The Arabidopsis tDR Ala forms G-quadruplex structures that can be unwound by the DExH1 DEA(D/H)-box RNA helicase.

As in many other organisms, tRNA-derived RNAs (tDRs) exist in plants and likely have multiple functions. We previously showed that tDRs are present in Arabidopsis under normal growth conditions, and that the ones originating from alanine tRNAs are the most abundant in leaves. We also showed that tDRs Ala of 20 nt produced from mature tRNAAla (AGC) can block in vitro protein translation. Here, we report that first, these tDRs Ala (AGC) can be found within peculiar foci in the cell that are neither P-bodies nor stress granules and, second, that they assemble into intermolecular RNA G-quadruplex (rG4) structures. Such tDR Ala rG4 structures can specifically interact with an Arabidopsis DEA(D/H) RNA helicase, the DExH1 protein, and unwind them. The rG4-DExH1 protein interaction relies on a glycine-arginine domain with RGG/RG/GR/GRR motifs present at the N-terminal extremity of the protein. Mutations on the four guanine residues located at the 5' extremity of the tDR Ala abolish its rG4 structure assembly, association with the DExH1 protein, and foci formation, but they do not prevent protein translation inhibition in vitro. Our data suggest that the sequestration of tDRs Ala into rG4 complexes might represent a way to modulate accessible and functional tDRs for translation inhibition within the plant cell via the activity of a specific RNA helicase, DExH1.

Development of a single-domain antibody to target a G-quadruplex located on the hepatitis B virus covalently closed circular DNA genome.

To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine-quadruplexes (G4s) are a secondary structure that can be adopted by DNA and play a significant role in regulating viral replication, transcription, and translation. Antibody-based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein, we describe the development of a humanized single-domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small-Angle X-ray Scattering. Lentiviral transduction of S10 into HepG2-NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next-generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10.

DNA topoisomerase 1 represses HIV-1 promoter activity through its interaction with a guanine quadruplex present in the LTR sequence.

BACKGROUND: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus. RESULTS: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes. CONCLUSIONS: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies.

Interaction of G-quadruplex Forming DNA Aptamers with PAMAM Dendrimers Studied by Dynamic Light Scattering and UV-VIS Spectrophotometry.

The complexes of G-quadruplex forming DNA thrombin binding aptamers (TBA) and polyamidoamine dendrimers (PAMAM) were studied with the aim to form a model targeted drug delivery system. Hydrodynamic diameter, zeta potential and melting temperature (Tm ) were investigated by dynamic light scattering and UV-VIS spectrophotometry. Non-covalent adsorption by means of electrostatic interaction between positively charged amino groups of dendrimers (+) and negatively charged phosphate groups of aptamers (-) has driven the formation of aggregates. The size of complexes was in the range of 0.2-2 µm and depended on the type of dispersant, charge ratio (+/-) and temperature. Raising the temperature increased the polydispersity, new smaller size distributions were observed indicating the G-quadruplex unfolding. The melting transition temperature of TBA aptamer was affected by the presence of amino-terminated PAMAM rather than carboxylated succinic acid PAMAM-SAH dendrimer, thus supporting the electrostatic nature of interaction that disturbed denaturation of target-specific quadruplex aptamer structure.

Non-duplex G-Quadruplex Structures Emerge as Mediators of Epigenetic Modifications.

The role of non-duplex DNA, the guanine-quadruplex structure in particular, is becoming widely appreciated. Increasing evidence in the last decade implicates quadruplexes in important processes such as transcription and replication. Interestingly, more recent work suggests roles for quadruplexes, in association with quadruplex-interacting proteins, in epigenetics through both DNA and histone modifications. Here, we review the effect of the quadruplex structure on post-replication epigenetic memory and quadruplex-induced promoter DNA/histone modifications. Furthermore, we highlight the epigenetic state of the telomerase promoter where quadruplexes could play a key regulatory role. Finally, we discuss the possibility that DNA structures such as quadruplexes, within a largely duplex DNA background, could act as molecular anchors for locally induced epigenetic modifications.

