DNA G-Quadruplexes as Targets for Natural Product Drug Discovery

DNA guanine(G)-quadruplexes(G4s)are unique secondary structures formed by two or more stacked Gtetrads in G-rich DNA sequences.These structures have been found to play a crucial role in highly transcribed genes,especially in cancer-related oncogenes,making them attractive targets for cancer therapeutics.Significantly,targeting oncogene promoter G4 structures has emerged as a promising strategy to address the challenge of undruggable and drug-resistant proteins,such as MYC,BCL2,KRAS,and EGFR.Natural products have long been an important source of drug discovery,particularly in the fields of cancer and infectious diseases.Noteworthy progress has recently been made in the discovery of naturally occurring DNA G4-targeting drugs.Numerous DNA G4s,such as MYC-G4,BCL2-G4,KRAS-G4,PDGFR-b-G4,VEGF-G4,and telomeric-G4,have been identified as potential targets of natural products,including berberine,telomestatin,quindoline,sanguinarine,isaindigotone,and many others.Herein,we summarize and evaluate recent advancements in natural and nature-derived DNA G4 binders,focusing on understanding the structural recognition of DNA G4s by small molecules derived from nature.We also discuss the challenges and opportunities associated with developing drugs that target DNA G4s.

Bloom's syndrome protein unfolding G-quadruplexes in two pathways

The Bloom helicase （BLM） gene product encodes a DNA helicase that functions in homologous recombination repair to prevent genomic instability. BLM is highly active in binding and unfolding G-quadruplexes （G4）, which are non- canonical DNA structures formed by Hoogsteen base-pairing in guanine-rich sequences. Here we use single-molecule fluorescence resonance energy transfer （smFRET） to study the molecular mechanism of BLM-catalysed G4 unfolding and show that BLM unfolds G4 in two pathways. Our data enable us to propose a model in which the HRDC domain functions as a regulator of BLM, depending on the position of the HRDC domain of BLM in action： when HRDC binds to the G4 sequence, BLM may hold G4 in the unfolded state; otherwise, it may remain on the unfolded G4 transiently so that G4 can refold immediately.

Interaction between human telomeric G-quadruplexes characterized by single molecule magnetic tweezers

Human telomeric G-quadruplex plays a crucial role in regulating the genome stability. Despite extensive studies on structures and kinetics of monomeric G-quadruplex, the interaction between G-quadruplexes is still in debate. In this work,we employ magnetic tweezers to investigate the folding and unfolding kinetics of two contiguous G-quadruplexes in 100-mM K~＋buffer. The interaction between G-quadruplexes and the consequent effect on the kinetics of G-quadruplex are revealed. The linker sequence between G-quadruplexes is further found to play an important role in the interaction between two G-quadruplexes. Our results provide a high-resolution insight into kinetics of multimeric G-quadruplexes and genome stability.

Hierarchical Self-assembly of G-Quadruplexes Based Hydrogel Consisting of Guanine and Peptide Epitope

Guanosine-based hydrogels have attracted considerable attention because of their simplicity and easy preparation.However,the sugar moiety limits its further applications because of the necessity of sugar in the hydrogel formation.This work reports a G-quadruplexes-based hydrogel consisting of guanine and peptide epitope to form a supramolecular hydrogel in the presence of metal cations.Using the metal ion-responsive peptide epitope from the ion channel to replace sugar motif at N9 position of guanosine results in a novel nucleopeptide.The results show that the gelation time,the diameter of nanofibers,the anisotropic property,and the mechanical property of the hydrogel can be simply controlled using metal cations.The magnesium and calcium ions direct the alignment of nanofibers to form anisotropic nano-bundles.The mechanistic studies indicate the formation of G-quadruplexes in the hydrogel.Compared to the storage modulus of nucleopeptide without the metal cation,adding zinc ions results in an over three-order increase in mechanical properties.Cytotoxicity experiment indicates the good biocompatibility of our hydrogel.Moreover,we demonstrate that the guanine-capped peptide could release STING agonist in a controlled manner.This work illustrates a simple way to modulate the property of the nucleopeptide hydrogel to develop soft materials.

