Structural Motifs at the Telomeres and Their Role in Regulatory Pathways.

Telomeres are specialized structures, found at the ends of linear chromosomes in eukaryotic cells, that play a crucial role in maintaining the stability and integrity of genomes. They are composed of repetitive DNA sequences, ssDNA overhangs, and several associated proteins. The length of telomeres is linked to cellular aging in humans, and deficiencies in their maintenance are associated with various diseases. Key structural motifs at the telomeres serve to protect vulnerable chromosomal ends. Telomeric DNA also has the ability to form diverse complex DNA higher-order structures, including T-loops, D-loops, R-loops, G-loops, G-quadruplexes, and i-motifs, in the complementary C-rich strand. While many essential proteins at telomeres have been identified, the intricacies of their interactions and structural details are still not fully understood. This Perspective highlights recent advancements in comprehending the structures associated with human telomeres. It emphasizes the significance of telomeres, explores various telomeric structural motifs, and delves into the structural biology surrounding telomeres and telomerase. Furthermore, telomeric loops, their topologies, and the associated proteins that contribute to the safeguarding of telomeres are discussed.

More than forty years of nucleic acid structural science.

As scientists who have worked with Stephen Neidle over many years and stages of his career, we present our perspective of his contributions to nucleic acid structural science. We trace some of the highlights of his research on nucleic acid drug interactions and the unique insights about the importance of hydration.

BMPQ-1 binds selectively to (3+1) hybrid topologies in human telomeric G-quadruplex multimers.

A single G-quadruplex forming sequence from the human telomere can adopt six distinct topologies that are inter-convertible under physiological conditions. This presents challenges to design ligands that show selectivity and specificity towards a particular conformation. Additional complexity is introduced in differentiating multimeric G-quadruplexes over monomeric species, which would be able to form in the single-stranded 3' ends of telomeres. A few ligands have been reported that bind to dimeric quadruplexes, but their preclinical pharmacological evaluation is limited. Using multidisciplinary approaches, we identified a novel quinoline core ligand, BMPQ-1, which bound to human telomeric G-quadruplex multimers over monomeric G-quadruplexes with high selectivity, and induced the formation of G-quadruplex DNA along with the related DNA damage response at the telomere. BMPQ-1 reduced tumor cell proliferation with an IC50 of ∼1.0 µM and decreased tumor growth rate in mouse by half. Biophysical analysis using smFRET identified a mixture of multiple conformations coexisting for dimeric G-quadruplexes in solution. Here, we showed that the titration of BMPQ-1 shifted the conformational ensemble of multimeric G-quadruplexes towards (3+1) hybrid-2 topology, which became more pronounced as further G-quadruplex units are added.

Towards the Inhibition of Protein-Protein Interactions (PPIs) in STAT3: Insights into a New Class of Benzothiadiazole Derivatives.

Signal transducer and activator of transcription 3 (STAT3) is a validated anticancer target due to the relationship between its constitutive activation and malignant tumors. Through a virtual screening approach on the STAT3-SH2 domain, 5,6-dimethyl-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide (1) was identified as a potential STAT3 inhibitor. Some benzothiadiazole derivatives were synthesized by employing a versatile methodology, and they were tested by an AlphaScreen-based assay. Among them, benzosulfamide 1 showed a significant activity with an IC50 = 15.8 ± 0.6 µM as a direct STAT3 inhibitor. Notably, we discovered that compound 1 was also able to interact with cysteine residues located around the SH2 domain. By applying mass spectrometry, liquid chromatography, NMR, and UV spectroscopy, an in-depth investigation was carried out, shedding light on its intriguing and unexpected mechanism of interaction.

Mechanistic In Situ and Ex Situ Studies of Phase Transformations in Molecular Co-Crystals.

Co-crystallisation is widely explored as a route to improve the physical properties of pharmaceutical active ingredients, but little is known about the fundamental mechanisms of the process. Herein, we apply a hyphenated differential scanning calorimetry-X-ray diffraction technique to mimic the commercial hot melt extrusion process, and explore the heat-induced synthesis of a series of new co-crystals containing isonicotinamide. These comprise a 1:1 co-crystal with 4-hydroxybenzoic acid, 2:1 and 1:2 systems with 4-hydroxyphenylacetic acid and a 1:1 crystal with 3,4-dihydroxyphenylactic acid. The formation of co-crystals during heating is complex mechanistically. In addition to co-crystallisation, conversions between polymorphs of the co-former starting materials and co-crystal products are also observed. A subsequent study exploring the use of inkjet printing and milling to generate co-crystals revealed that the synthetic approach has a major effect on the co-crystal species and polymorphs produced.

