A beginner's handbook to identify and characterize i-motif DNA.

Genomic DNA exhibits an innate ability to manifest diverse sequence-dependent secondary structures, serving crucial functions in gene regulation and cellular equilibrium. While extensive research has confirmed the formation of G-quadruplex structures by guanine-rich sequences in vitro and in cells, recent investigations have turned the quadruplex community's attention to the cytosine (C)-rich complementary strands that can adopt unique tetra-stranded conformation, termed as intercalated motif or i-motif. I-motifs are stabilized by hemi-protonated C:CH+ base pairs under acidic conditions. Initially, the in vivo occurrence of i-motifs was underestimated because their formation is favored at non-physiological pH. However, groundbreaking research utilizing the structure-specific iMab antibody and high-throughput sequencing have recently detected their conserved dispersion throughout the genome, challenging previous assumptions. Given the evolving nature of this research field, it becomes imperative to conduct independent in vitro experiments aimed at identifying potential i-motif formation in C-rich sequences and consolidating the findings to address the properties of i-motifs. This chapter serves as an introductory guide for the swift identification of novel i-motifs, where we present an experimental framework for investigating and characterizing i-motif sequences in vitro. In this chapter, we selected a synthetic oligonucleotide (C7T3) sequence and outlined appropriate methodologies for annealing the i-motif structure into suitable buffers. Then, we validated its formation by CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance) spectroscopy. Finally, we provided a thorough account of the step-by-step procedures to investigate the effect of i-motif formation on the stalling or retardation of DNA replication using high resolution primer extension assays.

A new G-quadruplex-specific photosensitizer inducing genome instability in cancer cells by triggering oxidative DNA damage and impeding replication fork progression.

Photodynamic therapy (PDT) ideally relies on the administration, selective accumulation and photoactivation of a photosensitizer (PS) into diseased tissues. In this context, we report a new heavy-atom-free fluorescent G-quadruplex (G4) DNA-binding PS, named DBI. We reveal by fluorescence microscopy that DBI preferentially localizes in intraluminal vesicles (ILVs), precursors of exosomes, which are key components of cancer cell proliferation. Moreover, purified exosomal DNA was recognized by a G4-specific antibody, thus highlighting the presence of such G4-forming sequences in the vesicles. Despite the absence of fluorescence signal from DBI in nuclei, light-irradiated DBI-treated cells generated reactive oxygen species (ROS), triggering a 3-fold increase of nuclear G4 foci, slowing fork progression and elevated levels of both DNA base damage, 8-oxoguanine, and double-stranded DNA breaks. Consequently, DBI was found to exert significant phototoxic effects (at nanomolar scale) toward cancer cell lines and tumor organoids. Furthermore, in vivo testing reveals that photoactivation of DBI induces not only G4 formation and DNA damage but also apoptosis in zebrafish, specifically in the area where DBI had accumulated. Collectively, this approach shows significant promise for image-guided PDT.

Parallel G-Quadruplex DNA Structures from Nuclear and Mitochondrial Genomes Trigger Emission Enhancement in a Nonfluorescent Nano-aggregated Fluorine-Boron-Based Dye.

Molecular self-assembly is a powerful tool for the development of functional nanostructures with adaptive optical properties. However, in aqueous solution, the hydrophobic effects in the monomeric units often afford supramolecular architectures with typical side-by-side π-stacking arrangement with compromised emissive properties. Here, we report on the role of parallel DNA guanine quadruplexes (G4s) as supramolecular disaggregating-capture systems capable of coordinating a zwitterionic fluorine-boron-based dye and promoting activation of its fluorescence signal. The dye's high binding affinity for parallel G4s compared to nonparallel topologies leads to a selective disassembly of the dye's supramolecular state upon contact with parallel G4s. This results in a strong and selective disaggregation-induced emission that signals the presence of parallel G4s observable by the naked eye and inside cells. The molecular recognition strategy reported here will be useful for a multitude of affinity-based applications with potential in sensing and imaging systems.

Correction: Light-induced in situ chemical activation of a fluorescent probe for monitoring intracellular G-quadruplex structures.

