Lung adenocarcinoma (LUAD) remains the leading cause of cancer deaths worldwide. Apurinic/apyrimidinic endonuclease 1 (APE1), an enzyme integral to DNA repair and redox signaling, is notably upregulated in LUAD. Here we reveal that APE1 amplification, primarily via allele duplication, strongly correlates with poor prognosis in LUAD patients. Using human LUAD cell lines and a KRAS-driven mouse model, we showed that APE1 deletion hampered cell proliferation and tumor growth, highlighting its role in tumorigenesis. Mechanistically, APE1 promoted the transcription of urea cycle genes CPS1 and ARG2 by modulating the presence of G-quadruplex (G4) structures in their promoter regions. APE1 loss disrupted the urea cycle and pyrimidine metabolism, inducing metabolic reprogramming and growth arrest, which could be rescued by CPS1 or pyrimidine restoration. These findings uncover APE1's role in transcriptional regulation of urea cycle metabolic reprogramming via G4 structure, providing a potential therapeutic target LUAD patients with elevated APE1 expression.

The vascular endothelial growth factor (VEGF) promoter region, which is involved in cancer progression, contains guanine-rich sequences capable of forming G-quadruplex (G4) structures. G4s play a critical role in transcriptional regulation and genomic stability and exhibit high structural polymorphism. The major VEGF G4 adopts a parallel topology involving the first four of five G-tracts (VEGF1234), while a potential "spare-tire" mechanism suggests the formation of VEGF1245 in response to oxidative damage. Here, we characterize this alternative G4 (VEGF1245), formed by excluding the third G-tract, using circular dichroism and nuclear magnetic resonance spectroscopy. Structural analysis reveals that VEGF1245 folds in a hybrid conformation. Different from the other five tracts containing G4s, for which various strand topologies can rapidly interconvert, VEGF1245 remains thermodynamically metastable and does not refold spontaneously into VEGF1234 at physiological temperatures. Further trapping of the VEGF1245 conformation by a photolabile protecting group and its in situ release documents that the transition to VEGF1234 requires elevated temperatures, implicating kinetic barriers in the refolding process and the delineation of VEGF1245 as a prominent metastable conformation. Our findings provide new insights into transcriptional regulation and DNA repair for cancer-related VEGF-G4.

Regulation of mitochondrial calcium overload and ferroptosis with mitochondria-targeting ligands is an attractive anticancer strategy but it remains a challenge. The aim of the present study was to demonstrate that a mitochondria-targeting and mtDNA G-quadruplex-binding ligand, BYB, induced mitochondrial calcium overload and ferroptosis in HeLa cells and showed potent in vitro and in vivo anticancer activity.

Cellular functions and molecular mechanism were studied using cell viability assay, live-cell imaging, western blotting, immunofluorescence, cell uptake, cell cycle arrest and apoptosis analysis, mitochondrial metabolism analysis, Comet assay, and wound-healing analysis. Pharmacokinetic studies were conducted in rat. In vivo antitumor activity was studied in a cervical cancer HeLa cell xenograft mouse model.

Cellular results showed that BYB induced mitochondrial calcium overload, attributed to ligand-induced mitochondrial dysfunction via the mechanism of inhibiting mitochondrial DNA replication and transcription. The expression of respiratory chain complexes was markedly downregulated in BYB-treated HeLa cells. The respiratory chain function was also dysregulated. Mitophagy and mitochondrial calcium overload were induced in BYB-treated HeLa cells. Mitochondrial calcium overload markedly induced mtROS production. The induced mtDNA stress activated cGAS-STING pathway, leading to autophagy-dependent ferroptosis. The antitumour efficacy of BYB, evaluated in a HeLa tumour xenograft mouse model, achieved over 60% tumour weight reduction.

BYB, via targeting mitochondria and mtDNA G-quadruplexes, induced mitochondrial calcium overload and ferroptosis, exhibited high in vivo antitumour efficacy and low toxicity. It shows high potential to be a mitochondria-targeting lead compound for chemical biology and drug discovery.

Mitochondria are central to cellular bioenergetics, with the unique ability to translate and transcribe a subset of their own proteome. Given the critical importance of energy production, mitochondria seem to utilize higher-order nucleic acid structures to regulate gene expression, much like nuclei. Herein, we introduce a tailored approach to probe the formation of such structures, specifically G-quadruplexes, within intact mitochondria by using sensitivity-enhanced dynamic nuclear polarization-supported solid-state NMR (DNP-ssNMR). We acquired NMR spectra on isolated intact isotopically labeled mitochondria treated with berberine, a known high-affinity G-quadruplex stabilizer. The DNP-ssNMR data revealed spectral changes in nucleic acid sugar correlations, increased signal intensity for guanosine carbons, and enhanced Hoogsteen hydrogen bond formation, providing evidence of in vivo G-quadruplex formation in mitochondria. Together, our workflow enables the study of mitochondrial nucleic acid-ligand interactions at endogenous concentrations within biologically relevant environments by DNP-ssNMR, thus paving the way for future research into mitochondrial diseases and their potential treatments.

The i-motif, a cytosine-rich DNA structure formed under acidic conditions, plays a pivotal role in regulating gene expression and holds significant therapeutic potential across various diseases. Found in the promoter regions of oncogenes such as Bcl-2, C-MYC, and KRAS, i-motifs dynamically interact with transcription factors and ligands to modulate oncogene activity. Their pH-sensitive nature enables innovative applications, including cellular pH sensors like the "i-switch" and drug delivery platforms such as DNA hydrogels that release therapeutics in acidic tumor microenvironments. However, challenges remain in developing specific ligands and detection methods. Advances in nanotechnology and multi-target therapies highlight the transformative potential of i-motifs in precision medicine. This review underscores the importance of i-motifs as therapeutic targets and tools, bridging fundamental research with clinical applications in oncology, metabolic disorders, and neurodegenerative diseases.

