipid content, and mechano-metabolic process are closely related. This network may be able to identify additional hotspots for pharmacological intervention and underscores the key role of KDM-mediated epigenetic modulation in PCa and CRPC.

Conformational plasticity of DNA secondary structures: probing the conversion between i-motif and hairpin species by circular dichroism and ultraviolet resonance Raman spectroscopies.

The promoter regions of important oncogenes such as BCL2 and KRAS contain GC-rich sequences that can form distinctive noncanonical DNA structures involved in the regulation of transcription: G-quadruplexes on the G-rich strand and i-motifs on the C-rich strand. Interestingly, BCL2 and KRAS promoter i-motifs are highly dynamic in nature and exist in a pH-dependent equilibrium with hairpin and even with hybrid i-motif/hairpin species. Herein, the effects of pH and presence of cell-mimicking molecular crowding conditions on conformational equilibria of the BCL2 and KRAS i-motif-forming sequences were investigated by ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectroscopies. Multivariate analysis of CD data was essential to model the presence and identity of the species involved. Analysis of UVRR spectra measured as a function of pH, performed also by the two-dimensional correlation spectroscopy (2D-COS) technique, showed the role of several functional groups in the DNA conformational transitions, and provided structural and dynamic information. Thus, the UVRR investigation of intramolecular interactions and of local and environmental dynamics in promoting the different species induced by the solution conditions provided valuable insights into i-motif conformational transitions. The combined use of the two spectroscopic tools is emphasized by the relevant possibility of working in the same DNA concentration range and by the heterospectral UVRR/CD 2D-COS analysis. The results of this study shed light on the factors that can influence at the molecular level the equilibrium between the different conformational species putatively involved in the oncogene expression.

Synthesis and Characterization of Bis-Triazolyl-Pyridine Derivatives as Noncanonical DNA-Interacting Compounds.

Besides the well-known double-helical conformation, DNA is capable of folding into various noncanonical arrangements, such as G-quadruplexes (G4s) and i-motifs (iMs), whose occurrence in gene promoters, replication origins, and telomeres highlights the breadth of biological processes that they might regulate. Particularly, previous studies have reported that G4 and iM structures may play different roles in controlling gene transcription. Anyway, molecular tools able to simultaneously stabilize/destabilize those structures are still needed to shed light on what happens at the biological level. Herein, a multicomponent reaction and a click chemistry functionalization were combined to generate a set of 31 bis-triazolyl-pyridine derivatives which were initially screened by circular dichroism for their ability to interact with different G4 and/or iM DNAs and to affect the thermal stability of these structures. All the compounds were then clustered through multivariate data analysis, based on such capability. The most promising compounds were subjected to a further biophysical and biological characterization, leading to the identification of two molecules simultaneously able to stabilize G4s and destabilize iMs, both in vitro and in living cells.

Structural Basis for Inhibition of ROS-Producing Respiratory Complex I by NADH-OH.

NADH:ubiquinone oxidoreductase, respiratory complex I, plays a central role in cellular energy metabolism. As a major source of reactive oxygen species (ROS) it affects ageing and mitochondrial dysfunction. The novel inhibitor NADH-OH specifically blocks NADH oxidation and ROS production by complex I in nanomolar concentrations. Attempts to elucidate its structure by NMR spectroscopy have failed. Here, by using X-ray crystallographic analysis, we report the structure of NADH-OH bound in the active site of a soluble fragment of complex I at 2.0 Šresolution. We have identified key amino acid residues that are specific and essential for binding NADH-OH. Furthermore, the structure sheds light on the specificity of NADH-OH towards the unique Rossmann-fold of complex I and indicates a regulatory role in mitochondrial ROS generation. In addition, NADH-OH acts as a lead-structure for the synthesis of a novel class of ROS suppressors.

Targeting of Telomeric Repeat-Containing RNA G-Quadruplexes: From Screening to Biophysical and Biological Characterization of a New Hit Compound.

DNA G-quadruplex (G4) structures, either within gene promoter sequences or at telomeres, have been extensively investigated as potential small-molecule therapeutic targets. However, although G4s forming at the telomeric DNA have been extensively investigated as anticancer targets, few studies focus on the telomeric repeat-containing RNA (TERRA), transcribed from telomeres, as potential pharmacological targets. Here, a virtual screening approach to identify a library of drug-like putative TERRA G4 binders, in tandem with circular dichroism melting assay to study their TERRA G4-stabilizing properties, led to the identification of a new hit compound. The affinity of this compound for TERRA RNA and some DNA G4s was analyzed through several biophysical techniques and its biological activity investigated in terms of antiproliferative effect, DNA damage response (DDR) activation, and TERRA RNA expression in high vs. low TERRA-expressing human cancer cells. The selected hit showed good affinity for TERRA G4 and no binding to double-stranded DNA. In addition, biological assays showed that this compound is endowed with a preferential cytotoxic effect on high TERRA-expressing cells, where it induces a DDR at telomeres, probably by displacing TERRA from telomeres. Our studies demonstrate that the identification of TERRA G4-targeting drugs with potential pharmacological effects is achievable, shedding light on new perspectives aimed at discovering new anticancer agents targeting these G4 structures.