Unlocking G-Quadruplexes as Targets and Tools against COVID-19

The applicability of G-quadruplexes(G4s)as antiviral targets,therapeutic agents and diagnostic tools for coronavirus disease 2019(COVID-19)is currently being evaluated,which has drawn the extensive attention of the scientific community.During the COVID-19 pandemic,research in this field is rapidly accumulating.In this review,we summarize the latest achievements and breakthroughs in the use of G4s as antiviral targets,therapeutic agents and diagnostic tools for COVID-19,particularly using G4 ligands.Finally,strength and weakness regarding G4s in anti-SARS-CoV-2 field are highlighted for prospective future projects.

Target-induced Trivalent G-quadruplex/hemin DNAzyme for Colorimetric Detection of Hg^(2+) Based on an Exonuclease III Assisted Catalytic Hairpin Assembly

Mercury ion(Hg^(2+)),a highly noxious of heavy metalion,has detrimental effects on the ecological environment and human health.Herein,we have developed an exonuclease III(Exo III)assisted catalytic hairpin assembly formation of a trivalent G-quadruplex/hemin DNAzyme for colorimetric detection of Hg^(2+).A hairpin DNA(Hr)was designed with thymine-Hg^(2+)-thymine pairs that catalyzed by Exo III is prompted to happen upon binding Hg^(2+).A released DNA fragment triggers the catalytic assembly of other three hairpins(H1,H2,and H3)to form many trivalent G-quadruplex/hemin DNA enzymes for signal output.The developed sensor shows a dynamic range from 2 pM to 2μM,with an impressively low detection limit of 0.32 pM for Hg^(2+)detection.Such a sensor also has good selectivity toward Hg^(2+)detection in the presence of other common metal ions.This strategy shows the great potential for visual detection with portable type.

一种新型脱氧核酶G-quadruplexe—DNAZYMES的构效关系研究

1962年DavidR.Davies首先发现富含鸟嘌呤的核酸分子可以通过hoogsteen氢键作用,形成一种由几个G-quarter片层堆叠构成的四链DNA结构,即G-quadruplexe结构。

G-quadruplexes in genomes of viruses infecting eukaryotes or prokaryotes are under different selection pressures from hosts

G-quadruplexes in viral genomes can be applied as the targets of antiviral therapies, which has attracted wide interest. However, it is still not clear whether the pervasive number of such elements in the viral world is the result of natural selection for functionality. In this study, we identified putative quadruplex-forming sequences(PQSs) across the known viral genomes and analyzed the abundance, structural stability, and conservation of viral PQSs. A Viral Putative G-quadruplex Database(http://jsjds.hzau.edu.cn/MBPC/Vi PGD/index.php/home/index) was constructed to collect the details of each viral PQS, which provides guidance for selecting the desirable PQS. The PQS with two putative G-tetrads(G2-PQS) was significantly enriched in both eukaryotic viruses and prokaryotic viruses, whereas the PQSs with three putative G-tetrads(G3-PQS) were only enriched in eukaryotic viruses and depleted in prokaryotic viruses. The structural stability of PQSs in prokaryotic viruses was significantly lower than that in eukaryotic viruses. Conservation analysis showed that the G2-PQS, instead of G3-PQS, was highly conserved within the genus. This suggested that the G2-quadruplex might play an important role in viral biology, and the difference in the occurrence of G-quadruplex between eukaryotic viruses and prokaryotic viruses may result from the different selection pressures from hosts.

Roles of flanking sequences in the binding between unimolecular parallel-stranded G-quadruplexes and ligands