Telomeric DNA forms G-quadruplex (G4) structures. These G4 structures are crucial for genomic stability and therapeutic targeting. Using time-resolved NMR and CD spectroscopy, we investigated how the ligand Phen-DC3 modulates the folding of the human telomeric repeat 23TAG DNA. The kinetics are modulated by the ligand and by the presence of potassium cations (K+). Ligand binding to G4 occurs via a triphasic process with fast and slow phases. Notably, for the G4 structure in the presence of K+, the slow rate is ten times slower than without K+. These findings offer key insights into the modulation of the complex folding landscape of G4s by ligands, advancing our understanding of G4-ligand interactions for potential therapeutic applications.

The study of G-quadruplex (G4) structures that form in DNA and RNA is a rapidly growing field, which has evolved from in vitro studies of isolated G4 sequences to genome-wide detection of G4s in a cellular context. This work has revealed the tangible and significant effects that G4s may have on biological regulation. This minireview describes recent progress in the design of photoluminescent intensity-independent optical probes for G4s. We discuss the design and use of probes based on fluorescence or phosphorescence lifetime, rather than intensity-based detection; spectral ratiometric probes; and fluorescent probes for single-molecule G4-detection. We argue that each of these modalities improve unbiased G4 detection in cellular experiments, overcoming problems associated with unknown cellular uptake of probes or their organelle concentration. We discuss the improvements offered by these types of probes, as well as limitations and future research directions needed to facilitate more robust research into G4 biology.

In bacteria, the regulation of gene expression involves complex networks that integrate both transcriptional and posttranscriptional mechanisms. At the transcriptional level, nucleoid-associated proteins (NAPs) such as H-NS, HU, Lrp, IHF, Fis and Hfq are key players as they not only compact bacterial DNA but also regulate transcription. Small noncoding RNAs (sRNAs), on the other hand, are known to affect bacterial gene expression posttranscriptionally by base pairing with the target mRNA, but they can also be involved in nucleoid condensation. Interestingly, certain NAPs also influence the function of sRNAs and, conversely, sRNAs themselves can modulate the activity of NAPs, creating a complex bidirectional regulatory network. Here, we summarise the current knowledge of the major NAPs, focusing on the specific role of Hfq. Examples of the regulation of NAPs by sRNAs, the regulation of sRNAs by NAPs and the role of sRNAs in nucleoid structuring are also discussed. This review focuses on the cross-talk between NAPs and sRNAs in an attempt to understand how this interplay works to orchestrate the functioning of the cell.

Nucleic acids have the potential to form not only duplexes, but also various non-canonical secondary structures in living cells. Non-canonical structures play regulatory functions mainly in the central dogma. Therefore, nucleic acid targeting molecules are potential novel therapeutic drugs that can target 'undruggable' proteins in various diseases. One of the concerns of small molecules targeting nucleic acids is selectivity, because nucleic acids have only four different building blocks. Three- and four-stranded non-canonical structures, triplexes and quadruplexes, respectively, are promising targets of small molecules because their three-dimensional structures are significantly different from the canonical duplexes, which are the most abundant in cells. Here, we describe some basic properties of the triplexes and quadruplexes and small molecules targeting the triplexes and tetraplexes.

Nucleic acids, which are fundamental to living organisms, play a crucial role in carrying and transmitting genetic information. Advances in molecular biology have led to the exploration of functional nucleic acids (FNAs), including aptamers, DNAzymes, and G-quadruplexes, known for specific recognition or catalysis. FNAs with high specificity, sequence programmability, modification ease and biocompatibility, have extensive applications in biosensing, environmental monitoring, drug delivery and cancer diagnosis. This review focuses on the structure and specific recognition principles of FNAs, followed by an exploration for biosensing and biomedical applications, offering insights into current challenges and future trends in FNAs as recognition elements.

i-Motifs, cytosine-tetrads, or C-quadruplexes are intercalated structures formed by base pairing between cytosine and protonated cytosine. These structures demonstrate increased stability in acidic environments due to the presence of the latter cytosinium group (i.e., the protonated cytosine). Research has shown that i-motifs are typically disrupted or destabilized at physiological pH levels (7.0-7.4), which makes their potential formation in the nucleus and their biological relevance uncertain. However, in 2018, it was demonstrated that i-motifs exist within the nucleus under physiological conditions, with various intracellular factors contributing to their stability. Identification of i-motifs in the nucleus and their association with gene promoters-particularly with those of proto-oncogenes-has generated significant interest in their potential regulatory functions. Additionally, recent studies suggest that i-motifs may function as switches for gene expression, influencing gene regulation through their folding and stabilization or unfolding and destabilization. This review aims to delve into these mechanisms to improve our understanding of the physiological significance of i-motifs.

We report the discovery of a novel higher-order RNA structure, RNA G-triplex (rG3), formed by the TERRA sequence. Through CD spectroscopy, NMR analysis, and molecular modeling, we confirmed its stable, parallel conformation. rG3 exhibits strong binding to thioflavin T (ThT), N-methyl mesoporphyrin IX (NMM), and hemin, showcasing its potential as a biosensing element. Additionally, CRISPR-Cas13a trans-cleaves rG3, demonstrating its utility as a sensitive reporter in diagnostic applications. These findings expand the structural diversity of RNA and suggest new avenues for RNA-based biosensors and CRISPR diagnostics.

