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Non-canonical G-quadruplex (G4) DNA structures play key roles in cellular regulation and are promising targets for cancer therapy. This study reports the design, synthesis, and biophysical evaluation of 15 novel pyridine bis-quinazoline derivatives for their ability to selectively bind and stabilize G4 DNA structures. The pyridine-bis-quinazoline central fragment was synthesized with various amine side chains via a 4–5 step sequence in high yields. Comprehensive analyses using different fluorescence resonance energy transfer (FRET) assays, fluorescence intercalator displacement (FID), circular dichroism (CD), and nuclear magnetic resonance (NMR) assays revealed strong G4 stabilization and selectivity over double-stranded DNA. The presence and composition of the aliphatic amine side chain proved critical and propylamine linkers exhibited superior performance, achieving ΔTm values exceeding 20 °C and dissociation constants in the nanomolar range. Structural preferences were observed for parallel and hybrid G4 topologies, and the ligands induced minimal conformational changes in G4 DNA upon binding. Finally, cell viability assays on HCT-8 and HepG2 cancer cell lines revealed that most ligands effectively entered the cells and decreased cancer cell viability in a dose-dependent manner. These findings underline the potential of pyridine bis-quinazoline derivatives as selective G4-stabilizing agents, paving the way for further exploration in anticancer drug development.

I-motif (iM) DNA structures are dynamic cytosine-rich secondary structures that are increasingly recognized for their roles in transcriptional regulation, genomic stability, and for their potential as therapeutic targets in cancer. Despite their significance, the development of selective small-molecule probes for iM DNA remains a challenge. In this study, a series of iminocoumarin-benzothaizole derivatives were designed, synthesized, and subjected to extensive screening to explore their interactions with various iM DNA constructs, including H-Telo, HRAS1, HRAS2, VEGF, and BCL2, as well as duplex DNA. This revealed compounds that display specific and strong interactions with H-Telo, HRAS1, or HRAS2 iM DNA structures depending on their substitution pattern. Detailed spectroscopic investigations revealed the details of how these compounds interact with the iM DNAs, resulting in hypochromic and bathochromic effects, fluorescence enhancements, and increased lifetimes. Furthermore, compounds with unique light-up properties in the presence of HRAS1, VEGF, and BCL2 iM DNA was identified, which has potential as a light-up probes for iM DNA studies in cellular environments. Additionally, circular dichroism (CD) and thermal melting studies confirmed that the compounds stabilized iM DNA without altering its topology, while FT-IR spectroscopy identified structural modifications in iM DNA upon binding. The synthesis of structurally diverse substituents, coupled with extensive spectroscopic, fluorescence, and thermodynamic screening, provided critical insights into structure–activity relationships. Overall, these findings highlight the potential of this compound class to be further developed as selective iM DNA-binding agents and light-up probes, paving the way for innovative diagnostic tools and therapeutic approaches targeting iM DNA in cancer and other diseases.

G-quadruplex (G4) DNA structures are noncanonical secondary structures found in key regulatory regions of the genome, including oncogenic promoters and telomeres. Small molecules, known as G4 ligands, capable of stabilizing G4s hold promise as chemical probes and therapeutic agents. Nevertheless, achieving precise specificity for individual G4 structures within the human genome remains a significant challenge. To address this, we expand upon G4-ligand-conjugated oligonucleotides (GL-Os), a modular platform combining the stabilizing properties of G4-ligands with the sequence specificity of guide DNA oligonucleotides. Central to this strategy is the linker that bridges the G4 ligand and the guide oligonucleotide. In this study, we develop multiple conjugation strategies for the GL-Os that enabled a systematic investigation of the linker in both chemical composition and length, enabling a thorough assessment of their impact on targeting oncogenic G4 DNA. Biophysical, biochemical, and computational evaluations revealed GL-Os with optimized linkers that exhibited enhanced binding to target G4s, even under thermal or structural stress. Notably, longer linkers broadened the range of targetable sequences without introducing steric hindrance, thereby enhancing the platform’s applicability across diverse genomic contexts. These findings establish GL-Os as a robust and versatile tool for the selective targeting of individual G4s. By facilitating precise investigations of G4 biology, this work provides a foundation for advancing G4-targeted therapeutic strategies and exploring their role in disease contexts.