G-quadruplexes attract more and more attention in recent years.Numerous small molecules which can induce or stabilize the formation of G-quadruplexes have been investigated on the purpose of anticancer drug development.As a motif existed in physiological condition,flanking sequences are an important part of G-quadruplexes but the study on the impact of flanking sequences on (G-quadruplex)-ligand binding is rarely reported.In this paper,the effects of flanking sequences on binding affinity between a series of unimolecular parallel-stranded G-quadruplex sequences derived from c-myc oncogene promoter (termed as c-myc G-quadruplexes) and their ligands are discussed in detail.The results showed that the flanking sequences on c-myc G-quadruplexes play key roles in (G-quadruplex)-ligand interaction.When a c-myc G-quadruplex is bound to its ligands,the flanking sequences might form a binding cavity above the terminal G-quartet,which could provide a suitable site for ligands to dock in.Moreover,the bases on flanking sequences could interact with ligand through π-π stacking,and finally form a sandwich-stacking mode (terminal G-quartet,ligand and bases on the flanking sequence).This mode could stabilize the (G-quadruplex)-ligand complex effectively and enhance the binding affinity dramatically.However,flanking sequences are also found to exhibit steric hindrance effect which could impede the (G-quadruplex)-ligand binding.

NDI-induced Topological Conversion of Human Telomeric G-Quadruplexes from Hybrid-2 to Parallel Form

G-Quadruplexes(GQs),which are formed by G-rich DNA sequences in human telomeres,have become an attractive target for cancer treatment.The ligands to stabilize the conformation of human telomeric GQs in vivo are particularly important for structure-based ligand design and drug development targeting the noncanonical DNA structure.Here we report the conformational conversion of Tel26 induced by a naphthalene diimide(NDI)ligand in K^(+)buffer,even at cellular physiological temperature(37℃)and under mimetic cellular crowding conditions created by Ficoll 70.We provide an insight into the dynamic conversion from initial hybrid-2 GQ topology to final parallel GQ topology.These results are helpful for the design of ligands with GQ conformation regulation.

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.

Application of G-quadruplex targets in gastrointestinal cancers:Advancements,challenges and prospects

Genomic instability and inflammation are considered to be two enabling characteristics that support cancer development and progression.G-quadruplex structure is a key element that contributes to genomic instability and inflammation.G-quadruplexes were once regarded as simply an obstacle that can block the transcription of oncogenes.A ligand targeting G-quadruplexes was found to have anticancer activity,making G-quadruplexes potential anticancer targets.However,further investigation has revealed that G-quadruplexes are widely distributed throughout the human genome and have many functions,such as regulating DNA replication,DNA repair,transcription,translation,epigenetics,and inflammatory response.G-quadruplexes play double regulatory roles in transcription and translation.In this review,we focus on G-quadruplexes as novel targets for the treatment of gastrointestinal cancers.We summarize the application basis of G-quadruplexes in gastrointestinal cancers,including their distribution sites,structural characteristics,and physiological functions.We describe the current status of applications for the treatment of esophageal cancer,pancreatic cancer,hepatocellular carcinoma,gastric cancer,colorectal cancer,and gastrointestinal stromal tumors,as well as the associated challenges.Finally,we review the prospective clinical applications of G-quadruplex targets,providing references for targeted treatment strategies in gastrointestinal cancers.

Investigation of formation of dimeric G-quadruplex of HIV-1 integrase inhibitor by nuclear magnetic resonance

In this research, an unusually dimeric G-quadruplex of d（GGGTGGGTGGGTGGGT） （SI）, the potent nanomolar HIV-1 integrase inhibitor, was detected by nuclear magnetic resonance （NMR）. This result has been confirmed by electrospray ionization mass spectrometry （ESI-MS） and circular dichroism （CD）.

Electrochemical potassium ion sensor based on DNA G-quadruplex conformation and gold nanoparticle amplification

A simple, rapid, highly sensitive electrochem-ical sensor for potassium ion （K^＋） based on the confor-mationai change of DNA sequence containing guanine-rich segments is presented. In the presence of K^＋, guanine-rich DNA sequence folds to G-quadruplex structure, allowing a ferrocene tag to transfer electrons to the electrode. Gold nanoparticles （AuNPs）, which are self-assembled on the surface of a bare gold electrode by using 4-aminothio-phenol as a medium, offer a big surface area to immobilize a large number of aptamers and improve the sensitivity of the sensor. The square-wave voltammetry peak current increases with K^＋ concentration. The plots of peak current against K^＋ concentration and the logarithm of K^＋ con- centration are linear over the range from 0.1 to 1.0 mmol·L^-1 and from 1 to 30 mmol·L^-1, respectively. A lower detection limit of 0.1 mmol·L^-1 K^＋ is obtained for AuNPs-modified sensor, which greatly surpasses that （100 mmol·L^-1） of the sensor without AuNPs modification by three orders of magnitude. Thus, the sensor with AuNPs amplification is expected to open new opportunities for highly sensitive detection of other biomolecules in the future.