Correction for 'Light-induced in situ chemical activation of a fluorescent probe for monitoring intracellular G-quadruplex structures' by Marco Deiana et al., Nanoscale, 2021, 13, 13795-13808, https://doi.org/10.1039/D1NR02855C.

Probing the folding pathways of four-stranded intercalated cytosine-rich motifs at single base-pair resolution.

Cytosine-rich DNA can fold into four-stranded intercalated structures called i-motifs (iMs) under acidic conditions through the formation of hemi-protonated C:C+ base pairs. However, the folding and stability of iMs rely on many other factors that are not yet fully understood. Here, we combined biochemical and biophysical approaches to determine the factors influencing iM stability under a wide range of experimental conditions. By using high-resolution primer extension assays, circular dichroism, and absorption spectroscopies, we demonstrate that the stabilities of three different biologically relevant iMs are not dependent on molecular crowding agents. Instead, some of the crowding agents affected overall DNA synthesis. We also tested a range of small molecules to determine their effect on iM stabilization at physiological temperature and demonstrated that the G-quadruplex-specific molecule CX-5461 is also a promising candidate for selective iM stabilization. This work provides important insights into the requirements needed for different assays to accurately study iM stabilization, which will serve as important tools for understanding the contribution of iMs in cell regulation and their potential as therapeutic targets.

Site-selected thionated benzothioxanthene chromophores as heavy-atom-free small-molecule photosensitizers for photodynamic therapy.

Photodynamic therapy is a clinically approved anticancer modality that employs a light-activated agent (photosensitizer) to generate cytotoxic reactive oxygen species (ROS). There is therefore a growing interest for developing innovative photosensitizing agents with enhanced phototherapeutic performances. Herein, we report on a rational design synthetic procedure that converts the ultrabright benzothioxanthene imide (BTI) dye into three heavy-atom-free thionated compounds featuring close-to-unit singlet oxygen quantum yields. In contrast to the BTI, these thionated analogs display an almost fully quenched fluorescence emission, in agreement with the formation of highly populated triplet states. Indeed, the sequential thionation on the BTI scaffold induces torsion of its skeleton reducing the singlet-triplet energy gaps and enhancing the spin-orbit coupling. These potential PSs show potent cancer-cell ablation under light irradiation while remaining non-toxic under dark condition owing to a photo-cytotoxic mechanism that we believe simultaneously involves singlet oxygen and superoxide species, which could be both characterized in vitro. Our study demonstrates that this simple site-selected thionated platform is an effective strategy to convert conventional carbonyl-containing fluorophores into phototherapeutic agents for anticancer PDT.

Light-induced in situ chemical activation of a fluorescent probe for monitoring intracellular G-quadruplex structures.

Light-activated functional materials capable of remote control over duplex and G-quadruplex (G4) nucleic acids formation at the cellular level are still very rare. Herein, we report on the photoinduced macrocyclisation of a helicenoid quinoline derivative of binaphthol that selectively provides easy access to an unprecedented class of extended heteroaromatic structures with remarkable photophysical and DNA/RNA binding properties. Thus, while the native bisquinoline precursor shows no DNA binding activity, the new in situ photochemically generated probe features high association constants to DNA and RNA G4s. The latter inhibits DNA synthesis by selectively stabilizing G4 structures associated with oncogenic promoters and telomere repeat units. Finally, the light sensitive compound is capable of in cellulo photoconversion, localizes primarily in the G4-rich sites of cancer cells, competes with a well-known G4 binder and shows a clear nuclear co-localization with the quadruplex specific antibody BG4. This work provides a benchmark for the future design and development of a brand-new generation of light-activated target-selective G4-binders.

The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization.