G-quadruplex (G4) DNAs are alternative nucleic acid structures, proposed to play important roles in regulating DNA replication, gene transcription, and translation. Several specialized DNA helicases are involved in cellular G4 metabolism, in some cases with redundant functions. Among them, human FANCJ/BRIP1, which has orthologs in all metazoans, is one of the most powerful G4 resolvases, believed to act mainly at DNA replication forks. Here, we tested the effects of a set of hydrazone-derivative G4 ligands in a FANCJ-knocked-out HeLa cell line and in a Caenorhabditis elegans strain, where DOG-1, a FANCJ ortholog, was disrupted, as a whole organism model system. Our results revealed that loss of FANCJ specifically sensitized cancer cells to FIM-15, a mono-guanylhydrazone derivative bearing the diimidazopyrimidine core, among the tested hydrazone-based compounds and induced enhanced DNA damage in different chromosomal sites including telomeric ends. Moreover, dietary administration of FIM-15 to dog-1 -/- nematodes stabilized G4 structures in gonadal cell nuclei and resulted in compromised embryonic development in the first-generation post-treatment. Collectively, our findings unveil a specific vulnerability of FANCJ-knocked-out cancer cells (and DOG-1-lacking worms) to G4 stabilization by the FIM-15 compound. This study provides an important proof-of-principle for use of G4 ligands in synthetic lethality-based therapeutic approaches targeting FANCJ-defective cancer cells.

G-quadruplexes (G4s) with continuous G-tracts are well-established regulators of gene expression and important therapeutic targets for various diseases. However, bioinformatics analyses have identified G4-like sequences containing interrupted G-tracts, incorporating non-G nucleotides as bulges (buG4s). Our findings show that the stability of buG4s is significantly influenced by the bulge position and size within the G-tract, with bulges at the 5' end exhibiting the highest stability. Moreover, a molecular crowding condition inducing by poly (ethylene glycol), providing a suitable intracellular environment, stabilizes buG4s, especially those with longer bulges, making their formation more pronounced. A transcription assay performed under crowding conditions revealed that the transcription arrested efficiency by buG4s is affected not only by stability but also by the position and size of the bulge. Based on these findings, we propose a model for the preliminary screening of buG4 sequences according to their stability, distinguishing functional sequences capable of transcriptional arrest (ΔG°37 ≤ -3.3 kcal·mol-1) from nonfunctional sequences (ΔG°37 > -3.3 kcal·mol-1). This provides valuable insight into estimating the efficiency of target buG4 sequences in either arresting or facilitating transcription, presenting a novel approach and emphasizing buG4s as emerging therapeutic targets.

Hydrogels are emerging as one of the most sought-after drug carriers due to their biocompatibility, high water content mimicking tissue-like environment, injectability, and stimuli responsiveness. Sustained drug release accompanied by targeted delivery to cancer cells can abate numerous adverse side effects of conventional chemotherapy. Folate receptors are overexpressed in various cancer cells, and their high binding affinity to folic acid (FA) makes folic acid-anchored drug carriers a specific targeting entity. Reports of folic acid-based hydrogels are still scarce, owing to their low solubility in water. In this study, we present a simple approach to generate a self-assembled supramolecular hydrogel by employing an amphiphilic low molecular weight gelator (LMWG), guanosine monophosphate (GMP), which noncovalently interacts and coassembles with FA. The hydrogel shows biocompatibility, thermoreversibility, self-healing, injectability, thixotropy, and self-adhesive properties. The hydrogel could encapsulate and release both hydrophilic (doxorubicin) and hydrophobic (curcumin) drugs in a sustained manner. In vitro studies on cancer cells showed that encapsulating the drugs within the hydrogel matrix resulted in enhanced uptake by the cancer cells, thereby increasing their therapeutic efficacy through upregulating tumor suppressor, apoptotic gene expression, and inhibiting cell proliferation markers. Thus, a straightforward fabrication procedure, cost-effectiveness, and treatment potency make the FA-GMP hydrogel a promising drug carrier for practical use in biomedical applications.

Certain proteins and synthetic covalent polymers experience aqueous phase transitions, driving functional self-assembly. Herein, we unveil the ability of supramolecular polymers (SPs) formed by G4.Cu+ to undergo heating-induced unexpected aqueous phase transitions. For the first time, guided by Cu+, guanosine (G) formed a highly stable G-quartet (G4.Cu+)/G-quadruplex as a non-canonical DNA secondary structure with temperature tolerance, distinct from the well-known G4.K+. The G4.Cu+ self-assembled in water through π-π stacking, metallophilic and hydrophobic interactions, forming thermally robust SPs. This enhanced stability is attributed to the stronger coordination of Cu+ to four carbonyl oxygens of G-quartet and the presence of Cu+- - -Cu+ attractive metallophilic interactions in Cu+-induced G-quadruplex, exhibiting a significantly higher interaction energy than K+ as determined computationally. Remarkably, the aqueous SP solution exhibited heating-induced phase transitions-forming a hydrogel through dehydration-driven crosslinking of SPs below cloud temperature (Tcp) and a hydrophobic collapse-induced solid precipitate above Tcp, showcasing a lower critical solution temperature (LCST) behavior. Notably, this LCST behavior of G4.Cu+ SP originates from biomolecular functionality rather than commonly exploited thermo-responsive oligoethylene glycols with supramolecular assemblies. Furthermore, exploiting the redox reversibility of Cu+/Cu2+, we demonstrated control over the assembly and disassembly of G-quartets/G-quadruplex and gelation reversibly.