Native de novo structural determinations of non-canonical nucleic acid motifs by X-ray crystallography at long wavelengths.

Obtaining phase information remains a formidable challenge for nucleic acid structure determination. The introduction of an X-ray synchrotron beamline designed to be tunable to long wavelengths at Diamond Light Source has opened the possibility to native de novo structure determinations by the use of intrinsic scattering elements. This provides opportunities to overcome the limitations of introducing modifying nucleotides, often required to derive phasing information. In this paper, we build on established methods to generate new tools for nucleic acid structure determinations. We report on the use of (i) native intrinsic potassium single-wavelength anomalous dispersion methods (K-SAD), (ii) use of anomalous scattering elements integral to the crystallization buffer (extrinsic cobalt and intrinsic potassium ions), (iii) extrinsic bromine and intrinsic phosphorus SAD to solve complex nucleic acid structures. Using the reported methods we solved the structures of (i) Pseudorabies virus (PRV) RNA G-quadruplex and ligand complex, (ii) PRV DNA G-quadruplex, and (iii) an i-motif of human telomeric sequence. Our results highlight the utility of using intrinsic scattering as a pathway to solve and determine non-canonical nucleic acid motifs and reveal the variability of topology, influence of ligand binding, and glycosidic angle rearrangements seen between RNA and DNA G-quadruplexes of the same sequence.

Near-infrared dual bioluminescence imaging in mouse models of cancer using infraluciferin.

Bioluminescence imaging (BLI) is ubiquitous in scientific research for the sensitive tracking of biological processes in small animal models. However, due to the attenuation of visible light by tissue, and the limited set of near-infrared bioluminescent enzymes, BLI is largely restricted to monitoring single processes in vivo. Here we show, that by combining stabilised colour mutants of firefly luciferase (FLuc) with the luciferin (LH2) analogue infraluciferin (iLH2), near-infrared dual BLI can be achieved in vivo. The X-ray crystal structure of FLuc with a high-energy intermediate analogue, 5'-O-[N-(dehydroinfraluciferyl)sulfamoyl] adenosine (iDLSA) provides insight into the FLuc-iLH2 reaction leading to near-infrared light emission. The spectral characterisation and unmixing validation studies reported here established that iLH2 is superior to LH2 for the spectral unmixing of bioluminescent signals in vivo; which led to this novel near-infrared dual BLI system being applied to monitor both tumour burden and CAR T cell therapy within a systemically induced mouse tumour model.

X-Ray Crystallographic Studies of G-Quadruplex Structures.

The application of X-ray crystallographic methods toward a structural understanding of G-quadruplex (G4) motifs at atomic level resolution can provide researchers with exciting opportunities to explore new structural arrangements of putative G4 forming sequences and investigate their recognition by small molecule compounds. The crowded and ordered crystalline environment requires the self-assembly of stable G4 motifs, allowing for an understanding of their inter- and intramolecular interactions in a packed environment, revealing thermodynamically stable topologies. Additionally, crystallographic data derived from these experiments in the form of electron density provides valuable opportunities to visualize various solvent molecules associated with G4s along with the geometries of the metal ions associated within the central channel-elements critical to the understanding G4 stability and topology. Now, with the advent of affordable, commercially sourced and purified synthetic DNA and RNA molecules suitable for immediate crystallization trials, and combined with the availability of specialized and validated crystallization screens, researchers can now undertake in-house crystallization trials without the need for local expertise. When this is combined with access to modern synchrotron platforms that offer complete automation of the data collection process-from the receipt of crystals to delivery of merged and scaled data for the visualization of electron density-the application of X-ray crystallographic techniques is made open to nonspecialist researchers. In this chapter we aim to provide a simple how-to guide to enable the reader to undertake crystallographic experiments involving G4s, encompassing the design of oligonucleotide sequences, fundamentals of the crystallization process and modern strategies used in setting up successful crystallization trials. We will also describe data collection strategies, phasing, electron density visualization, and model building. We will draw on our own experiences in the laboratory and hopefully build an appreciation of the utility of the X-ray crystallographic approaches to investigating G4s.

Holliday Junctions Formed from Human Telomeric DNA.