The identification of G-quadruplex (G4) binding proteins and insights into their mechanism of action are important for understanding the regulatory functions of G4 structures. Here, we performed an unbiased affinity-purification assay coupled with mass spectrometry and identified 30 putative G4 binding proteins from the fission yeast Schizosaccharomyces pombe. Gene ontology analysis of the molecular functions enriched in this pull-down assay included mRNA binding, RNA helicase activity, and translation regulator activity. We focused this study on three of the identified proteins that possessed putative arginine-glycine-glycine (RGG) domains, namely the Stm1 homolog Oga1 and the DEAD box RNA helicases Dbp2 and Ded1. We found that Oga1, Dbp2, and Ded1 bound to both DNA and RNA G4s in vitro. Both Dbp2 and Ded1 bound to G4 structures through the RGG domain located in the C-terminal region of the helicases, and point mutations in this domain weakened the G4 binding properties of the helicases. Dbp2 and Ded1 destabilized less thermostable G4 RNA and DNA structures, and this ability was independent of ATP but dependent on the RGG domain. Our study provides the first evidence that the RGG motifs in DEAD box helicases are necessary for both G4 binding and G4 destabilization.

A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures.

G-quadruplex (G4) DNA structures are widespread in the human genome and are implicated in biologically important processes such as telomere maintenance, gene regulation, and DNA replication. Guanine-rich sequences with potential to form G4 structures are prevalent in the promoter regions of oncogenes, and G4 sites are now considered as attractive targets for anticancer therapies. However, there are very few reports of small "druglike" optical G4 reporters that are easily accessible through one-step synthesis and that are capable of discriminating between different G4 topologies. Here, we present a small water-soluble light-up fluorescent probe that features a minimalistic amidinocoumarin-based molecular scaffold that selectively targets parallel G4 structures over antiparallel and non-G4 structures. We showed that this biocompatible ligand is able to selectively stabilize the G4 template resulting in slower DNA synthesis. By tracking individual DNA molecules, we demonstrated that the G4-stabilizing ligand perturbs DNA replication in cancer cells, resulting in decreased cell viability. Moreover, the fast-cellular entry of the probe enabled detection of nucleolar G4 structures in living cells. Finally, insights gained from the structure-activity relationships of the probe suggest the basis for the recognition of parallel G4s, opening up new avenues for the design of new biocompatible G4-specific small molecules for G4-driven theranostic applications.

Unravelling the cellular emission fingerprint of the benchmark G-quadruplex-interactive compound Phen-DC3.

Phen-DC3 is among the most commonly used G-quadruplex (G4)-stabilizers in vitro and in cells. Here, we show that the G4-interactive binding interactions enable one to tune the optical properties of Phen-DC3 allowing the detection of G4 structures in cancer cells. This work opens up new directions for the use of Phen-DC3 as a selective G4 fluorescent reporter.

Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication.

G-quadruplex (G4) structures are stable non-canonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancer-associated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.

A site-specific self-assembled light-up rotor probe for selective recognition and stabilization of c-MYC G-quadruplex DNA.

Direct and unambiguous evidence of the formation of G-quadruplexes (G4s) in human cells have shown their implication in several key biological events and has emphasized their role as important targets for small-molecule cancer therapeutics. Here, we report on the first example of a self-assembled molecular-rotor G4-binder able to discriminate between an extensive panel of G4 and non-G4 structures and to selectively light-up (up to 64-fold), bind (nanomolar range), and stabilize the c-MYC promoter G4 DNA. In particular, association with the c-MYC G4 triggers the disassembly of its supramolecular state (disaggregation-induced emission, DIE) and induces geometrical restrictions (motion-induced change in emission, MICE) leading to a significant enhancement of its emission yield. Moreover, this optical reporter is able to selectively stabilize the c-MYC G4 and inhibit DNA synthesis. Finally, by using confocal laser-scanning microscopy (CLSM) we show the ability of this compound to localize primarily in the subnuclear G4-rich compartments of cancer cells. This work provides a benchmark for the future design and development of a new generation of smart sequence-selective supramolecular G4-binders that combine outstanding sensing and stability properties, to be utilized in anti-cancer therapy.

The Relation Between Position and Chemical Composition of Bis-Indole Substituents Determines Their Interactions with G-Quadruplex DNA.

G-quadruplex (G4) DNA structures are linked to fundamental biological processes and human diseases, which has triggered the development of compounds that affect these DNA structures. However, more knowledge is needed about how small molecules interact with G4 DNA structures. This study describes the development of a new class of bis-indoles (3,3-diindolyl-methyl derivatives) and detailed studies of how they interact with G4 DNA using orthogonal assays, biophysical techniques, and computational studies. This revealed compounds that strongly bind and stabilize G4 DNA structures, and detailed binding interactions which for example, show that charge variance can play a key role in G4 DNA binding. Furthermore, the structure-activity relationships generated opened the possibilities to replace or introduce new substituents on the core structure, which is of key importance to optimize compound properties or introduce probes to further expand the possibilities of these compounds as tailored research tools to study G4 biology.