G-Quadruplex Recognition by DARPIns through Epitope/Paratope Analogy.

We investigated the mechanisms leading to the specific recognition of Guanine Guadruplex (G4) by DARPins peptides, which can lead to the design of G4 s specific sensors. To this end we carried out all-atom molecular dynamic simulations to unravel the interactions between specific nucleic acids, including human-telomeric (h-telo), Bcl-2, and c-Myc, with different peptides, forming a DARPin/G4 complex. By comparing the sequences of DARPin with that of a peptide known for its high affinity for c-Myc, we show that the recognition cannot be ascribed to sequence similarity but, instead, depends on the complementarity between the three-dimensional arrangement of the molecular fragments involved: the α-helix/loops domain of DARPin and the G4 backbone. Our results reveal that DARPins tertiary structure presents a charged hollow region in which G4 can be hosted, thus the more complementary the structural shapes, the more stable the interaction.

G-quadruplex-based CpG oligodeoxynucleotide/DOTAP complex strongly stimulates immunity in CpG motif-specific and loop-length-dependent manners.

Guanine-quadruplex (G4) oligodeoxynucleotides (ODNs) that contain unmethylated cytosine-phosphate-guanine motifs (G4 CpG ODN) with phosphodiester backbones are safer than the phosphorothioate (PT)-modified CpG ODNs recently used as vaccine adjuvants. However, cellular uptake and the nuclease stability of G4 CpG ODNs are still insufficient, resulting in lower immunostimulatory activity than PT-modified CpG ODNs. We aimed to enhance the immunostimulatory properties of G4 CpG ODNs by complexing with the cationic liposome 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). The complex acquired nuclease resistance and improved cellular uptake. The immunostimulatory activity of the G4 CpG ODN-DOTAP lipoplexes was enhanced to a level comparable to that of PT-modified ODNs. In addition, the lipoplexes based on unmodified G4 CpG ODNs demonstrated CpG motif-specific immunostimulant activity, although PT-modified ODNs lacking the CpG motif could activate human immune cells. Interestingly, G4 CpG ODN-DOTAP lipoplexes induced interferon-α production in a loop-length dependent manner.

Electronic Level Structure of Novel Guanine Octuplex DNA Single Molecules.

Throughout the past few decades, guanine quadruplex DNA structures have attracted much interest both from a fundamental material science perspective and from a technologically oriented perspective. Novel guanine octuplex DNA, formed from coiled quadruplex DNA, was recently discovered as a stable and rigid DNA-based nanostructure. A detailed electronic structure study of this new nanomaterial, performed by scanning tunneling spectroscopy on a subsingle-molecule level at cryogenic temperature, is presented herein. The electronic levels and lower energy gap of guanine octuplex DNA compared to quadruplex DNA dictate higher transverse conductivity through guanine octads than through guanine tetrads.

Diversity of Parallel Guanine Quadruplexes Induced by Guanine Substitutions.

Recently, we reported an inhibitory effect of guanine substitutions on the conformational switch from antiparallel to parallel quadruplexes (G4) induced by dehydrating agents. As a possible cause, we proposed a difference in the sensitivity of parallel and antiparallel quadruplexes to the guanine substitutions in the resulting thermodynamic stability. Reports on the influence of guanine substitutions on the biophysical properties of intramolecular parallel quadruplexes are rare. Moreover, such reports are often complicated by the multimerisation tendencies of parallel quadruplexes. To address this incomplete knowledge, we employed circular dichroism spectroscopy (CD), both as stopped-flow-assisted fast kinetics measurements and end-point measurements, accompanied by thermodynamic analyses, based on UV absorption melting profiles, and electrophoretic methods. We showed that parallel quadruplexes are significantly more sensitive towards guanine substitutions than antiparallel ones. Furthermore, guanine-substituted variants, which in principle might correspond to native genomic sequences, distinctly differ in their biophysical properties, indicating that the four guanines in each tetrad of parallel quadruplexes are not equal. In addition, we were able to distinguish by CD an intramolecular G4 from intermolecular ones resulting from multimerisation mediated by terminal tetrad association, but not from intermolecular G4s formed due to inter-strand Hoogsteen hydrogen bond formation. In conclusion, our study indicates significant variability in parallel quadruplex structures, otherwise disregarded without detailed experimental analysis.