Mycobacterium tuberculosis (Mtb) contains potential G-quadruplex (PGQ) motifs in the genes espK and cyp51, which are crucial for the bacteria's virulence within host cells. Aminoglycoside molecules are commonly used as antibiotics for ribosomal targets. This study provides insight into the interactions between these aminoglycosides and Mtb-PGQ sequences (espK and cyp51), shedding light on the structural and thermodynamic dynamics of their binding. This study demonstrates the stability, affinity, and conformation of Mtb-PGQ in the presence of neomycin and streptomycin. Ultraviolet-visible spectroscopy (UV-vis), circular dichroism spectroscopy (CD), CD thermal melting, isothermal titration calorimetry (ITC), and fluorescence intercalator displacement (FID) assays were used to comprehensively examine these interactions. Our results reveal that neomycin with Mtb-PGQexhibits hypochromism accompanied by a 4-5 nm red shift in the UV-visible absorption titration, whereas streptomycin exhibits a hypochromic shift without changes in the maximum wavelength. Notably, neomycin shows a nonlinear binding isotherm, suggesting the involvement of more than one binding process in the formation of neomycin.Mtb-PGQ complexes. Scatchard plot analysis indicates higher binding affinity values for neomycin compared with weaker affinity of streptomycin. CD studies reveal that neomycin decreases the ellipticity of Mtb-PGQ with a red shift while retaining the parallel topology, ultimately enhancing the thermal stability of both espK and cyp51. In contrast, streptomycin destabilizes the cells. ITC analysis reveals that neomycin exhibits the strongest binding affinity for cyp51, with the relative order being NEO-cyp51 > NEO-espk > STR-cyp51 > STR-espk. Moreover, thermodynamic analysis reveals that neomycin possesses a unique dual mode of binding through grooves as well as stacking. FID studies further confirm a lower DC50 value for neomycin than for streptomycin, suggesting that neomycin is a strong displacer of thiazole orange. Thus, the results show that neomycin with amino groups selectively recognizes the grooves of cyp51 over espK.

As artificial water channels have received widespread attention, various types of artificial water channels have been reported. However, apart from I-quartet channels, the development of 1D water channels with water wires constructed from small molecules has rarely been reported, because of the difficulty in precisely tuning the dipolar water molecules. Inspired by G-quartet functionalization strategies, this study explored C8 modifications of our previously reported molecule, 2-amino-2'-fluoro-2'-deoxyadenosine (2FA), known for its strong hydration properties and self-assembly capabilities, and investigated its potential for constructing nucleoside-based water channel-like structures. Among all derivatives, 2-amino-8-(4-aminophenyl)-2'-deoxy-2'-fluoro-D-adenosine can form an S-shaped channel as a tetramer, incorporating water wire arrays in the solid state. Such water channel-like structures in nucleoside self-assemblies provide new insights into the development of novel nucleoside-based supramolecular water channel materials.

Membrane-less organelles, formed by liquid-liquid phase separation, participate in many vital cellular processes and have received extensive attention recently. A notable form of noncanonical nucleic acid secondary structure, G-quadruplex (G4), interacts with the scaffolding proteins in these membrane-less organelles and becomes an integral part of this condensed phase. However, the structure and stability features of the integrated G4 remain poorly characterized. Herein, we employed NMR along with other biophysical methods to investigate the conformation of a G4 within condensates formed by a disordered protein known as DDX4N1. We discovered that the human telomeric sequence MHT24, which forms a G4 structure in a non-condensed phase solution of protein DDX4N1, unfolds when it is within DDX4N1 condensates due to phase separation. Our findings provide an instance of a protein acquiring new functionality through phase separation process, which deepen our understanding of how protein condensates regulate G4 structure and their functions.

RNA G-quadruplexes (rG4s) are non-canonical secondary nucleic acid structures found in the transcriptome. They play crucial roles in gene regulation by interacting with G4-binding proteins (G4BPs) in cells. rG4-G4BP complexes have been associated with human diseases, making them important targets for drug development. Generating innovative tools to disrupt rG4-G4BP interactions will provide a unique opportunity to explore new biological mechanisms and potentially treat related diseases. Here, we have rationally designed and developed a series of rG4-based proteolytic targeting chimeras (rG4-PROTACs) aimed at degrading G4BPs, such as DHX36, a specific G4BP that regulates gene expression by binding to and unraveling rG4 structures in messenger RNAs (mRNAs). Our comprehensive data and systematic analysis reveals that rG4-PROTACs predominantly and selectively degrade DHX36 through a proteosome-dependent mechanism, which promotes the formation of the rG4 structure in mRNA, leading to the translation inhibition of rG4-containing transcripts. Notably, rG4-PROTACs inhibit rG4-mediated APP protein expression, and impact the proliferative capacity of skeletal muscle stem cells by negatively regulating Gnai2 protein expression. In summary, rG4-PROTACs provide a new avenue to understand rG4-G4BP interactions and the biological implications of dysregulated G4BPs, promoting the development of PROTACs technology based on the non-canonical structure of nucleic acids.

As global food production continues to surge, the widespread use of herbicides has also increased concurrently, posing challenges like health risks and environmental pollution. Traditional detection methods for pesticide residues, such as diquat (DQ), were hampered by limitations like high expenses, lengthy detection times and complex operations, restricting their practical application in rapid clinical diagnosis.

