Single-molecule approaches to study G-quadruplex, R-loop, and protein interactions.

The formation of membraneless organelles via liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as a central mechanism of cellular compartmentalization to finely regulate biological processes. DDX3X, a member of the DEAD-box RNA helicase family, is one of the global regulators of RNA-containing phase-separated organelles. While the importance of DDX3X in organelle formation is well-recognized, the molecular mechanisms underlying its RNA-driven LLPS remain poorly understood. In this study, we focused on the dynamic interactions between the N-terminal intrinsically disordered region (N-IDR) of DDX3X and G-quadruplex (GQ) RNA, which is a key regulator of physiological membraneless organelle assembly owing to its unique ability to promote LLPS. Using solution nuclear magnetic resonance spectroscopy, we identified hotspot regions for self-assembly within the N-IDR. These regions comprise charged stretches interspersed with key aromatic residues, whose interactions drive LLPS through a combination of electrostatic and π-interactions. Binding of GQ RNA effectively strengthens intermolecular interactions involving the arginine-rich segments of the N-IDR, providing molecular insights into its RNA-driven LLPS. We further discuss the functional implications of GQ-specific granule formation under stress conditions, highlighting the potential roles of DDX3X-GQ RNA interplay in cellular translational regulation.

While it is well established that the Zα domains of ADAR1 and ZBP1 proteins bind Z-form-prone nucleic acids (Z-NAs), it has also been shown that the Zα domain of ADAR1 binds DNA G-quadruplexes (GQs). However, no binding partner of the structurally homologous Zβ domain of ADAR1 has been identified to date. Based on AlphaFold and molecular dynamics simulations, it has recently been suggested that Zβ of ADAR1 targets its substrate by recognizing GQs. Here, we provide the first experimental evidence for Zβ binding to select GQ RNA and DNA in vitro, with structural specificity and nanomolar affinity. We also demonstrate that the Zα domains of ZBP1 and ADAR1 bind to both DNA and RNA GQs with similar affinity. These findings extend the range of potential functional roles for these proteins and open new hypotheses for testing in cells.

Conformational flexibility of natural enzymes, characterized by marginal stability, displays functional variations. In this work, we uncovered that artificial DNAzymes made of G-quadruplex (GQ)-hemin complexes also display this marginal stability phenomenon. Through single-molecule fluorescent MT-HILO (Magnetic Tweezers coupled with Highly Inclined and Laminated Optical sheet), we recorded the highest peroxidase activity among all known natural or artificial enzymes when the GQ mechanophore inside the DNAzymes was destabilized by an external force. We name enzymes with such force-responsive marginal stability "mechanozymes" to reflect the mechanical modulation of enzymatic activities. To set the stage for mechanical modulation on mechanozymes beyond the single-molecule level, ultrasonication was applied to enhance the catalytic function of a large ensemble of mechanozymes by weakening their GQ mechanophore structures. This work not only established marginal stability in artificial enzymes for the first time, but it also provided unprecedented mechanical modulation on catalytic activities, both of which are expected to have profound ramifications for catalysis exploited across the fields of chemistry and biosciences.

Small bioactive molecules show significant propensity to form noncovalent addition complexes with guanine quadruplexes, G4. The stabilization energies of these complexes have been computed precisely at the sufficiently high 6-31G** basis set level of density functional quantum chemical theory, DFT. A decisive factor in present model computations is the adopted size of G4 models, whether these consist simply of stacked quanine quartets, or also involve (deoxy)ribose-phosphate fragments of proper nucleic acids. The challenge is in the preservation of physico-chemical accuracy of DFT computations with increasing sizes of models, involving upwards of 120 atoms for the simplest two-layer G4, plus at least 60 pentose-phosphate linker atoms per each pair of guanine quartets. Bioactive ligand sizes add to the requirements for further rigorous analyses of the roles of G4 complexes in biological processes, which thus remain necessarily open-ended.