The continued advancement of oligonucleotide-based strategies in research and therapeutics relies on expanding the repertoire of chemical modifications to overcome persistent challenges, such as improving cellular uptake and delivery. Addressing these obstacles requires innovative bioconjugation approaches that integrate seamlessly with oligonucleotide modalities. Here, we report the development of a novel phosphotriester trifunctional probe based on the H-phosphonate derivative ammonium (9H-fluoren-9-yl)methyl, introducing significant advancements in synthetic phosphate chemistry. This platform supports robust and versatile chemical transformations, enabling the incorporation of diverse functionalities, such as biotin, fluorescent markers, G4-stabilizing ligands, and azido groups, into oligonucleotide backbones. The resulting multifunctional probes are compatible with different conjugation strategies and phosphorothioate modifications, allowing late-stage functionalization in solution without requiring solid-phase synthesis. We demonstrate the utility of this approach through the synthesis of G4-ligand-conjugated oligonucleotides (GL-Os) designed to target individual G4 structures. However, the strategy's adaptability ensures compatibility with a wide range of oligonucleotide-based applications that benefit from the addition of functional probes. This flexibility broadens accessibility and applicability, facilitating the development of oligonucleotide tools for advanced chemical biology studies, including fluorescence-based imaging and pull-down experiments.

G-Quadruplex (G4) DNA structures are important regulatory elements in central biological processes. Small molecules that selectively bind and stabilize G4 structures have therapeutic potential, and there are currently >1000 known G4 ligands. Despite this, only two G4 ligands ever made it to clinical trials. In this work, we synthesized several heterocyclic G4 ligands and studied their interactions with G4s (e.g., G4s from the c-MYC, c-KIT, and BCL-2 promoters) using biochemical assays. We further studied the effect of selected compounds on cell viability, the effect on the number of G4s in cells, and their pharmacokinetic properties. This identified potent G4 ligands with suitable properties and further revealed that the dispersion component in arene-arene interactions in combination with electron-deficient electrostatics is central for the ligand to bind with the G4 efficiently. The presented design strategy can be applied in the further development of G4-ligands with suitable properties to explore G4s as therapeutic targets.

G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions, such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of the G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a long-standing problem in the field is how to achieve specificity among these different G4 structures. Here, we developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4-Ligand-conjugated oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amount of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.

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.

G-quadruplex (G4) DNA structures are involved in central biological processes such as DNA replication and transcription. These DNA structures are enriched in promotor regions of oncogenes and are thus promising as novel gene silencing therapeutic targets that can be used to regulate expression of oncoproteins and in particular those that has proven hard to drug with conventional strategies. G4 DNA structures in general have a well-defined and hydrophobic binding area that also is very flat and featureless and there are ample examples of G4 ligands but their further progression towards drug development is limited. In this study, we use synthetic organic chemistry to equip a drug-like and low molecular weight central fragment with different side chains and evaluate how this affect the compound's selectivity and ability to bind and stabilize G4 DNA. Furthermore, we study the binding interactions of the compounds and connect the experimental observations with the compound's structural conformations and electrostatic potentials to understand the basis for the observed improvements. Finally, we evaluate the top candidates' ability to selectively reduce cancer cell growth in a 3D co-culture model of pancreatic cancer which show that this is a powerful approach to generate highly active and selective low molecular weight G4 ligands with a promising therapeutic window.

G-quadruplex (G4) DNA structures are implicated in central biological processes and are considered promising therapeutic targets because of their links to human diseases such as cancer. However, functional details of how, when, and why G4 DNA structures form in vivo are largely missing leaving a knowledge gap that requires tailored chemical biology studies in relevant live-cell model systems. Towards this end, we developed a synthetic platform to generate complementary chemical probes centered around one of the most effective and selective G4 stabilizing compounds, Phen-DC3. We used a structure-based design and substantial synthetic devlopments to equip Phen-DC3 with an amine in a position that does not interfere with G4 interactions. We next used this reactive handle to conjugate a BODIPY fluorophore to Phen-DC3. This generated a fluorescent derivative with retained G4 selectivity, G4 stabilization, and cellular effect that revealed the localization and function of Phen-DC3 in human cells. To increase cellular uptake, a second chemical probe with a conjugated cell-penetrating peptide was prepared using the same amine-substituted Phen-DC3 derivative. The cell-penetrating peptide conjugation, while retaining G4 selectivity and stabilization, increased nuclear localization and cellular effects, showcasing the potential of this method to modulate and direct cellular uptake e.g. as delivery vehicles. The applied approach to generate multiple tailored biochemical tools based on the same core structure can thus be used to advance the studies of G4 biology to uncover molecular details and therapeutic approaches. This journal is