In light of the pressing necessity for the identification of minute pesticide residues and the intrinsic constraints of small molecule analysis, a novel chromophotometric biosensor targeting small molecules was developed based on bi-epitopes on single antibody to immobilize two DQ-PAL, inhibiting the hybridization of DQ-PAL. Accordingly, the free DQ-PAL could hybridize with each other to form a G-quadruplex for a highly selective analysis of DQ with a detection limit of 26.3 pg/mL and 10 pg/mL by chromophotometric and image colorimetric method respectively. Furthermore, this designed biosensor has been successfully applied to evaluate the levels of DQ residues in real samples, providing an efficient solution for the biological analysis of small molecule targets and enhancing food safety concerning pesticide residues.

In comparison to conventional techniques, this biosensor has the advantages of user-friendly operations, portability, high sensitivity, low detection limit and minimal background interference, making it well-suited for clinical diagnostics. At the same time, this technology provides a new idea for the rapid in vitro detection of biological small molecules, and shows great potential applications in agricultural residue-related food safety.

The human genome contains numerous repetitive nucleotide sequences that display a propensity to fold into non-canonical DNA structures including G-quadruplexes (G4s). G4s have both positive and negative impacts on various aspects of nucleic acid metabolism including DNA replication, DNA repair and RNA transcription. Poly (ADP-ribose) polymerase (PARP1), an important anticancer drug target, has been recently shown to bind a subset of G4s, and to undergo auto-PARylation. The mechanism of this interaction, however, is poorly understood. Utilizing Mass Photometry (MP) and single-molecule total internal reflection fluorescence microscopy (smTIRFM), we demonstrate that PARP1 dynamically interacts with G4s with a 1:1 stoichiometry. Interaction of a single PARP1 molecule with nicked DNA or DNA containing G4 and a primer-template junction is sufficient to activate robust auto-PARylation resulting in the addition of poly (ADP-ribose) chains with molecular weight of several hundred kDa. Pharmacological PARP inhibitors EB-47, Olaparib and Veliparib differently affect PARP1 retention on G4-containing DNA compared to nicked DNA.

Natural channel proteins allow the selective permeation of ions, water or other nutritious entities across bilayer membranes, facilitating various essential physiological functions in living systems. Inspired by nature, chemists endeavor to simulate the structural features and transport behaviors of channel proteins through biomimetic strategies. In this review, we start from introducing the inherent traits of channel proteins such as their crystal structures, functions and mechanisms. Subsequently, different kind of synthetic ion channels including their design principles, dynamic regulations and therapeutic applications were carefully reviewed. Finally, the potential challenges and opportunities in this research field were also carefully discussed. It is anticipated that this review could provide some inspiring ideas and future directions towards the construction of novel bionic ion channels with higher-level structures, properties, functions and practical applications.

Retroviruses are among the most extensively studied viral families, both historically and in contemporary research. They are primarily investigated in the fields of viral oncogenesis, reverse transcription mechanisms, and other infection-specific aspects. These include the integration of endogenous retroviruses (ERVs) into host genomes, a process widely utilized in genetic engineering, and the ongoing search for HIV/AIDS treatment. G-quadruplexes (G4) have emerged as potential therapeutic targets in antiviral therapy and have been identified in important regulatory regions of viral genomes. In this study, we examine the presence of potential G-quadruplex-forming sequences (PQS) across all currently available unique retroviral genomes. Given that these retroviral genomes typically consist of single-stranded RNA (ssRNA) molecules, we also investigated whether the localization of PQSs is strand-dependent. This is particularly relevant since antisense transcripts have been detected in HIV, and ERV integration into the host genome involves reverse transcription from genomic positive strand ssRNA to double-stranded DNA (dsDNA), implicating both strands in this process. We show that in most mammalian retroviruses, including human retroviruses, PQSs are significantly more prevalent on the negative (antisense) strand, with some notable exceptions such as HIV-1. In sharp contrast, avian retroviruses exhibit a higher prevalence of PQSs on the positive (sense) strand.

I-motifs are non-canonical DNA structures with recognized biological significance and a proven utility in material engineering. Consequently, understanding and control of i-motif properties is essential to sustain progress across both disciplines. In this work, we systematically investigate how proximity to the most common form of DNA, a double-stranded duplex, influences the thermodynamic and kinetic properties of adjacent i-motifs. We demonstrate that double-stranded stems in i-motif loops promote kinetic trapping of very stable and persistent partially folded conformations. Further, we investigate pathways toward rational control over a folding topology makeup.

Quadruplexes formed by guanine derivatives or guanine-rich nucleic acids are involved in metabolism and genetic storage of many living organisms, they are used in DNA nanotechnologies or as biosensors. Since many quadruplex geometries are possible the determination of their structures in aqueous solutions is difficult. Raman optical activity (ROA) can make it easier: For guanosine monophosphate (GMP), we observed a distinct change of the spectra upon its condensation and quadruplex formation. The vibrational bands become more numerous, stronger, and narrower. In particular, a huge ROA signal appears below 200 cm-1. The aggregation can be induced by high concentration, low temperature, or by a metal ion. We focused on well-defined quadruplexes stabilized by potassium, where using molecular dynamics and density functional theory the spectra and particular features related to GMP geometric parameters could be understood. The simulations explain the main experimental trends and confirm that the ROA spectroscopy is sensitive to fine structural details, including guanine base twist in the quadruplex helix.

