Non-canonical Nucleic Acid Structures Research Articles

Differences in Conformational Sampling and Intrinsic Electric Fields Drive Ion Binding in Telomeric and TERRA G-Quadruplexes.

The formation of G-quadruplexes (GQs) occurs in guanine-rich sequences of DNA and RNA, producing highly stable and structurally diverse noncanonical nucleic acid structures. GQs play crucial roles in regulating transcription, translation, and replication and maintaining the genome, among others; thus, changes to their structures can lead to diseases such as cancer. Previous studies using polarizable molecular dynamics simulations have shown differences in ion binding properties between telomeric and telomeric repeat-containing RNA GQs despite architectural similarities. Here, we used volume-based metadynamics and repulsive potential simulations in conjunction with polarizable force fields to quantify the impact of ion binding on the GQ dynamics and ion binding free energies. Furthermore, we describe how GQs exert electric fields on their surroundings to link dynamics with variations in the electronic structure. Our findings provide new insights into the energetic, physical, and conformational properties of GQs and expose subtle but important differences between DNA and RNA GQs with the same fold.

Theoretical Assessment of the Ligand/Metal/Quadruplex Recognition in the Non-Canonical Nucleic Acids Structures.

Quadruplexes (GQs), peculiar DNA/RNA motifs concentrated in specific genomic regions, play a vital role in biological processes including telomere stability and, hence, represent promising targets for anticancer therapy. GQs are formed by folding guanine-rich sequences into square planar G-tetrads which stack onto one another. Metal cations, most often potassium, further stabilize the architecture by coordinating the lone electron pairs of the O atoms. The presence of additional nucleic acid bases, however, has been recently observed experimentally and contributes substantially to the structural heterogeneity of quadruplexes. Therefore, it is of paramount significance to understand the factors governing the underlying complex processes in these structures. The current study employs DFT calculations to model the interactions between metal cations (K+, Na+, Sr2+) and diverse tetrads composed of a guanine layer in combination with a guanine (G)-, adenine (A)-, cytosine (C)-, thymine (T)-, or uracil (U)-based tetrad layer. Moreover, the addition of 4-(3,4-dihydroisoquinolin-2-yl)-2-(quinolin-2-yl)quinazoline to the modeled quadruplexes as a possible mechanism of its well-exerted antitumor effect is assessed. The calculations imply that the metal cation competition and ligand complexation are influenced by the balance between electronic and implicit/explicit solvation effects, the composition of the tetrad layers, as well as by the solvent exposure to the surrounding environment expressed in terms of different dielectric constant values. The provided results significantly enhance our understanding of quadruplex diversity, ligand recognition, and the underlying mechanisms of stabilization at an atomic level.

Complexity of Guanine Quadruplex Unfolding Pathways Revealed by Atomistic Pulling Simulations.

Guanine quadruplexes (GQs) are non-canonical nucleic acid structures involved in many biological processes. GQs formed in single-stranded regions often need to be unwound by cellular machinery, so their mechanochemical properties are important. Here, we performed steered molecular dynamics simulations of human telomeric GQs to study their unfolding. We examined four pulling regimes, including a very slow setup with pulling velocity and force load accessible to high-speed atomic force microscopy. We identified multiple factors affecting the unfolding mechanism, i.e.,: (i) the more the direction of force was perpendicular to the GQ channel axis (determined by GQ topology), the more the base unzipping mechanism happened, (ii) the more parallel the direction of force was, GQ opening and cross-like GQs were more likely to occur, (iii) strand slippage mechanism was possible for GQs with an all-anti pattern in a strand, and (iv) slower pulling velocity led to richer structural dynamics with sampling of more intermediates and partial refolding events. We also identified that a GQ may eventually unfold after a force drop under forces smaller than those that the GQ withstood before the drop. Finally, we found out that different unfolding intermediates could have very similar chain end-to-end distances, which reveals some limitations of structural interpretations of single-molecule spectroscopic data.