Quinazoline Ligands Induce Cancer Cell Death through Selective STAT3 Inhibition and G-Quadruplex Stabilization.

The signal transducer and activator of transcription 3 (STAT3) protein is a master regulator of most key hallmarks and enablers of cancer, including cell proliferation and the response to DNA damage. G-Quadruplex (G4) structures are four-stranded noncanonical DNA structures enriched at telomeres and oncogenes' promoters. In cancer cells, stabilization of G4 DNAs leads to replication stress and DNA damage accumulation and is therefore considered a promising target for oncotherapy. Here, we designed and synthesized novel quinazoline-based compounds that simultaneously and selectively affect these two well-recognized cancer targets, G4 DNA structures and the STAT3 protein. Using a combination of in vitro assays, NMR, and molecular dynamics simulations, we show that these small, uncharged compounds not only bind to the STAT3 protein but also stabilize G4 structures. In human cultured cells, the compounds inhibit phosphorylation-dependent activation of STAT3 without affecting the antiapoptotic factor STAT1 and cause increased formation of G4 structures, as revealed by the use of a G4 DNA-specific antibody. As a result, treated cells show slower DNA replication, DNA damage checkpoint activation, and an increased apoptotic rate. Importantly, cancer cells are more sensitive to these molecules compared to noncancerous cell lines. This is the first report of a promising class of compounds that not only targets the DNA damage cancer response machinery but also simultaneously inhibits the STAT3-induced cancer cell proliferation, demonstrating a novel approach in cancer therapy.

A Light-up Logic Platform for Selective Recognition of Parallel G-Quadruplex Structures via Disaggregation-Induced Emission.

The design of turn-on dyes with optical signals sensitive to the formation of supramolecular structures provides fascinating and underexplored opportunities for G-quadruplex (G4) DNA detection and characterization. Here, we show a new switching mechanism that relies on the recognition-driven disaggregation (on-signal) of an ultrabright coumarin-quinazoline conjugate. The synthesized probe selectively lights-up parallel G4 DNA structures via the disassembly of its supramolecular state, demonstrating outputs that are easily integrable into a label-free molecular logic system. Finally, our molecule preferentially stains the G4-rich nucleoli of cancer cells.

Identification of putative G-quadruplex DNA structures in S. pombe genome by quantitative PCR stop assay.

In order to understand in which biological processes the four-stranded G-quadruplex (G4) DNA structures play a role, it is important to determine which predicted regions can actually adopt a G4 structure. Here, to identify DNA regions in Schizosaccharomyces pombe that fold into G4 structures, we first optimized a quantitative PCR (qPCR) assay using the G4 stabilizer, PhenDC3. We call this method the qPCR stop assay, and used it to screen for G4 structures in genomic DNA. The presence of G4 stabilizers inhibited DNA amplification in 14/15 unexplored genomic regions in S. pombe that encompassed predicted G4 structures, suggesting that at these sites the stabilized G4 structure formed an obstacle for the DNA polymerase. Furthermore, the formation of G4 structures was confirmed by complementary in vitro assays. In vivo, the S. pombe G4 unwinder Pif1 helicase, Pfh1, was associated with tested G4 sites, suggesting that the G4 structures also formed in vivo. Thus, we propose that the confirmed G4 structures in S. pombe form an obstacle for replication in vivo, and that the qPCR stop assay is a method that can be used to identify G4 structures. Finally, we suggest that the qPCR stop assay can also be used for identifying G4 structures in other organisms, as well as being adapted to screen for novel G4 stabilizers.

The Pif1 signature motif of Pfh1 is necessary for both protein displacement and helicase unwinding activities, but is dispensable for strand-annealing activity.