Non-canonical DNA structures, such as the G-quadruplex (G4) and i-motif (iM), are formed at guanine- and cytosine-rich sequences, respectively, in living cells and involved in regulating various biological processes during the cell cycle. Therefore, the formation and resolution of these non-canonical structures must be dynamically regulated by physiological conditions or factors that can bind G4 and iM structures. Although many G4 binding proteins responsible for tuning the G4 structure have been discovered, the structural regulation of iM by iM-binding proteins remains enigmatic. In this study, we developed a protein-labeling DNA probe bearing an alkyne moiety through a reactive linker, for proximity-labeling of nucleic acid-binding proteins, and searched for new iM-binding proteins. Alkyne-modified proteins in the nuclear extract of HeLa cells were labeled with biotin via a click reaction and then captured with streptavidin-coated magnetic beads. This fingerprint-targeting enrichment, followed by proteome analyses, identified new candidate proteins that potentially bind to the iM structure, in addition to the reported iM-binding proteins. Among the newly identified candidates, we characterized a nucleolar protein, nucleolin, that binds to the iM structure and relaxes it, while nucleolin stabilizes the G4 structure.

The functional effects of an RNA can arise from complex three-dimensional folds known as tertiary structures. However, predicting the tertiary structure of an RNA and whether an RNA adopts distinct tertiary conformations remains challenging. To address this, we developed BASH MaP, a single-molecule dimethyl sulfate (DMS) footprinting method and DAGGER, a computational pipeline, to identify alternative tertiary structures adopted by different molecules of RNA. BASH MaP utilizes potassium borohydride to reveal the chemical accessibility of the N7 position of guanosine, a key mediator of tertiary structures. We used BASH MaP to identify diverse conformational states and dynamics of RNA G-quadruplexes, an important RNA tertiary motif, in vitro and in cells. BASH MaP and DAGGER analysis of the fluorogenic aptamer Spinach reveals that it adopts alternative tertiary conformations which determine its fluorescence states. BASH MaP thus provides an approach for structural analysis of RNA by revealing previously undetectable tertiary structures.

Sequence-selective G-quadruplex ligands are valuable for controlling gene expression. Here, we established a new fluorescence displacement assay using a NRAS G-quadruplex selective fluorescent probe to identify sequence-selective DNA G-quadruplex ligands. These sequence-selective NRAS G-quadruplex ligands retained their binding affinity even in the presence of excessive human telomeric DNA G-quadruplex and regulated enzymatic activities in a sequence-selective manner.

Regulating gene expression in plant development and environmental responses is vital for mitigating the effects of climate change on crop growth and productivity. The eukaryotic genome largely shows the canonical B-DNA structure that is organized into nucleosomes with histone modifications shaping the epigenome. Nuclear proteins and RNA interactions influence chromatin conformations and dynamically modulate gene activity. Non-B DNA conformations and their transitions introduce novel aspects to gene expression modulation, particularly in response to environmental shifts. We explore the current understanding of non-B DNA structures in plant genomes, their interplay with epigenomics and gene expression, and advances in methods for their mapping and characterization. The exploration of so far uncharacterized non-B DNA structures remains an intriguing area in plant chromatin research and offers insights into their potential role in gene regulation.

The construction of supramolecular aerogels still faces great challenges. Herein, we present a novel bio-based supramolecular aerogel derived from G-Quadruplex self-assembly of guanosine (G), boric acid (B) and sodium alginate (SA) and the obtained GBS aerogels exhibit superior flame-retardant and thermal insulating properties. The entire process involves environmentally friendly aqueous solvents and freeze-drying. Benefiting from the supramolecular self-assembly and interpenetrating dual network structures, GBS aerogels exhibit unique structures and sufficient self-supporting capabilities. The resulting GBS aerogels exhibit overall low densities (36.5-52.4 mg/cm3), and high porosities (>95 %). Moreover, GBS aerogels also illustrate excellent flame retardant and thermal insulating properties. With an oxygen index of 47.0-51.1 %, it can easily achieve a V-0 rating and low heat, smoke release during combustion. This work demonstrates the preparation of intrinsic flame-retardant aerogels derived from supramolecular self-assembly and dual cross-linking strategies, and is expected to provide an idea for the realization and application of novel supramolecular aerogel materials.

Artificial metallo-nucleases (AMNs) are small molecule DNA cleavage agents, also known as DNA molecular scissors, and represent an important class of chemotherapeutic with high clinical potential. This review provides a primary level of exploration on the concepts key to this area including an introduction to DNA structure, function, recognition, along with damage and repair mechanisms. Building on this foundation, we describe hybrid molecules where AMNs are covalently attached to directing groups that provide molecular scissors with enhanced or sequence specific DNA damaging capabilities. As this research field continues to evolve, understanding the applications of AMNs along with synthetic conjugation strategies can provide the basis for future innovations, particularly for designing new artificial gene editing systems.

Novel strategies against parasitic infections are of great importance. Here, we describe a G4 DNA ligand with subnanomolar antiparasitic activity against T. brucei and a remarkable selectivity index (IC50 MRC-5/T. brucei) of 2285-fold. We also correlate the impact of small structural changes to G4 binding activity and antiparasitic activity.

Nuclear localization of the metabolic enzyme PKM2 is widely observed in various cancer types. We identify nuclear PKM2 as a non-canonical RNA-binding protein (RBP) that specifically interacts with folded RNA G-quadruplex (rG4) structures in precursor mRNAs (pre-mRNAs). PKM2 occupancy at rG4s prevents the binding of repressive RBPs, such as HNRNPF, and promotes the expression of rG4-containing pre-mRNAs (the "rG4ome"). We observe an upregulation of the rG4ome during epithelial-to-mesenchymal transition and a negative correlation of rG4 abundance with patient survival in different cancer types. By preventing the nuclear accumulation of PKM2, we could repress the rG4ome in triple-negative breast cancer cells and reduce migration and invasion of cancer cells in vitro and in xenograft mouse models. Our data suggest that the balance of folded and unfolded rG4s controlled by RBPs impacts gene expression during tumor progression.