Cells have evolved inherent mechanisms, like homologous recombination (HR), to repair damaged DNA. However, repairs at telomeres can lead to genomic instability, often associated with cancer. While most rapidly dividing cells employ telomerase, the others maintain telomere length through HR-dependent alternative lengthening of telomeres (ALT) pathways. Here we describe the crystal structures of Holliday junction intermediates of the HR-dependent ALT mechanism. Using an extended human telomeric repeat, we also report the crystal structure of two Holliday junctions in close proximity, which associate together through strand exchange to form a hemicatenated double Holliday junction. Our combined structural results demonstrate that ACC nucleotides in the C-rich lagging strand (5'-CTAACCCTAA-3') at the telomere repeat sequence constitute a conserved structural feature that constrains crossover geometry and is a preferred site for Holliday junction formation in telomeres.

A high-throughput fluorescence polarization assay for discovering inhibitors targeting the DNA-binding domain of signal transducer and activator of transcription 3 (STAT3).

Anti-cancer drug discovery efforts to directly inhibit the signal transducer and activator of transcription 3 (STAT3) have been active for over a decade following the discovery that 70% of cancers exhibit elevated STAT3 activity. The majority of research has focused on attenuating STAT3 activity through preventing homo-dimerization by targeting the SH2 or transcriptional activation domains. Such dimerization inhibitors have not yet reached the market. However, an alternative strategy focussed on preventing STAT3 DNA-binding through targeting the DNA-binding domain (DBD) offers new drug design opportunities. Currently, only EMSA and ELISA-based methods have been implemented with suitable reliability to characterize STAT3 DBD inhibitors. Herein, we present a new orthogonal, fluorescence polarization (FP) assay suitable for high-throughput screening of molecules. This assay, using a STAT3127-688 construct, was developed and optimized to screen molecules that attenuate the STAT3:DNA association with good reliability (Z' value > 0.6) and a significant contrast (signal-to-noise ratio > 15.0) at equilibrium. The assay system was stable over a 48 hour period. Significantly, the assay is homogeneous and simple to implement for high-throughput screening compared to EMSA and ELISA. Overall, this FP assay offers a new way to identify and characterize novel molecules that inhibit STAT3:DNA association.

A naphthalene diimide G-quadruplex ligand inhibits cell growth and down-regulates BCL-2 expression in an imatinib-resistant gastrointestinal cancer cell line.

Gastro-intestinal tumours (GISTs) are driven by aberrant expression of the c-KIT oncoprotein. They can be effectively treated by the kinase inhibitor imatinib, which locks the c-KIT kinase domain into an inactive conformation. However resistance to imatinib, driven by active-site mutations, is a recurrent clinical challenge, which has been only partly met by the subsequent development of second and third-generation c-KIT inhibitors. It is reported here that a tetra-substituted naphthalene diimide derivative, which is a micromolar inhibitor of cell growth in a wild-type patient-derived GIST cell line, has a sub-micromolar activity in two distinct patient-derived imatinib-resistant cell lines. The compound has been previously shown to down-regulate expression of the c-KIT protein in a wild-type GIST cell line. It does not affect c-KIT protein expression in a resistant cell line to the same extent, whereas it profoundly down-regulates the expression of the anti-apoptopic protein BCL-2. It is proposed that the mechanism of action involves targeting quadruplex nucleic acid structures, and in particular those in the BCL-2 gene and its RNA transcript. The BCL-2 protein is up-regulated in the GIST-resistant cell line, and is strongly down-regulated after treatment. The compound strongly stabilises a range of G-quadruplexes including a DNA one from the BCL-2 promoter and an RNA quadruplex from its 5'-UTR region. A reporter assay construct incorporating the 5'-UTR quadruplex sequence demonstrates down-regulation of BCL-2 expression.

G-quadruplex-binding small molecules ameliorate C9orf72 FTD/ALS pathology in vitro and in vivo.

Intronic GGGGCC repeat expansions in C9orf72 are the most common known cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), which are characterised by degeneration of cortical and motor neurons, respectively. Repeat expansions have been proposed to cause disease by both the repeat RNA forming foci that sequester RNA-binding proteins and through toxic dipeptide repeat proteins generated by repeat-associated non-ATG translation. GGGGCC repeat RNA folds into a G-quadruplex secondary structure, and we investigated whether targeting this structure is a potential therapeutic strategy. We performed a screen that identified three structurally related small molecules that specifically stabilise GGGGCC repeat G-quadruplex RNA We investigated their effect in C9orf72 patient iPSC-derived motor and cortical neurons and show that they significantly reduce RNA foci burden and the levels of dipeptide repeat proteins. Furthermore, they also reduce dipeptide repeat proteins and improve survival in vivo, in GGGGCC repeat-expressing Drosophila Therefore, small molecules that target GGGGCC repeat G-quadruplexes can ameliorate the two key pathologies associated with C9orf72 FTD/ALS These data provide proof of principle that targeting GGGGCC repeat G-quadruplexes has therapeutic potential.