Multiple sclerosisis a fatal neurodegenerative disease that progressesby eroding the myelin sheath and exposing the neuron, leading to neuronaldegradation and death. While multiple sclerosis remains without aneffective treatment or cure, studies have identified genes that aredysregulated in multiple sclerosis patients and predicted to be involvedwith disease progression. These genes are primarily involved in controllingDNA methylation, a process required for regulating gene expressionthat is critical for cellular health. Having identified potentialgenetic risk factors, current research focuses on how to manipulatethe expression of these genes, offsetting DNA methylation errors inpatients by targeting DNA secondary structure formation. Serine hydroxymethyltransferase1 (SHMT1) is a key player in DNA methylation and was determined tobe upregulated in multiple sclerosis patients. Here, we characterizedhybrid 3 + 1 G-quadruplex (GQ) and i-motif (iM) structures in the SHMT1 DNA 5′ untranslated region and a parallel GQin the corresponding mRNA. Additionally, we found that the GQ/iM structuressuppress the mRNA levels and protein expression of a reporter gene.Together, these data suggest that GQ/iM structures are necessary for SHMT1 regulation, which could serve as a target for therapeuticintervention for multiple sclerosis patients.

SARS-CoV-2 RNA contains guanine-rich sequences that form secondary structures known as G quadruplexes (G4s). The SARS-CoV-2 nonstructural protein (NSP13) resolves G4s due to its helicase and ATPase activity, a process essential for viral replication. Here, we tested the effects of synthetic G4s on SARS-CoV-2 replication. In agreement, a synthetic G4 DNA 20 mer, consisting exclusively of guanines linked by a phosphorothioate backbone (designated GQ20-PTO), inhibited the replication of various SARS-CoV-2 variants in human lung cell cultures. Mechanistically, GQ20-PTO bound to NSP13 and inhibited its helicase and ATPase activity. Independent of its antiviral effects, GQ20-PTO additionally suppressed IFNβ and IL-6 (but not TNFα) signaling and the formation of reactive oxygen species, processes known to contribute to hyperinflammation in severe COVID-19. Hence, G4 quadruplexes like GQ20-PTO represent a novel class of DNA-based compounds for COVID-19 treatment with the potential to interfere with both SARS-CoV-2 replication and the uncontrolled inflammation associated with life-threatening COVID-19.

Wash-free super-resolution sensing of telomeric G-quadruplex/t-loop states in living cells using a cyclometalated Ir(III) probe.

G-quadruplexes (GQs) are important therapeutic targets in cancer due to their diverse biochemical roles. Molecular dynamics (MD) simulation is an essential tool for the property research of GQs. However, conventional MD simulations often suffer from overestimated ion repulsion, unstable loop regions and bifurcated hydrogen bonds in G-tetrads. To address the above issues, we developed a polarized nucleic acid-specific charge (PNC) model based on fragment quantum chemistry. MD simulations compared standard non-polarized force fields (bsc0, bsc1, OL15, OL21) against PNC-based polarized versions in GQ and hairpin systems. In the 1JRN GQ system, non-polarized force fields showed a higher RMSD/RMSF, bifurcated hydrogen bonds and losses of coordinating ions — issues observed across five K[Formula: see text] and four Na[Formula: see text] parameter sets. In contrast, MD simulations under PNC maintained stable loop structures, preserved central K[Formula: see text]/Na[Formula: see text] ions within the channel, and reproduced crystal-like hydrogen bonding. Furthermore, in the MD simulations of the 5M1W system, where a single-stranded DNA folds into a hairpin structure, which is a key intermediate likely involved in GQs folding, compared within non-polarized force fields, the free energy landscapes generated using PNC-based force fields show improvements to varying degrees. Overall, for GQ or hairpin systems, the inclusion of polarization effects should be considered indispensable in MD simulations, as it is critical for maintaining structural stability and yielding a more reliable conformational landscape for evaluating thermodynamic properties to a certain extent.