Effect of interaction between loop bases and ions on stability of G-quadruplex DNA

G-quadruplexes(GQs) are guanine-rich, non-canonical nucleic acid structures that play fundamental roles in biological processes. The topology of GQs is associated with the sequences and lengths of DNA, the types of linking loops, and the associated metal cations. However, our understanding on the basic physical properties of the formation process and the stability of GQs is rather limited. In this work, we employed ab initio, molecular dynamics(MD), and steered MD(SMD)simulations to study the interaction between loop bases and ions, and the effect on the stability of G-quadruplex DNA, the Drude oscillator model was used in MD and SMD simulations as a computationally efficient manner method for modeling electronic polarization in DNA ion solutions. We observed that the binding energy between DNA bases and ions(K^(+)/Na^(+))is about the base stacking free energies indicates that there will be a competition among the binding of M^(+)-base, H-bonds between bases, and the base-stacking while ions were bound in loop of GQs. Our SMD simulations indicated that the side loop inclined to form the base stacking while the loop sequence was Thy or Ade, and the cross-link loop upon the G-tetrads was not easy to form the base stacking. The base stacking side loop complex K+was found to have a good stabilization synergy. Although a stronger interaction was observed to exist between Cyt and K+, such an interaction was unable to promote the stability of the loop with the sequence Cyt.

Structure-based simulations complemented by conventional all-atom simulations to provide new insights into the folding dynamics of human telomeric G-quadruplex

The hybrid atomistic structure-based model has been validated to be effective in investigation of G-quadruplex folding.In this study,we performed large-scale conventional all-atom simulations to complement the folding mechanism of human telomeric sequence Htel24 revealed by a multi-basin hybrid atomistic structure-based model.Firstly,the real time-scale of folding rate,which cannot be obtained from the structure-based simulations,was estimated directly by constructing a Markov state model.The results show that Htel24 may fold as fast as on the order of milliseconds when only considering the competition between the hybrid-1 and hybrid-2 G-quadruplex conformations.Secondly,in comparison with the results of structure-based simulations,more metastable states were identified to participate in the formation of hybrid-1 and hybrid-2 conformations.These findings suggest that coupling the hybrid atomistic structure-based model and the conventional all-atom model can provide more insights into the folding dynamics of DNA G-quadruplex.As a result,the multiscale computational framework adopted in this study may be useful to study complex processes of biomolecules involving large conformational changes.

To unwind the biological knots:The DNA/RNA G-quadruplex resolvase RHAU(DHX36)in development and disease

The G-quadruplex(G4)sequences are short fragments of 4-i nterval triple guanine(G)with frequent and ubiquitous distribution in the genome and RNA transcripts.The G4sequences are usually folded into secondary“knot”structure via Hoogsteen hydrogen bond to exert negative regulation on a variety of biological processes,including DNA replication and transcription,mRNA translation,and telomere maintenance.Recent structural biological and mouse genetics studies have demonstrated that RHAU(DHX36)can bind and unwind the G4“knots”to modulate embryonic development and postnatal organ function.Deficiency of RHAU gives rise to embryonic lethality,impaired organogenesis,and organ dysfunction.These studies uncovered the pivotal G4 resolvase function of RHAU to release the G4 barrier,which plays fundamental roles in development and physiological homeostasis.This review discusses the latest advancements and findings in deciphering RHAU functions using animal models.