Selectivity of major isoquinoline alkaloids from Chelidonium majus towards telomeric G-quadruplex: A study using a transition-FRET (t-FRET) assay.

BACKGROUND: Natural bioproducts are invaluable resources in drug discovery. Isoquinoline alkaloids of Chelidonium majus constitute a structurally diverse family of natural products that are of great interest, one of them being their selectivity for human telomeric G-quadruplex structure and telomerase inhibition. METHODS: The study focuses on the mechanism of telomerase inhibition by stabilization of telomeric G-quadruplex structures by berberine, chelerythrine, chelidonine, sanguinarine and papaverine. Telomerase activity and mRNA levels of hTERT were estimated using quantitative telomere repeat amplification protocol (q-TRAP) and qPCR, in MCF-7 cells treated with different groups of alkaloids. The selectivity of the main isoquinoline alkaloids of Chelidonium majus towards telomeric G-quadruplex forming sequences were explored using a sensitive modified thermal FRET-melting measurement in the presence of the complementary oligonucleotide CT22. We assessed and monitored G-quadruplex topologies using circular dichroism (CD) methods, and compared spectra to previously well-characterized motifs, either alone or in the presence of the alkaloids. Molecular modeling was performed to rationalize ligand binding to the G-quadruplex structure. RESULTS: The results highlight strong inhibitory effects of chelerythrine, sanguinarine and berberine on telomerase activity, most likely through substrate sequestration. These isoquinoline alkaloids interacted strongly with telomeric sequence G-quadruplex. In comparison, chelidonine and papaverine had no significant interaction with the telomeric quadruplex, while they strongly inhibited telomerase at transcription level of hTERT. Altogether, all of the studied alkaloids showed various levels and mechanisms of telomerase inhibition. CONCLUSIONS: We report on a comparative study of anti-telomerase activity of the isoquinoline alkaloids of Chelidonium majus. Chelerythrine was most effective in inhibiting telomerase activity by substrate sequesteration through G-quadruplex stabilization. GENERAL SIGNIFICANCE: Understanding structural and molecular mechanisms of anti-cancer agents can help in developing new and more potent drugs with fewer side effects. Isoquinolines are the most biologically active agents from Chelidonium majus, which have shown to be telomeric G-quadruplex stabilizers and potent telomerase inhibitors.

Structural Insights into the Quadruplex-Duplex 3' Interface Formed from a Telomeric Repeat: A Potential Molecular Target.

We report here on an X-ray crystallographic and molecular modeling investigation into the complex 3' interface formed between putative parallel stranded G-quadruplexes and a duplex DNA sequence constructed from the human telomeric repeat sequence TTAGGG. Our crystallographic approach provides a detailed snapshot of a telomeric 3' quadruplex-duplex junction: a junction that appears to have the potential to form a unique molecular target for small molecule binding and interference with telomere-related functions. This unique target is particularly relevant as current high-affinity compounds that bind putative G-quadruplex forming sequences only rarely have a high degree of selectivity for a particular quadruplex. Here DNA junctions were assembled using different putative quadruplex-forming scaffolds linked at the 3' end to a telomeric duplex sequence and annealed to a complementary strand. We successfully generated a series of G-quadruplex-duplex containing crystals, both alone and in the presence of ligands. The structures demonstrate the formation of a parallel folded G-quadruplex and a B-form duplex DNA stacked coaxially. Most strikingly, structural data reveals the consistent formation of a TAT triad platform between the two motifs. This triad allows for a continuous stack of bases to link the quadruplex motif with the duplex region. For these crystal structures formed in the absence of ligands, the TAT triad interface occludes ligand binding at the 3' quadruplex-duplex interface, in agreement with in silico docking predictions. However, with the rearrangement of a single nucleotide, a stable pocket can be produced, thus providing an opportunity for the binding of selective molecules at the interface.

G-quadruplexes: Emerging roles in neurodegenerative diseases and the non-coding transcriptome.

G-rich sequences in DNA and RNA have a propensity to fold into stable secondary structures termed G-quadruplexes. G-quadruplex forming sequences are widespread throughout the human genome, within both, protein coding and non-coding genes, and regulatory regions. G-quadruplexes have been implicated in multiple cellular functions including chromatin epigenetic regulation, DNA recombination, transcriptional regulation of gene promoters and enhancers, and translation. Here we will review the evidence for the occurrence of G-quadruplexes both in vitro and in vivo; their role in neurological diseases including G-quadruplex-forming repeat expansions in the C9orf72 gene in frontotemporal dementia and amyotrophic lateral sclerosis and loss of the G-quadruplex binding protein FMRP in the intellectual disability fragile X syndrome. We also review mounting evidence that supports a role for G-quadruplexes in regulating the processing or function of a range of non-coding RNAs. Finally we will highlight current perspectives for therapeutic interventions that target G-quadruplexes.