This article provides a comprehensive examination of non-canonical DNA structures, particularly focusing on G-quadruplexes (G4s) and i-motifs. G-quadruplexes, four-stranded structures formed by guanine-rich sequences, are stabilized by Hoogsteen hydrogen bonds and monovalent cations like potassium. These structures exhibit diverse topologies and are implicated in critical genomic regions such as telomeres and promoter regions of oncogenes, playing significant roles in gene expression regulation, genome stability, and cellular aging. I-motifs, formed by cytosine-rich sequences under acidic conditions and stabilized by hemiprotonated cytosine-cytosine (C:C+) base pairs, also contribute to gene regulation despite being less prevalent than G4s. This review highlights the factors influencing the stability and dynamics of these structures, including sequence composition, ionic conditions, and environmental pH. Molecular dynamics simulations and high-resolution structural techniques have been pivotal in advancing our understanding of their folding and unfolding mechanisms. Additionally, the article discusses the therapeutic potential of small molecules designed to selectively bind and stabilize G4s and i-motifs, with promising implications for cancer treatment. Furthermore, the structural properties of these DNA forms are explored for applications in nanotechnology and molecular devices. Despite significant progress, challenges remain in observing these structures in vivo and fully elucidating their biological functions. The review underscores the importance of continued research to uncover new insights into the genomic roles of G4s and i-motifs and their potential applications in medicine and technology. This ongoing research promises exciting developments in both basic science and applied fields, emphasizing the relevance and future prospects of these intriguing DNA structures.

G-quadruplex DNA structures (G4s) form within single-stranded DNA in nucleosome-free chromatin. As G4s modulate gene expression and genomic stability, genome-wide mapping of G4s has generated strong research interest. Recently, the Cleavage Under Targets and Tagmentation (CUT&Tag) method was performed with the G4-specific BG4 antibody to target Tn5 transposase to G4s. While this method generated a novel high-resolution map of G4s, we unexpectedly observed a strong correlation between the genome-wide signal distribution of BG4 CUT&Tag and accessible chromatin. To examine whether untargeted Tn5 cutting at accessible chromatin contributes to BG4 CUT&Tag signal, we examined the genome-wide distribution of signal from untargeted (i.e. negative control) CUT&Tag datasets. We observed that untargeted CUT&Tag signal distribution was highly similar to both that of accessible chromatin and of BG4 CUT&Tag. We also observed that BG4 CUT&Tag signal increased at mapped G4s, but this increase was accompanied by a concomitant increase in untargeted CUT&Tag at the same loci. Consequently, enrichment of BG4 CUT&Tag over untargeted CUT&Tag was not increased at mapped G4s. These results imply that either the vast majority of accessible chromatin regions contain mappable G4s or that the presence of G4s within accessible chromatin cannot reliably be determined using BG4 CUT&Tag alone.

G-quadruplexes are noncanonical structures of nucleic acids formed mainly by G-rich sequences and play crucial roles in important cellular processes. They are also increasingly used in nanotechnology for their valuable properties. Various unexpected structures of G-quadruplexes have been solved recently, including a stable G-quadruplex lacking one guanine in the G-tetrad core, harboring a vacant site. In this study, we demonstrate the interlocking of two intramolecular G-quadruplexes: one containing a vacant site (4n - 1) and the other with an unbound guanine (4n + 1). These G-quadruplexes interact through a G-triad-G connection with unprecedented 5'-3' stacking. Using these interconnection properties, we have identified a sequence capable of self-assembling into G-wires in K+ solutions with potential nanotechnological applications.

As with all new fields of discovery, work on the biological role of G-quadruplexes (GQs) has produced a number of results that at first glance are quite baffling, sometimes because they do not fit well together, but mostly because they are different from commonly held expectations. Like other classes of flipons, those that form G-quadruplexes have a repeat sequence motif that enables the fold. The canonical DNA motif (G3N1-7)3G3, where N is any nucleotide and G is guanine, is a feature that is under active selection in avian and mammalian genomes. The involvement of G-flipons in genome maintenance traces back to the invertebrate Caenorhabditis elegans and to ancient DNA repair pathways. The role of GQs in transcription is supported by the observation that yeast Rap1 protein binds both B-DNA, in a sequence-specific manner, and GQs, in a structure-specific manner, through the same helix. Other sequence-specific transcription factors (TFs) also engage both conformations to actuate cellular transactions. Noncoding RNAs can also modulate GQ formation in a sequence-specific manner and engage the same cellular machinery as localized by TFs, linking the ancient RNA world with the modern protein world. The coevolution of noncoding RNAs and sequence-specific proteins is supported by studies of early embryonic development, where the transient formation of G-quadruplexes coordinates the epigenetic specification of cell fate.

Local variation of DNA structure and its dynamic nature play an essential role in the regulation of important biological processes. One of the most prominent noncanonical structures are G-quadruplexes, which form in vivo within guanine-rich regions and have been demonstrated to be involved in the regulation of transcription, translation and telomere maintenance. We provide an analysis of G-quadruplex formation in sequences with five and six guanine residues long G-tracts, which have emerged from the investigation of the gapless human genome and are associated with genes related to cancer and neurodegenerative diseases. We systematically explored the effect of G-tract and loop elongations by means of NMR and CD spectroscopy and polyacrylamide electrophoresis. Despite both types of elongation leading up to structural polymorphism, we successfully determined the topologies of four out of eight examined sequences, one of which contributes to a very scarce selection of currently known intramolecular four G-quartet structures in potassium solutions. We demonstrate that examined sequences are incompatible with five or six G-quartet structures with propeller loops, although the compatibility with other loop types cannot be factored out. Lastly, we propose a novel approach towards specific G-quadruplex targeting that could be implemented in structures with more than four G-quartets.