G-quadruplexes (G4s) are specialized nucleic acid structures extensively formed throughout the genome, with particular enrichment in regulatory regions such as telomeres, promoters, and transcriptional enhancers. These four-stranded assemblies are involved in multiple chromosomal processes, including DNA replication, transcription, maintenance of genomic stability, and epigenetic regulation, and are closely associated with cancer biology. Due to their unusual thermodynamic stability, G4s serve as physical barriers to DNA/RNA unwinding, thereby impeding replication, transcription, and translation and compromising genome integrity. To mitigate this threat, cells have evolved dedicated helicases that can actively resolve G4 structures. In this review, we summarize recent structural advances-primarily derived from protein crystallography-regarding the mechanisms by which helicases unwind G4 quadruplexes. The insights presented herein establish a framework for elucidating the molecular basis of G4 unfolding and for the rational design of small-molecule G4 ligands and therapeutic agents. Additionally, we explore the applications of G4 helicases in nanopore sequencing, which aim to enhance sequencing accuracy, throughput, and continuity.

The role of alternative nucleic acid structures (ANS) in biology is an area of increasing interest. These non-canonical structures include the Z-DNA and Z-RNA duplexes (ZNA), the three-stranded triplex, the four-stranded G-quadruplex (GQ), and i-motifs. Previously, the biological relevance of ANS was dismissed. Their formation in vitro often required non-physiological conditions, and there was no genetic evidence for their function. Further, structural studies confirmed that sequence-specific transcription factors (TFs) bound B-DNA. In contrast, ANS are formed dynamically by a subset of repeat sequences, called flipons. The flip requires energy, but not strand cleavage. Flipons are enriched in promoters where they modulate transcription. Here, computational modeling based on AlphaFold V3 (AF3), under optimized conditions, reveals that known B-DNA-binding TFs also dock to ANS, such as ZNA and GQ. The binding of HLH and bZIP homodimers to Z-DNA is promoted by methylarginine modifications. Heterodimers only bind preformed Z-DNA. The interactions of TFs with ANS likely enhance genome scanning to identify cognate B-DNA-binding sites in active genes. Docking of TF homodimers to Z-DNA potentially facilitates the assembly of heterodimers that dissociate and are stabilized by binding to a cognate B-DNA motif. The process enables rapid discovery of the optimal heterodimer combinations required to regulate a nearby promoter.

Expansion of d(GGGGC)n repeat in the C9ORF72 gene is causal for Amyotrophic Lateral Sclerosis (ALS) and Frontal Temporal Dementia (FTD). Proposed mechanisms include Repeat-Associated Non-AUG translation or the formation of G-quadruplexes (GQ) that disrupt translation, induce protein aggregation, sequester RNA processing factors, or alter RNA editing. Here, I show, using AlphaFold V3 (AF3) modeling, that the TAR DNA-binding protein (TDP-43) docks to a complex of GQ and hemin. TDP-43 methionines lie over hemin and likely squelch the generation of superoxide by the porphyrin-bound Fe. These TDP-43 methionines are frequently altered in ALS patients. Tau protein, a variant of which causes ALS, also binds to GQ and heme and positions methionines to detoxify peroxides. Full-length Tau, which is often considered prone to aggregation and a prion-like disease agent, can bind to an array composed of multiple GQs as a fully folded protein. In ALS and FTD, loss-of-function variants cause an uncompensated surplus of superoxide, which sparks neuronal cell death. In Alzheimer's Disease (AD) patients, GQ and heme complexes bound by β-amyloid 42 (Aβ4) are also likely to generate superoxides. Collectively, these neuropathologies have proven difficult to treat. The current synthesis provides a framework for designing future therapeutics.