A Double-Strand Break Does Not Promote Neisseria gonorrhoeae Pilin Antigenic Variation.

The major subunit of the type IV pilus (T4p) of Neisseria gonorrhoeae undergoes antigenic variation (AV) dependent on a guanine quadruplex (G4) DNA structure located upstream of the pilin gene. Since the presence of G4 DNA induces genome instability in both eukaryotic and prokaryotic chromosomes, we tested whether a double-strand break (DSB) at the site of the pilE G4 sequence could substitute for G4-directed pilin AV. The G4 motif was replaced by an I-SceI cut site, and the cut site was also introduced to locations near the origin of replication and the terminus. Expression of the I-SceI endonuclease from an irrelevant chromosomal site confirmed that the endonuclease functions to induce double-strand breaks at all three locations. No antigenic variants were detected when the G4 was replaced with the I-SceI cut site, but there was a growth defect from having a DSB in the chromosome, and suppressor mutations that were mainly deletions of the cut site and/or the entire pilE gene accumulated. Thus, the pilE G4 does not act to promote pilin AV by generating a DSB but requires either a different type of break, a nick, or more complex interactions with other factors to stimulate this programmed recombination system.IMPORTANCENeisseria gonorrhoeae, the causative agent of gonorrhea, possesses a DNA recombination system to change one of its surface-exposed antigens. This recombination system, known as antigenic variation, uses an alternate DNA structure to initiate variation. The guanine quadruplex DNA structure is known to cause nicks or breaks in DNA; however, much remains unknown about how this structure functions in cells. We show that inducing a break by different means does not allow antigenic variation, indicating that the DNA structure may have a more complicated role.

Guanine Quadruplex DNA Regulates Gamma Radiation Response of Genome Functions in the Radioresistant Bacterium Deinococcus radiodurans.

Guanine quadruplex (G4) DNA/RNA are secondary structures that regulate the various cellular processes in both eukaryotes and bacteria. Deinococcus radiodurans, a Gram-positive bacterium known for its extraordinary radioresistance, shows a genomewide occurrence of putative G4 DNA-forming motifs in its GC-rich genome. N-Methyl mesoporphyrin (NMM), a G4 DNA structure-stabilizing drug, did not affect bacterial growth under normal conditions but inhibited the postirradiation recovery of gamma-irradiated cells. Transcriptome sequencing analysis of cells treated with both radiation and NMM showed repression of gamma radiation-responsive gene expression, which was observed in the absence of NMM. Notably, this effect of NMM on the expression of housekeeping genes involved in other cellular processes was not observed. Stabilization of G4 DNA structures mapped at the upstream of recA and in the encoding region of DR_2199 had negatively affected promoter activity in vivo, DNA synthesis in vitro and protein translation in Escherichia coli host. These results suggested that G4 DNA plays an important role in DNA damage response and in the regulation of expression of the DNA repair proteins required for radioresistance in D. radioduransIMPORTANCEDeinococcus radiodurans can recover from extensive DNA damage caused by many genotoxic agents. It lacks LexA/RecA-mediated canonical SOS response. Therefore, the molecular mechanisms underlying the regulation of DNA damage response would be worth investigating in this bacterium. D. radiodurans genome is GC-rich and contains numerous islands of putative guanine quadruplex (G4) DNA structure-forming motifs. Here, we showed that in vivo stabilization of G4 DNA structures can impair DNA damage response processes in D. radiodurans Essential cellular processes such as transcription, DNA synthesis, and protein translation, which are also an integral part of the double-strand DNA break repair pathway, are affected by the arrest of G4 DNA structure dynamics. Thus, the role of DNA secondary structures in DNA damage response and radioresistance is demonstrated.