Evolutionary selection on synonymous codons in RNA G-quadruplex structural region

To investigate how synonymous codons have been adapted to the formation of ribonucleic acid(RNA)G-quadruplex(rG4)structure,a computational searching algorithm G4Hunter was applied to detect rG4 structures in protein-coding sequences of mRNAs in five eukaryotic species.The native sequences forming rG4s were then compared with randomized sequences to evaluate selection on synonymous codons.Factors that may influence the formation of rG4 were also investigated,and the selection pressures of rG4 in different gene regions were compared to explore its potential roles in gene regulation.The results show universal selective pressure acts on synonymous codons in rG4 regions to facilitate rG4 formation in five eukaryotic organisms.While G-rich codon combinations are preferred in the rG4 structural region,C-rich codon combinations are selectively unfavorable for rG4 formation.Gene's codon usage bias,nucleotide composition,and evolutionary rate can account for the selective variations on synonymous codons among rG4 structures within a species.Moreover,rG4 structures in the translational initiation region showed significantly higher selective pressures than those in the translational elongation region.

Synthesis, Crystal Structure and Interactions with Different G-quadruplex DNA and ctDNA of Zn-CAP

In this study, one mononuclear zinc（II） complex with 1,2-bis CAP （（5-chlorosalicylidene amino）-phenylene）： C22C13N2035Znl5 H0125 （Zn-CAP） was synthesized. The binding properties of Zn-CAP with G-quadruplex DNA and ctDNA （calf thymus DNA） were examined by fluorescence, CD （circular dichroism） spectroscopic and FRET （fluorescence resonance energy transfer） assay. In the fluorescence emission spectral analysis, the addition of three series of G-quadruplex DNA （G4-HTG21, G4-Pu27 and G4-c-kit-l） into the Zn-CAP solution induced moderate or add hypochromicity with total quenching ratios of 10.73%, 15.07% and 8.59% in the presence of K＋ were achieved, respectively. While the addition of ctDNA under same condition only caused 7.08% quenching on the fluorescence emission of Zn-CAP. In the CD spectral analysis, the interaction with Zn-CAP could induce significant spectral changes on the CD absorption of G4-HTG21, G4-Pu27 and G4-c-kit-1, with 106.00%, 93.06%, 113.47% increment at 232 nm absorption, along with a 81.11%, 92.80%, 83.72% decrement at 295 nm or 270 nm absorption, which demonstrated that the antiparallel structure of G-quadruplex DNA is more stable in the presence of Zn-CAP. Comparatively, the addition of Zn-CAP could induce significant spectral changes on the CD absorption of double helix ctDNA, with 64.17% decrement on the positive peak absorption, along with a 90.91% increment on the negative peak absorption. On the other hand, in the FRET-melting assay analysis, it was clear that Zn-CAP at 0.5 equivalences could raise the melting temperature of G-quadruplex （F2 IT or FPul 8T） by 3.45℃ and 15.85℃, indicating an obvious stabilization effect of Zn-CAP on G-quadruplex in Pu27. All the results indicated that Zn-CAP exhibited higher binding affinity and binding intensity to G-quadruplex DNA than ctDNA, especially G-quadruplex Pu27.

The parallel tetrameric DNA G-quadruplex formed by the two-repeat C9orf72 GGGGCC sequence in solution Dedicated to Professor Chaohui Ye on the occasion of his 80th birthday

The abnormal expansion of G-rich hexanucleotide repeat,GGGGCC(G4C2),in chromosome 9 open reading frame 72(C9orf72)is known to be the prevailing genetic cause of two fatal degenerative neurological diseases,amyotrophic lateral sclerosis(ALS)and frontotemporal dementia(FTD).It is well known that the DNA G4C2 repeat expansion with different lengths can form G-quadruplexes which affect gene transcription related to ALS/FTD,therefore it is crucial to understand DNA G4C2 G-quadruplex structures.Herein,by utilizing nuclear magnetic resonance(NMR)spectroscopy,we examined DNA G-quadruplex structure adopted by two G4C2 hexanucleotide repeats with an inosine substitution at position 4,d(G4C2)2-I4.We show that d(G4C2)2-I4 folds into an eight-layer parallel tetrameric G-quadruplex containing two parallel dimeric G-quadruplexes stacking together through p-p interaction via 50-to-50 mode in solution.Each dimeric G-quadruplex unit involves two propeller loops composed of two cytosine bases.This result is consistent with the observation in the crystal structure of d(G4C2)2.Our work not only sheds light on the structural diversity of G-quadruplexes adopted by d(G4C2)n but also provides a structural basis for drug design in treatment of ALS and FTD.