A regular thymine tetrad and a peculiar supramolecular assembly in the first crystal structure of an all-LNA G-quadruplex.

Locked nucleic acids (LNAs) are formed by bicyclic ribonucleotides where the O2' and C4' atoms are linked through a methylene bridge and the sugar is blocked in a 3'-endo conformation. They represent a promising tool for therapeutic and diagnostic applications and are characterized by higher thermal stability and nuclease resistance with respect to their natural counterparts. However, structural descriptions of LNA-containing quadruplexes are rather limited, since few NMR models have been reported in the literature. Here, the first crystallographically derived model of an all-LNA-substituted quadruplex-forming sequence 5'-TGGGT-3' is presented refined at 1.7 Šresolution. This high-resolution crystallographic analysis reveals a regular parallel G-quadruplex arrangement terminating in a well defined thymine tetrad at the 3'-end. The detailed picture of the hydration pattern reveals LNA-specific features in the solvent distribution. Interestingly, two closely packed quadruplexes are present in the asymmetric unit. They face one another with their 3'-ends giving rise to a compact higher-order structure. This new assembly suggests a possible way in which sequential quadruplexes can be disposed in the crowded cell environment. Furthermore, as the formation of ordered structures by molecular self-assembly is an effective strategy to obtain nanostructures, this study could open the way to the design of a new class of LNA-based building blocks for nanotechnology.

Crystal structure of a promoter sequence in the B-raf gene reveals an intertwined dimer quadruplex.

The sequence d(GGGCGGGGAGGGGGAAGGGA) occurs in the promoter region of the B-raf gene. An X-ray crystallographic study has found that this forms an unprecedented dimeric quadruplex arrangement, with a core of seven consecutive G-quartets and an uninterrupted run of six potassium ions in the central channel of the quadruplex. Analogy with previously reported promoter quadruplexes had initially suggested that in common with these a monomeric quadruplex was to be expected. The structure has a distorted G·C·G·C base quartet at one end and four flipped-out adenosine nucleosides at the other. The only loops in the structure are formed by the cytosine and by the three adenosines within the sequence, with all of the guanosines participating in G-quartet formation. Solution UV and circular dichroism data are in accord with a stable quadruple arrangement being formed. 1D NMR data, together with gel electrophoresis measurements, are consistent with a dimer being the dominant species in potassium solution. A single-chain intramolecular quadruplex has been straightforwardly constructed using molecular modeling, by means of a six-nucleotide sequence joining 3' and 5' ends of each strand in the dimer. A human genomic database search has revealed a number of sequences containing eight or more consecutive short G-tracts, suggesting that such intramolecular quadruplexes could be formed within the human genome.

Observation of unphosphorylated STAT3 core protein binding to target dsDNA by PEMSA and X-ray crystallography.

The STAT3 transcription factor plays a central role in a wide range of cancer types where it is over-expressed. Previously, phosphorylation of this protein was thought to be a prerequisite for direct binding to DNA. However, we have now shown complete binding of a purified unphosphorylated STAT3 (uSTAT3) core directly to M67 DNA, the high affinity STAT3 target DNA sequence, by a protein electrophoretic mobility shift assay (PEMSA). Binding to M67 DNA was inhibited by addition of increasing concentrations of a phosphotyrosyl peptide. X-ray crystallography demonstrates one mode of binding that is similar to that known for the STAT3 core phosphorylated at Y705.

Nanofiber-based delivery of therapeutic peptides to the brain.

The delivery of therapeutic peptides and proteins to the central nervous system is the biggest challenge when developing effective neuropharmaceuticals. The central issue is that the blood-brain barrier is impermeable to most molecules. Here we demonstrate the concept of employing an amphiphilic derivative of a peptide to deliver the peptide into the brain. The key to success is that the amphiphilic peptide should by design self-assemble into nanofibers wherein the active peptide epitope is tightly wrapped around the nanofiber core. The nanofiber form appears to protect the amphiphilic peptide from degradation while in the plasma, and the amphiphilic nature of the peptide promotes its transport across the blood-brain barrier. Therapeutic brain levels of the amphiphilic peptide are achieved with this strategy, compared with the absence of detectable peptide in the brain and the consequent lack of a therapeutic response when the underivatized peptide is administered.