Formation of G-wires, bimolecular and tetramolecular quadruplex: Cation-induced structural polymorphs of G-rich DNA sequence of human SYTX gene.

An exceptional property of auto-folding into a range of intra- as well as intermolecular quadruplexes by guanine-rich oligomers (GROs) of promoters, telomeres and various other genomic locations is still one of the most attractive areas of research at present times. The main reason for this attention is due to their established in vivo existence and biological relevance. Herein, the structural status of a 20-nt long G-rich sequence with two G5 stretches (SG20) is investigated using various biophysical and biochemical techniques. Bioinformatics analysis suggested the presence of a 17-nt stretch of this SG20 sequence in the intronic region of human SYTX (Synaptotagmin 10) gene. The SYTX gene helps in sensing out the Ca2+ ion, causing its intake in the pre-synaptic neuron. A range of various topologies like bimolecular, tetramolecular and guanine-wires (nano-wires) was exhibited by the studied sequence, as a function of cations (Na+ /K+ ) concentration. UV-thermal denaturation, gel electrophoresis, and circular dichroism (CD) spectroscopy showed correlations and established a cation-dependent structural switch. The G-wire formation, in the presence of K+ , may further be explored for its possible relevance in nano-biotechnological applications.

Spectroscopic insights into quadruplexes of five-repeat telomere DNA sequences upon G-block damage.

BACKGROUND: The DNA lesions, resulting from oxidative damage, were shown to destabilize human telomere four-repeat quadruplex and to alter its structure. Long telomere DNA, as a repetitive sequence, offers, however, other mechanisms of dealing with the lesion: extrusion of the damaged repeat into loop or shifting the quadruplex position by one repeat. METHODS: Using circular dichroism and UV absorption spectroscopy and polyacrylamide electrophoresis, we studied consequences of lesions at different positions of the model five-repeat human telomere DNA sequences on the structure and stability of their quadruplexes in sodium and in potassium. RESULTS: The repeats affected by lesion are preferentially positioned as terminal overhangs of the core quadruplex structurally similar to the four-repeat one. Forced affecting of the inner repeats leads to presence of variety of more parallel folds in potassium. In sodium the designed models form mixture of two dominant antiparallel quadruplexes whose population varies with the position of the affected repeat. The shapes of quadruplex CD spectra, namely the height of dominant peaks, significantly correlate with melting temperatures. CONCLUSION: Lesion in one guanine tract of a more than four repeats long human telomere DNA sequence may cause re-positioning of its quadruplex arrangement associated with a shift of the structure to less common quadruplex conformations. The type of the quadruplex depends on the loop position and external conditions. GENERAL SIGNIFICANCE: The telomere DNA quadruplexes are quite resistant to the effect of point mutations due to the telomere DNA repetitive nature, although their structure and, consequently, function might be altered.

Exploring the biological roles of DHX36, a DNA/RNA G-quadruplex helicase, highlights functions in male infertility: A comprehensive review.

It is estimated that 15 % of couples at reproductive age worldwide suffer from infertility, approximately 50 % of cases are caused by male factors. Significant progress has been made in the diagnosis and treatment of male infertility through assisted reproductive technology and molecular genetics methods. However, there is still inadequate research on the underlying mechanisms of gene regulation in the process of spermatogenesis. Guanine-quadruplexes (G4s) are a class of non-canonical secondary structures of nucleic acid commonly found in genomes and RNAs that play important roles in various biological processes. Interestingly, the DEAH-box helicase 36 (DHX36) displays high specificity for the G4s which can unwind both DNA G4s and RNA G4s enzymatically and is highly expressed in testis, thereby regulating multiple cellular functions including transcription, pre-mRNA splicing, translation, telomere maintenance, genomic stability, and RNA metabolism in development and male infertility. This review provides an overview of the roles of G4s and DHX36 in reproduction and development. We mainly focus on the potential role of DHX36 in male infertility. We also discuss possible future research directions regarding the mechanism of spermatogenesis mediated by DHX36 through G4s in spermatogenesis-related genes and provide new targets for gene therapy of male infertility.