Pfh1, the sole member of the Pif1 helicases in Schizosaccharomyces pombe, is multifunctional and essential for maintenance of both the nuclear and mitochondrial genomes. However, we lack mechanistic insights into the functions of Pfh1 and its different motifs. This paper is specifically concerned with the importance of the Pif1 signature motif (SM), a 23 amino acids motif unique to Pif1 helicases, because a single amino acid substitution in this motif is associated with increased risk of breast cancer in humans and inviability in S. pombe. Here we show that the nuclear isoform of Pfh1 (nPfh1) unwound RNA/DNA hybrids more efficiently than DNA/DNA, suggesting that Pfh1 resolves RNA/DNA structures like R-loops in vivo. In addition, nPfh1 displaced proteins from DNA and possessed strand-annealing activity. The unwinding and protein displacement activities were dependent on the SM because nPfh1 without a large portion of this motif (nPfh1-Δ21) or with the disease/inviability-linked mutation (nPfh1-L430P) lost these properties. Unexpectedly, both nPfh1-L430P and nPfh1-Δ21 still displayed binding to G-quadruplex DNA and demonstrated strand-annealing activity. Misregulated strand annealing and binding of nPfh1-L430P without unwinding are perhaps the reasons that cells expressing this allele are inviable.

Flexible Versus Rigid G-Quadruplex DNA Ligands: Synthesis of Two Series of Bis-indole Derivatives and Comparison of Their Interactions with G-Quadruplex DNA.

Small molecules that target G-quadruplex (G4) DNA structures are not only valuable to study G4 biology but also for their potential as therapeutics. This work centers around how different design features of small molecules can affect the interactions with G4 DNA structures, exemplified by the development of synthetic methods to bis-indole scaffolds. Our synthesized series of bis-indole scaffolds are structurally very similar but differ greatly in the flexibility of their core structures. The flexibility of the molecules proved to be an advantage compared to locking the compounds in the presumed bioactive G4 conformation. The flexible derivatives demonstrated similar or even improved G4 binding and stabilization in several orthogonal assays even though their entropic penalty of binding is higher. In addition, molecular dynamics simulations with the c-MYC G4 structure showed that the flexible compounds adapt better to the surrounding. This was reflected by an increased number of both stacking and polar interactions with both the residues in the G4 DNA structure and the DNA residues just upstream of the G4 structure.

The functions of the multi-tasking Pfh1Pif1 helicase.

Approximately, 1% of the genes in eukaryotic genomes encode for helicases, which make the number of helicases expressed in the cell considerably high. Helicases are motor proteins that participate in many central aspects of the nuclear and mitochondrial genomes, and based on their helicase motif conservation, they are divided into different helicase families. The Pif1 family of helicases is an evolutionarily conserved helicase family that is associated with familial breast cancer in humans. The Schizosaccharomyces pombe Pfh1 helicase belongs to the Pif1 helicase family and is a multi-tasking helicase that is important for replication fork progression through natural fork barriers, for G-quadruplex unwinding, and for Okazaki fragment maturation, and these activities are potentially shared by the human Pif1 helicase. This review discusses the known functions of the Pfh1 helicase, the study of which has led to a better understanding of nucleic acid metabolism in eukaryotes.

Identification of Compounds that Selectively Stabilize Specific G-Quadruplex Structures by Using a Thioflavin†T-Displacement Assay as a Tool.

Small molecules are used in the G-quadruplex (G4) research field in vivo and in vitro, and there are increasing demands for ligands that selectively stabilize different G4 structures. Thioflavin T (ThT) emits an enhanced fluorescence signal when binding to G4 structures. Herein, we show that ThT can be competitively displaced by the binding of small molecules to G4 structures and develop a ThT-displacement high-throughput screening assay to find novel and selective G4-binding compounds. We screened approximately 28 000 compounds by using three different G4 structures and identified eight novel G4 binders. Analysis of the structural conformation and stability of the G4 structures in presence of these compounds demonstrated that the four compounds enhance the thermal stabilization of the structures without affecting their structural conformation. In addition, all four compounds also increased the G4-structure block of DNA synthesis by Taq DNA polymerase. Also, two of these compounds showed selectivity between certain Schizosaccharomyces pombe G4 structures, thus suggesting that these compounds or their analogues can be used as selective tools for G4 DNA studies.