Development and application of metallo-phthalocyanines as potent G-quadruplex DNA binders and photosensitizers

Metallo-phthalocyanines (MPc) are common photosensitizers with ideal photophysical and photochemical properties. Also, these molecules have shown to interact with non-canonical nucleic acid structures, such as G-quadruplexes, and modulate oncogenic expression in cancer cells. Herein, we report the synthesis and characterisation of two metallo-phthalocyanines containing either zinc (ZnPc) or nickel (NiPc) in the central aromatic core and four alkyl ammonium lateral chains. The interaction of both molecules with G-quadruplex DNA was assessed by UV–Vis, fluorescence and FRET melting experiments. Both molecules bind strongly to G-quadruplexes and stabilise these structures, being NiPc the most notable G-quadruplex stabiliser. In addition, the photosensitizing ability of both metal complexes was explored by the evaluation of the singlet oxygen generation and their photoactivation in cells. Only ZnPc showed a high singlet oxygen generation either by direct observation or by indirect evaluation using a DPBF dye. The cellular evaluation showed mainly cytoplasmic localization of ZnPc and a decrease of the IC50 values of the cell viability of ZnPc upon light activation of two orders of magnitude.Graphical abstractTwo metallo-phthalocyanines containing zinc and nickel within the aromatic core have been investigated as G-quadruplex stabilizers and photosensitizers. NiPc shows a high G4 binding but negligible photosensitizing ability while ZnPc exhibits a moderate binding to G-quadruplex together with a high potency to generate singlet oxygen and photocytotoxicity. The interaction with G4s and capacity to be photosensitized is associated with the geometry adopted by the central metal core of the phthalocyanine scaffold.

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GENE THERAPY Reactions ShareShare onFacebookTwitterWechatLinked InRedditEmail C&EN, 2023, 101 (18), p 3June 5, 2023Cite this:C&EN 101, 18, 3(Credit: C&EN)Figure1of2(Credit: C&EN)Figure1of2Letters to the editorPeptide nucleic acids targeting noncanonical DNA and RNAAs a molecular biologist, I always enjoy reading articles concerning DNA and RNA. Your excellent article in the April 3, 2023, issue of C&EN (page 18) titled “The Promise of Peptide Nucleic Acids” discussed an important topic relating to gene therapy—that is, antisense therapeutics and gene- editing tools. Peptide nucleic acids (PNAs) can open a double-stranded (ds) DNA molecule and inhibit its gene expression. PNAs bind to DNA and RNA with high specificity and selectivity, resulting in PNA-DNA and PNA-RNA hybrid structures. Therefore, they have exciting potential as diagnostic tools and therapeutic agents.However, only briefly discussed in the article is the possibility of developing PNAs to target nucleic acid secondary structures such as RNA hairpins. More focus needs to be placed on all the other nucleic acid secondary structures, going beyond canonical right-handed ds-B-DNA, right-handed ds-A-RNA, and single-stranded (such as messenger RNA) nucleic acids based on nucleotide sequences—that is, the hybridization of base pairs.Researchers need to consider targeting exotic, noncanonical nucleic acids—that is, alternative and multistranded DNA and RNA structures. Local transitions in the structure of canonical ds-B-DNA are functionally significant biological molecules, regulating many cellular processes, such as transcription and translation. These exotic nucleic acid structures have also been connected to genetic instability in the presence or deficiency of DNA damage, with or without DNA replication. DNA and RNA possess dynamic structural variability resulting in a variety of unusual nucleic acid structures, such as cruciform DNA, DNA-RNA hybrids, slipped-strand DNA, and parallel-stranded DNA.Expanding the therapeutic capabilities of PNAs beyond canonical B-DNA would expand PNAs’ use in therapeutics. The expansion should include alternative ds-DNA structures such as left-handed ds-Z-DNA and left-handed ds-Z-RNA. It should also include multistranded DNA structures such as triplex DNA (intermolecular and intramolecular, like H-DNA), G4-quadruplex DNA, and i-motif DNA.If possible, targeting noncanonical nucleic acid structures with PNAs would increase the chance of developing novel therapeutics that can target all genomic DNA and RNA structures in genes and telomeres. We are witnessing the transition of gene medicine from an experimental technology into a real-world, practical approach for developing nucleic acid–based therapeutics, such as used for gene silencing or gene editing (like CRISPR) of pathological conditions.Using PNAs as therapeutic tools for targeting both canonical and noncanonical nucleic acids will allow for their full potential and optimal expansion of gene therapy.Claude GagnaBronxville, New YorkRecognizing nitrogen-fixing bacteria’s potential to reduce fertilizer useIn the article titled “Growing More Food with Less Chemicals,” which appeared in the May 8, 2023, issue of C&EN (page 28), there was a glaring omission.What was left out was the research and development of genetically modified nitrogen-fixing bacteria that can be applied to seed, foliage, or soil to reduce fertilizer use.Herbert B. ScherMoraga, California

Presence, Location and Conservation of Putative G-Quadruplex Forming Sequences in Arboviruses Infecting Humans.