The intricate relationship of G-Quadruplexes and bacterial pathogenicity islands.

The dynamic interplay between guanine-quadruplex (G4) structures and pathogenicity islands (PAIs) represents a captivating area of research with implications for understanding the molecular mechanisms underlying pathogenicity. This study conducted a comprehensive analysis of a large-scale dataset from reported 89 pathogenic strains of bacteria to investigate the potential interactions between G4 structures and PAIs. G4 structures exhibited an uneven and non-random distribution within the PAIs and were consistently conserved within the same pathogenic strains. Additionally, this investigation identified positive correlations between the number and frequency of G4 structures and the GC content across different genomic features, including the genome, promoters, genes, tRNA, and rRNA regions, indicating a potential relationship between G4 structures and the GC-associated regions of the genome. The observed differences in GC content between PAIs and the core genome further highlight the unique nature of PAIs and underlying factors, such as DNA topology. High-confidence G4 structures within regulatory regions of Escherichia coli were identified, modulating the efficiency or specificity of DNA integration events within PAIs. Collectively, these findings pave the way for future research to unravel the intricate molecular mechanisms and functional implications of G4-PAI interactions, thereby advancing our understanding of bacterial pathogenicity and the role of G4 structures in pathogenic diseases.

G-quadruplex ligands as potent regulators of lysosomes.

Guanine-quadruplex structures (G4) are unusual nucleic acid conformations formed by guanine-rich DNA and RNA sequences and known to control gene expression mechanisms, from transcription to protein synthesis. So far, a number of molecules that recognize G4 have been developed for potential therapeutic applications in human pathologies, including cancer and infectious diseases. These molecules are called G4 ligands. When the biological effects of G4 ligands are studied, the analysis is often limited to nucleic acid targets. However, recent evidence indicates that G4 ligands may target other cellular components and compartments such as lysosomes and mitochondria. Here, we summarize our current knowledge of the regulation of lysosome by G4 ligands, underlying their potential functional impact on lysosome biology and autophagic flux, as well as on the transcriptional regulation of lysosomal genes. We outline the consequences of these effects on cell fate decisions and we systematically analyzed G4-prone sequences within the promoter of 435 lysosome-related genes. Finally, we propose some hypotheses about the mechanisms involved in the regulation of lysosomes by G4 ligands.

Substantial dimerized G-quadruplex signal units engineered by cutting-mediated exponential rolling circle amplification for ultrasensitive and label-free detection of exosomes.

Sensitive and accurate determination of tumor-derived exosomes from complicated biofluids is an important prerequisite for early tumor diagnosis through exosome-based liquid biopsy. Herein, a label-free fluorescence immunoassay protocol for ultrasensitive detection of exosomes was developed by engineering substantial dimerized guanine-quadruplex (Dimer-G4) signal units via in situ cutting-mediated exponential rolling circle amplification (CM-ERCA). First, exosomes were captured and enriched via immunomagnetic separation. Then, molecular recognition was built by the formation of antibody-aptamer sandwich immunocomplex through the specific binding of the designed aptamer-primers with the targeted exosomes. The accuracy of exosome detection was significantly improved by the specific recognition of two typical exosomal protein markers simultaneously. Eventually, in situ CM-ERCA was triggered by a perfect match between the multifunctional circular DNA template and the aptamer-primer on exosomal surface. Amplicons of CM-ERCA loaded with Dimer-G4 were exponentially accumulated during continuous cyclic amplification, dramatically lighting up the thioflavin T (ThT) and generating substantial Dimer-G4 signal units. As a result, ultrasensitive detection of exosomes with the detection limit down to 2.4 × 102 particles/mL was achieved due to the fluorescence enhancement of substantial Dimer-G4 signal units, which is ahead of most of available fluorescence-based methods reported currently. In addition, the intense fluorescence emission and favorable anti-interference of the proposed immunoassay supports identification of exosomes direct in human serums, overcoming the limitations of conventional G4/ThT in serum analysis and revealing its potential for exosome-based liquid biopsy.