The melanocyte lineage-determining Microphthalmia-associated transcription factor (MITF) drives proliferation and survival of melanocytic melanoma cells through regulation of both coding genes and long non-coding RNAs (LncRNAs). Here we characterize LINC00520 (hereafter called LncRNA ENhancer of Translation, LENT) regulated by MITF and strongly expressed in melanocytic melanoma cells. LENT is essential for the proliferation and survival of cultured melanocytic melanoma cells and xenograft tumors. LENT interacts with the G4 quadruplex resolvase DHX36, and both associate with the ribosome in the 80S and light polysome fractions. LENT modulates DHX36 association with a collection of mRNAs regulating their engagement with polysomes and fine-tuning their subsequent translation. These mRNAs encode proteins involved in endoplasmic reticulum (ER) and mitochondrial homeostasis as well as autophagy. Consequently, LENT silencing leads to extensive autophagy and mitophagy, compromised oxidative metabolic capacity, accompanied by an accumulation and mis-localization of mitochondrial proteins leading to proteotoxic stress and apoptosis. The LENT-DHX36 axis therefore fine-tunes translation of proteins involved in ER and mitochondrial homeostasis, suppressing autophagy and promoting survival and proliferation of melanoma cells.

Human chromosomes terminate in 50-300 nucleotide (nt) long single-stranded telomeric overhangs composed of repeating d(TTAGGG) sequences, which can fold into tandem G-quadruplex (GQ) structures that protect chromosome ends. Stabilization of GQs by small-molecule ligands inhibits telomerase activity, motivating extensive efforts to develop GQ-targeting anti-cancer therapeutics. However, how interactions between successive GQs and bound ligands influence small-molecule accessibility remains poorly understood. Here, we employ single-molecule fluorescence microscopy and stepwise photobleaching analysis to quantify the binding stoichiometry of a fluorescently-labeled oxazole telomestatin derivative (L1Cy5-7OTD) to telomeric overhangs capable of forming 1-6 GQs (30-162 nt long), spanning much of the physiologically relevant range. We find that longer overhangs accommodate more ligands on average but exhibit consistently lower binding stoichiometry than the theoretical maximum, saturating at six molecules even in constructs with twelve binding sites. This trend was further supported by experiments showing increased L1Cy5-7OTD binding when the inter-GQ spacer was extended from 3-nt to 9-nt. This effect was independently confirmed by ensemble fluorescence enhancement experiments utilizing N-methyl mesoporphyrin IX (NMM) as a ligand. Complementary modeling with a one-dimensional lattice model describing equilibrium ligand binding to partially ordered telomeric overhangs revealed positive cooperativity between folding of successive GQs, negative cooperativity between ligands bound opposing (top and bottom) faces of successive GQs, and reduced binding affinity to GQs located at the junction of double stranded and single stranded telomeres. Together, these findings demonstrate how telomeric overhang architecture governs ligand accessibility and provide mechanistic insight to guide the rational design of GQ-targeting anticancer agents.

DNA G-quadruplexes: Structural and functional insights.

Telomeres are transcribed into the long non-coding RNA TERRA, which is essential for telomere protection and maintenance. In cancer cells, telomere lengthening occurs via telomerase reactivation or the alternative lengthening of telomeres (ALT). TERRA is highly overexpressed in ALT cells and directly influences this process. However, due to the lack of efficient tools to investigate TERRA biology, its role in cancer progression and its potential as a therapeutic target remains unclear. Both telomeric DNA and TERRA form noncanonical structures called G-quadruplexes (GQs) on their G-rich strands, which can be the targets of GQ ligands. Using a ligand-based virtual screening of FDA-approved drugs, we identified novel TERRA GQ ligands capable of stabilizing TERRA binding to chromatin. This interaction increased telomeric DNA:RNA hybrids, induced telomeric defects, and elevated ALT-associated PML bodies formation in both telomerase- and ALT-positive cancer cells in an RNAseH1 dependent manner. These ligands also partly increased C-circle levels. In vitro, these ligands recognized and stabilized DNA:RNA GQ hybrids, revealing a novel mechanism of TERRA binding to telomeric DNA, which may contribute to replication stress, sister-telomere disjunction impairment, and enhanced ALT activity, offering new insights into TERRA’s multifaceted role in telomere dynamics and its implications for cancer biology.