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4s are found in the human genome and in the genomes of human pathogens, where they are involved in the regulation of gene expression and genome replication. G4s have been proposed as novel pharmacological targets in humans and their exploitation for antiviral therapy is an emerging research topic. Here, we report on the presence, conservation and localization of putative G4-forming sequences (PQSs) in human arboviruses. The prediction of PQSs was performed on more than twelve thousand viral genomes, belonging to forty different arboviruses that infect humans, and revealed that the abundance of PQSs in arboviruses is not related to the genomic GC content, but depends on the type of nucleic acid that constitutes the viral genome. Positive-strand ssRNA arboviruses, especially Flaviviruses, are significantly enriched in highly conserved PQSs, located in coding sequences (CDSs) or untranslated regions (UTRs). In contrast, negative-strand ssRNA and dsRNA arboviruses contain few conserved PQSs. Our analyses also revealed the presence of bulged PQSs, accounting for 17-26% of the total predicted PQSs. The data presented highlight the presence of highly conserved PQS in human arboviruses and present non-canonical nucleic acid-structures as promising therapeutic targets in arbovirus infections.

Stability of Human Telomeric G-Quadruplexes Complexed with Photosensitive Ligands and Irradiated with Visible Light.

Guanine-rich DNA sequences can fold into non-canonical nucleic acid structures called G-quadruplexes (G4s). These nanostructures have strong implications in many fields, from medical science to bottom-up nanotechnologies. As a result, ligands interacting with G4s have attracted great attention as candidates in medical therapies, molecular probe applications, and biosensing. In recent years, the use of G4-ligand complexes as photopharmacological targets has shown significant promise for developing novel therapeutic strategies and nanodevices. Here, we studied the possibility of manipulating the secondary structure of a human telomeric G4 sequence through the interaction with two photosensitive ligands, DTE and TMPyP4, whose response to visible light is different. The effect of these two ligands on G4 thermal unfolding was also considered, revealing the occurrence of peculiar multi-step melting pathways and the different attitudes of the two molecules on the quadruplex stabilization.

Single-molecule studies on co-transcriptional R-loop formation at DNA damage site.

R-loop, a non-canonical nucleic acid structure, is composed of an RNA-DNA hybrid and a single-stranded DNA(ssDNA). This abnormal structure can form through transcription. R-loop can play a role in some biological processes such as gene regulation, but R-loop has been considered a putative risk factor causing genome instability. Recent studies, however, suggest that R-loop formation is also required for certain DNA double-strand break (DSB) repair processes implying even positive aspects of R-loop in light of the maintenance of genome integrity. Here, we show that E. coli RNA polymerases have an unexpected ability to create R-loop when they encounter DSB during transcription. The efficiency of DSB induced R-loop formation under single-round transcription was quantitatively measured. We also observed the propagation of RNA-DNA hybrids from break sites to upstream. These extended RNA-DNA hybrids hindered the next round of transcription. Furthermore, we studied whether other types of genetic lesions and abnormal structures could induce co-transcriptional R-loop formation.

Dynamics and conformational heterogeneity of the PIM1 G-quadruplex from polarizable molecular dynamics simulations.