Influence of the Charge Ratio of Guanine-Quadruplex Structure-Based CpG Oligodeoxynucleotides and Cationic DOTAP Liposomes on Cytokine Induction Profiles.

Cationic liposomes, specifically 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes, serve as successful carriers for guanine-quadruplex (G4) structure-based cytosine-guanine oligodeoxynucleotides (CpG ODNs). The combined benefits of CpG ODNs forming a G4 structure and a non-viral vector carrier endow the ensuing complex with promising adjuvant properties. Although G4-CpG ODN-DOTAP complexes show a higher immunostimulatory effect than naked G4-CpG ODNs, the effects of the complex composition, especially charge ratios, on the production of the pro-inflammatory cytokines interleukin (IL)-6 and interferon (IFN)-α remain unclear. Here, we examined whether charge ratios drive the bifurcation of cytokine inductions in human peripheral blood mononuclear cells. Linear CpG ODN-DOTAP liposome complexes formed micrometer-sized positively charged agglomerates; G4-CpG ODN-DOTAP liposome complexes with low charge ratios (0.5 and 1.5) formed ~250 nm-sized negatively charged complexes. Notably, low-charge-ratio (0.5 and 1.5) complexes induced significantly higher IL-6 and IFN-α levels simultaneously than high-charge-ratio (2 and 2.5) complexes. Moreover, confocal microscopy indicated a positive correlation between the cellular uptake of the complex and amount of cytokine induced. The observed effects of charge ratios on complex size, surface charge, and affinity for factors that modify cellular-uptake, intracellular-activity, and cytokine-production efficiency highlight the importance of a rational complex design for delivering and controlling G4-CpG ODN activity.

DNA/RNA helicase DHX36 is required for late stages of spermatogenesis.

Spermatogenesis is a highly complex developmental process that typically consists of mitosis, meiosis, and spermiogenesis. DNA/RNA helicase DHX36, a unique guanine-quadruplex (G4) resolvase, plays crucial roles in a variety of biological processes. We previously showed that DHX36 is highly expressed in male germ cells with the highest level in zygotene spermatocytes. Here, we deleted Dhx36 in advanced germ cells with Stra8-GFPCre and found that a Dhx36 deficiency in the differentiated spermatogonia leads to meiotic defects and abnormal spermiogenesis. These defects in late stages of spermatogenesis arise from dysregulated transcription of G4-harboring genes, which are required for meiosis. Thus, this study reveals that Dhx36 plays crucial roles in late stages of spermatogenesis.

Early steps of oxidative damage in DNA quadruplexes are position-dependent: Quantum mechanical and molecular dynamics analysis of human telomeric sequence containing ionized guanine.

Guanine radical cation (G•+) is a key intermediate in many oxidative processes occurring in nucleic acids. Here, by combining mixed Quantum Mechanical/Molecular Mechanics calculations and Molecular Dynamics (MD) simulations, we study how the structural behaviour of a tract GGG(TTAGGG)3 (hereafter Tel21) of the human telomeric sequence, folded in an antiparallel quadruple helix, changes when one of the G bases is ionized to G•+ (Tel21+). Once assessed that the electron-hole is localized on a single G, we perform MD simulations of twelve Tel21+ systems, differing in the position of G•+ in the sequence. When G•+ is located in the tetrad adjacent to the diagonal loop, we observe substantial structural rearrangements, which can decrease the electrostatic repulsion with the inner Na+ ions and increase the solvent exposed surface of G•+. Analysis of solvation patterns of G•+ provides new insights on the main reactions of G•+, i.e. the deprotonation at two different sites and hydration at the C8 atom, the first steps of the processes producing 8oxo-Guanine. We suggest the main structural determinants of the relative reactivity of each position and our conclusions, consistent with the available experimental trends, can help rationalizing the reactivity of other G-quadruplex topologies.