Telomeres, crucial for chromosomal integrity, have been related to aging and cancer formation, mainly through regulating G-quadruplex structures. G-quadruplexes are structural motifs that can arise as secondary structures of nucleic acids, especially in guanine-rich DNA and RNA regions. Targeting these structures by small compounds shows promise in the selective suppression of cell growth, opening up novel possibilities for anticancer treatment. A comprehensive investigation of the many structural forms of G-quadruplex ligands is required to create ground-breaking anticancer drugs. Recent research into using specific benzimidazole molecules in stabilizing telomeric DNA into G-quadruplex structures has highlighted their ability to influence oncogene expression and demonstrate antiproliferative characteristics against cancer cells. This review describes the benzimidazole derivative, designed to enhance the stability of the G-quadruplex structure DNA to suppress the activity of telomerase enzyme, exhibiting promising potential for anticancer therapy.

RNA G-quadruplexes (rG4s) are noncanonical secondary structures formed by guanine-rich sequences that are found in different regions of RNA molecules. These structures have been implicated in diverse biological processes, including translation, splicing, and RNA stability. Recent studies have suggested that rG4s play a role in the cellular response to stress. This review summarizes the current knowledge on rG4s under stress, focusing on their formation, regulation, and potential functions in stress response pathways. We discuss the molecular mechanisms that regulate the formation of rG4 under different stress conditions and the impact of these structures on RNA metabolism, gene expression, and cell survival. Finally, we highlight the potential therapeutic implications of targeting rG4s for the treatment of stress-related diseases through modulating cell survival.

G-quadruplexes (G4s) form throughout the genome and influence important cellular processes. Their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected role for the double-stranded DNA (dsDNA) translocase helicase-like transcription factor (HLTF) in responding to G4s. We show that HLTF, which is enriched at G4s in the human genome, can directly unfold G4s in vitro and uses this ATP-dependent translocase function to suppress G4 accumulation throughout the cell cycle. Additionally, MSH2 (a component of MutS heterodimers that bind G4s) and HLTF act synergistically to suppress G4 accumulation, restrict alternative lengthening of telomeres, and promote resistance to G4-stabilizing drugs. In a discrete but complementary role, HLTF restrains DNA synthesis when G4s are stabilized by suppressing primase-polymerase (PrimPol)-dependent repriming. Together, the distinct roles of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability.

The functional effects of an RNA can arise from complex three-dimensional folds known as tertiary structures. However, predicting the tertiary structure of an RNA and whether an RNA adopts distinct tertiary conformations remains challenging. To address this, we developed BASH MaP, a single-molecule dimethyl sulfate (DMS) footprinting method and DAGGER, a computational pipeline, to identify alternative tertiary structures adopted by different molecules of RNA. BASH MaP utilizes potassium borohydride to reveal the chemical accessibility of the N7 position of guanosine, a key mediator of tertiary structures. We used BASH MaP to identify diverse conformational states and dynamics of RNA G-quadruplexes, an important RNA tertiary motif, in vitro and in cells. BASH MaP and DAGGER analysis of the fluorogenic aptamer Spinach reveals that it adopts alternative tertiary conformations which determine its fluorescence states. BASH MaP thus provides an approach for structural analysis of RNA by revealing previously undetectable tertiary structures.

In billion years of evolution, eukaryotes preserved the chromosome ends with arrays of guanine repeats surrounded by thymines and adenines, which can form stacks of four-stranded planar structure known as G-quadruplex (G4). The rationale behind the evolutionary conservation of the G4 structure at the telomere remained elusive. Our recent study has shed light on this matter by revealing that telomere G4 undergoes oscillation between at least two distinct folded conformations. Additionally, tumor suppressor BRCA2 exhibits a unique mode of interaction with telomere G4. To elaborate, BRCA2 directly interacts with G-triplex (G3)-derived intermediates that form during the interconversion of the two different G4 states. In doing so, BRCA2 remodels the G4, facilitating the restart of stalled replication forks. In this review, we succinctly summarize the findings regarding the dynamicity of telomeric G4, emphasize its importance in maintaining telomere replication homeostasis, and the physiological consequences of losing G4 dynamicity at the telomere.

G-quadruplex (G4) sequences, which can fold into higher-order G4 structures, are abundant in the human genome and are over-represented in the promoter regions of many genes involved in human cancer initiation, progression, and metastasis. They are plausible targets for G4-binding small molecules, which would, in the case of promoter G4s, result in the transcriptional downregulation of these genes. However, structural information is currently available on only a very small number of G4s and their ligand complexes. This limitation, coupled with the currently restricted information on the G4-containing genes involved in most complex human cancers, has led to the development of a phenotypic-led approach to G4 ligand drug discovery. This approach was illustrated by the discovery of several generations of tri- and tetra-substituted naphthalene diimide (ND) ligands that were found to show potent growth inhibition in pancreatic cancer cell lines and are active in in vivo models for this hard-to-treat disease. The cycles of discovery have culminated in a highly potent tetra-substituted ND derivative, QN-302, which is currently being evaluated in a Phase 1 clinical trial. The major genes whose expression has been down-regulated by QN-302 are presented here: all contain G4 propensity and have been found to be up-regulated in human pancreatic cancer. Some of these genes are also upregulated in other human cancers, supporting the hypothesis that QN-302 is a pan-G4 drug of potential utility beyond pancreatic cancer.