Reproductive cancers, such as triple-negative breast cancer (TNBC), are often challenging to treat due to a lack of specific chemotherapeutic targets. Overproduction of PIM1, an anti-apoptotic protein, is common in TNBC. Thus, counteracting this overproduction could serve as a viable therapeutic strategy against TNBC. The promoter region of the PIM1 gene can adopt two G-quadruplexes (GQs) with distinct topologies. GQs are higher-order, noncanonical nucleic acid structures that are rich in guanine and have become intensely explored as potential novel drug targets due to their proposed involvement in transcriptional regulation and other critical cellular processes. Two GQ-duplex hybrid structures have been found to exist in equilibrium in the promoter region of PIM1. The dominant GQ structure, form 1, contains conventional guanine tetrads and a duplex loop region of three Watson-Crick base pairs, while form 2 adopts a fold containing a mixed guanine-cytosine (GCGC) tetrad via slippage of two canonical Watson-Crick base pairs, and also contains a duplex loop consisting of two additional Watson-Crick pairs. We performed unbiased simulations and Gaussian-accelerated molecular dynamics simulations of both GQs with the Drude-2017 polarizable force field to better understand the properties of these GQs and how they might interconvert. Given the role of ion binding and noncanonical base pairing in stabilizing GQ structures, we monitored ion coordination and electronic properties (base dipole moments and electric fields). We observed an increased dipole moment for cytosine bases in the GCGC mixed tetrad of form 2, suggesting electronic plasticity as a factor in structural rearrangement between forms 1 and 2. We also report on conformational sampling in each structure to provide atomistic details that will be essential in the future design of more specific chemotherapeutic agents that target PIM1 on the nucleic acid level.

Computational Methods to Study DNA:DNA:RNA Triplex Formation by lncRNAs.

Long non-coding RNAs (lncRNAs) impact cell function via numerous mechanisms. In the nucleus, interactions between lncRNAs and DNA and the consequent formation of non-canonical nucleic acid structures seems to be particularly relevant. Along with interactions between single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA), such as R-loops, ssRNA can also interact with double-stranded DNA (dsDNA) to form DNA:DNA:RNA triplexes. A major challenge in the study of DNA:DNA:RNA triplexes is the identification of the precise RNA component interacting with specific regions of the dsDNA. As this is a crucial step towards understanding lncRNA function, there exist several computational methods designed to predict these sequences. This review summarises the recent progress in the prediction of triplex formation and highlights important DNA:DNA:RNA triplexes. In particular, different prediction tools (Triplexator, LongTarget, TRIPLEXES, Triplex Domain Finder, TriplexFFP, TriplexAligner and Fasim-LongTarget) will be discussed and their use exemplified by selected lncRNAs, whose DNA:DNA:RNA triplex forming potential was validated experimentally. Collectively, these tools revealed that DNA:DNA:RNA triplexes are likely to be numerous and make important contributions to gene expression regulation.

Antiviral effects of G-quadruplex binding ligands against human herpesviruses

G-quadruplexes (G4s) are noncanonical secondary nucleic acid structures constituted by stacking of guanine rich planar shaped tetrad formations that form a complex. G4s are implicated for various important roles in key cellular processes transcription, translation, telomere maintenance, epigenetic regulation, replication, and recombination. G-quadruplexes were first discovered as important structures in oncology, but for the past decade its relevance in viruses is becoming more evident. Human herpesviruses are DNA viruses of the Herpesviridae family and are unique in characteristic with two types of infection which can be distinguished by lytic and latency establishment in the host. During latency the virus maintains lifelong dormancy and intermittently undergoes reactivation, causing the host medical problems. Recently there are increasing number of reports regarding role of G4s in viral genomes and the potential antiviral efficacy of G4 ligands, including G4s in latency. Many results suggest viral G4s play significant roles in the virus life cycle and treatment of G4 ligands exhibit antiviral activities in both lytic and latent infections. In this review, the importance of G4s in herpesvirus genomes will be introduced with the potent G4 ligands used to study these mechanisms and finally explain the distinct functional properties of each G4 ligands.

Structural Polymorphism of Guanine Quadruplex-Containing Regions in Human Promoters.

Intramolecular guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by four guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4-forming DNA sequences are enriched in gene promoters and are implicated in the control of gene expression. Most G4-forming DNA contains more G residues than can simultaneously be incorporated into the core resulting in a variety of different possible G4 structures. Although this kind of structural polymorphism is well recognized in the literature, there remain unanswered questions regarding possible connections between G4 polymorphism and biological function. Here we report a detailed bioinformatic survey of G4 polymorphism in human gene promoter regions. Our analysis is based on identifying G4-containing regions (G4CRs), which we define as stretches of DNA in which every residue can form part of a G4. We found that G4CRs with higher degrees of polymorphism are more tightly clustered near transcription sites and tend to contain G4s with shorter loops and bulges. Furthermore, we found that G4CRs with well-characterized biological functions tended to be longer and more polymorphic than genome-wide averages. These results represent new evidence linking G4 polymorphism to biological function and provide new criteria for identifying biologically relevant G4-forming regions from genomic data.