Multiple sclerosis (MS) is a fatal neurodegenerative disease that progresses by eroding the myelin sheath and exposing the neuron, leading to neuronal degradation and death. While MS remains without an effective treatment or cure, studies have identified genes that are dysregulated in MS patients and predicted to be involved with disease progression. These genes are primarily involved in controlling DNA methylation: a process required for regulating gene expression, which is critical for cellular health. Having identified potential genetic risk factors, research is focused on how to manipulate the expression of these genes by offsetting DNA methylation errors in patients through the targeting of DNA and RNA secondary structure formation. Serine hydroxy methyltransferase 1 (SHMT1), a key player in DNA methylation, was determined to be upregulated in MS patients. Here, we identified and characterized a hybrid 3+1 G-quadruplex (GQ) and i-motif (iM) structures in the SHMT1 DNA 5' untranslated region and a parallel GQ in the corresponding mRNA. Additionally, we found that the GQ/iM structures suppress the mRNA levels and protein expression of a reporter gene. Together, these data suggest that GQ/iM structures are necessary for SHMT1 regulation, which could serve as a target for therapeutic intervention for MS patients.

G4-quadruplex (G4) is a noncanonical nucleic acid secondary structure that forms in single-stranded DNA or RNA. G4 structures are involved in various cellular processes, including transcription, translation, and DNA replication. It has been shown that G4 formation in gene promoters can inhibit promoter activation. Recently, a positive role of G4 in promoters has also been discovered, but evidence in viral promoters remains limited. In this study, we demonstrate that a G4 structure forms in the human cytomegalovirus UL146 promoter and acts as a binding site for the viral transactivator IE2, which plays a key role in viral gene expression. Using recombinant viruses, we confirm that IE2 binding to G4 is necessary for efficient UL146 transcription during virus infection. This study provides evidence that G4 structures in promoters can positively regulate viral gene expression and uncovers a novel mechanism by which IE2 activates gene expression.

G-quadruplex (GQ)/hemin DNAzymes represent a class of high-activity artificial peroxidases that have been widely utilized in various redox reactions. However, the limited thermal stability of GQ/hemin systems restricts their application in high-temperature biocatalysis. Herein, we developed a thermally activated peroxidase-mimetic DNAzyme constructed by G-quadruplex nanowire (GQwire) and hemin. A remarkable temperature-enhanced activity is shown on a model reaction of 2,2’-azino-bis(3-ethylben zothiozoline-6-sulfonic acid) (ABTS) oxidation by H2O2, achieving a 4.7-fold increase in catalytic activity and a 3.6-fold increase in cumulative product yield at 70 °C compared to 25°C. Structural analysis reveals that this unique thermal activation arises from the two-step thermal transition of GQwire architecture. GQwire disassembled from nanowire to oligomeric GQ rods that provides additional terminal binding sites for hemin and consequently enhancing catalytic activity at elevated temperatures. This thermal-activated DNAzyme provides an effective strategy for fabricating biocatalysts that expands their potentials in industrial manufacture.

1 While it is well established that the Zα domains of ADAR1 and ZBP1 proteins bind Z-form-prone nucleic acids (Z-NAs), it has also been shown that the Zα domain of ADAR1 binds DNA G-Quadruplexes (GQ). However, no binding partner of the structurally homologous Zβ domain of ADAR1 has been identified to date. Based on AlphaFold and molecular dynamics simulations, it has recently been suggested that the Zβ domain of ADAR1 targets its substrate by recognizing GQs. Here, we provide the first experimental evidence for Zβ domain binding to select G-quadruplex RNA and DNA in vitro , with structural specificity and low micromolar affinity. We also demonstrate that the Zα domains of ZBP1 bind to both DNA and RNA GQs with similar affinity. These findings extend the range of potential functional roles for these proteins and open new hypotheses for testing in cells.