Can G-quadruplex become a promising target in HBV therapy?

The chronic infection with hepatitis B virus (HBV) is an important health problem that affects millions of people worldwide. Current therapies for HBV always suffer from a poor response rate, common side effects, and the need for lifelong treatment. Novel therapeutic targets are expected. Interestingly, non-canonical structures of nucleic acids play crucial roles in the regulation of gene expression. Especially the formation of G-quadruplexes (G4s) in G-rich strands has been demonstrated to affect many bioprocesses including replication, transcription, and translation, showing great potential as targets in anticancer and antiviral therapies. In this review, we summarize recent antiviral studies about G4s and discuss the potential roles of G4 structures in antiviral therapy for HBV.

RNA as a Major-Groove Ligand: RNA-RNA and RNA-DNA Triplexes Formed by GAA and UUC or TTC Sequences.

Friedreich's ataxia is associated with noncanonical nucleic acid structures that emerge when GAA:TTC repeats in the first intron of the FXN gene expand beyond a critical number of repeats. Specifically, the noncanonical repeats are associated with both triplexes and R-loops. Here, we present an in silico investigation of all possible triplexes that form by attaching a third RNA strand to an RNA:RNA or DNA:DNA duplex, complementing previous DNA-based triplex studies. For both new triplexes results are similar. For a pyridimine UUC+ third strand, the parallel orientation is stable while its antiparallel counterpart is unstable. For a neutral GAA third strand, the parallel conformation is stable. A protonated GA+A third strand is stable in both parallel and antiparallel orientations. We have also investigated Na+ and Mg2+ ion distributions around the triplexes. The presence of Mg2+ ions helps stabilize neutral, antiparallel GAA triplexes. These results (along with previous DNA-based studies) allow for the emergence of a complete picture of the stability and structural characteristics of triplexes based on the GAA and TTC/UUC sequences, thereby contributing to the field of trinucleotide repeats and the associated unusual structures that trigger expansion.

Quindoline-derivatives display potent G-quadruplex-mediated antiviral activity against herpes simplex virus 1

G-quadruplexes (G4s) are non-canonical nucleic acid structures that regulate key biological processes, from transcription to genome replication both in humans and viruses. The herpes simplex virus-1 (HSV-1) genome is prone to form G4s that, along with proteins, regulate its viral cycle. General G4 ligands have been shown to hamper the viral cycle, pointing to viral G4s as original antiviral targets. Because cellular G4s are also normally present in infected cells, the quest for improved anti-HSV-1 G4 ligands is still open. Here, we evaluated a series of new quindoline-derivatives which showed high binding to and stabilization of the viral G4s. They displayed nanomolar-range anti-HSV-1 activity paralleled by negligible cytotoxicity in human cells, thus proving remarkable selectivity. The best-in-class compound inhibited the viral life cycle at the early times post infection up to the step of viral genome replication. In infected human cells, it reduced expression of ICP4, the main viral transcription factor, by stabilizing the G4s embedded in ICP4 promoter. Quindoline-derivatives thus emerge as a new class of G4 ligands with potent dual anti HSV-1 activity.

RHAU Peptides Specific for Parallel G-Quadruplexes: Potential Applications in Chemical Biology.

G-quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine (G)-rich sequences, which are ubiquitously found in the human genome and transcriptome. Targeting G4s by specific ligands provides a powerful tool to monitor and regulate G4s-associated biological processes. RHAU peptides, derived from the G4-binding motif of "RNA Helicase associated with AU-rich element" (RHAU), have emerged as extraordinary ligands for specific recognition of parallel G4s. This review highlights the significances of recent studies investigating potential applications of the engineered RHAU peptides incorporated to different functional moieties.

Indoloquinoline-Mediated Targeted Downregulation of KRAS through Selective Stabilization of the Mid-Promoter G-Quadruplex Structure.

KRAS is a well-validated anti-cancer therapeutic target, whose transcriptional downregulation has been demonstrated to be lethal to tumor cells with aberrant KRAS signaling. G-quadruplexes (G4s) are non-canonical nucleic acid structures that mediate central dogmatic events, such as DNA repair, telomere elongation, transcription and splicing events. G4s are attractive drug targets, as they are more globular than B-DNA, enabling more selective gene interactions. Moreover, their genomic prevalence is increased in oncogenic promoters, their formation is increased in human cancers, and they can be modulated with small molecules or targeted nucleic acids. The putative formation of multiple G4s has been described in the literature, but compounds with selectivity among these structures have not yet been able to distinguish between the biological contribution of the predominant structures. Using cell free screening techniques, synthesis of novel indoloquinoline compounds and cellular models of KRAS-dependent cancer cells, we describe compounds that choose between KRAS promoter G4near and G4mid, correlate compound cytotoxic activity with KRAS regulation, and highlight G4mid as the lead molecular non-canonical structure for further targeting efforts.

Targeting the RNA G-Quadruplex and Protein Interactome for Antiviral Therapy.

In recent years, G-quadruplexes (G4s), types of noncanonical four-stranded nucleic acid structures, have been identified in many viruses that threaten human health, such as HIV and Epstein-Barr virus. In this context, G4 ligands were designed to target the G4 structures, among which some have shown promising antiviral effects. In this Perspective, we first summarize the diversified roles of RNA G4s in different viruses. Next, we introduce small-molecule ligands developed as G4 modulators and highlight their applications in antiviral studies. In addition to G4s, we comprehensively review the medical intervention of G4-interacting proteins from both the virus (N protein, viral-encoded helicases, severe acute respiratory syndrome-unique domain, and Epstein-Barr nuclear antigen 1) and the host (heterogeneous nuclear ribonucleoproteins, RNA helicases, zinc-finger cellular nucelic acid-binding protein, and nucleolin) by inhibitors as an alternative way to disturb the normal functions of G4s. Finally, we discuss the challenges and opportunities in G4-based antiviral therapy.

Human Virus Genomes Are Enriched in Conserved Adenine/Thymine/Uracil Multiple Tracts That Pause Polymerase Progression

The DNA secondary structures that deviate from the classic Watson and Crick base pairing are increasingly being reported to form transiently in the cell and regulate specific cellular mechanisms. Human viruses are cell parasites that have evolved mechanisms shared with the host cell to support their own replication and spreading. Contrary to human host cells, viruses display a diverse array of nucleic acid types, which include DNA or RNA in single-stranded or double-stranded conformations. This heterogeneity improves the possible occurrence of non-canonical nucleic acid structures. We have previously shown that human virus genomes are enriched in G-rich sequences that fold in four-stranded nucleic acid secondary structures, the G-quadruplexes.Here, by extensive bioinformatics analysis on all available genomes, we showed that human viruses are enriched in highly conserved multiple A (and T or U) tracts, with such an array that they could in principle form quadruplex structures. By circular dichroism, NMR, and Taq polymerase stop assays, we proved that, while A/T/U-quadruplexes do not form, these tracts still display biological significance, as they invariably trigger polymerase pausing within two bases from the A/T/U tract. “A” bases display the strongest effect. Most of the identified A-tracts are in the coding strand, both at the DNA and RNA levels, suggesting their possible relevance during viral translation. This study expands on the presence and mechanism of nucleic acid secondary structures in human viruses and provides a new direction for antiviral research.

Heterogeneous hydration patterns of G-quadruplex DNA

G-quadruplexes (GQs) are guanine-rich, non-canonical nucleic acid structures that play fundamental roles in biological processes. Their structure and function are strongly influenced by their hydration shells. Although extensively studied through various experimental and computational methods, hydration patterns near DNA remain under debate due to the chemically and topologically heterogeneous nature of the exposed surface. In this work, we employed all-atom molecular dynamics (MD) simulation to study the hydration patterns of GQ DNA. The Drude oscillator model was used in MD simulation as a computationally efficient method for modeling electronic polarization in DNA ion solutions. Hydration structure was analyzed in terms of radial distribution functions and high-density three-dimensional hydration sites. Analysis of hydration dynamics focused on self-diffusion rates and orientation time correlation at different structural regions of GQ DNA. The results show highly heterogeneous hydration patterns in both structure and dynamics; for example, there are several insular high-density sites in the inner channel, and ‘spine of water’ in the groove. For water inside the loop, anomalous diffusion is present over a long time scale, but for water around the phosphate group and groove, diffusion becomes normal after ∼ 30 ps. These essentially correspond to deeply buried structural water and strong interaction with DNA, respectively.