Structure Of DNA Duplex Research Articles

Ultrasensitive Electrochemiluminescence Biosensor Based on DNA-Bio-Bar-Code and Hybridization Chain Reaction Dual Signal Amplification for Exosomes Detection.

Exosomes have received considerable attention as potent reference markers for the diagnosis of various neoplasms due to their close and direct relationship with the proliferation, adhesion, and migration of tumor. The ultrasensitive detection of cancer-derived low-abundance exosomes is imperative, but still a great challenge. Herein, we report an electrochemiluminescence (ECL) biosensor based on the DNA-bio-bar-code and hybridization chain reaction (HCR)-mediated dual signal amplification for the ultrasensitive detection of cancer-derived exosomes. In this system, two types of aptamers were modified on the magnetic nanoprobe (MNPs) and gold nanoparticles (AuNPs) with numerous bio-bar-code DNA, respectively, which formed "sandwich" structures in the presence of specific target exosomes. The "sandwich" structures were separated under magnetic field, and the numerous bio-bar-code DNA were released by dissolving AuNPs. The released bio-bar-code DNA triggered the HCR procedure to produce a good deal of long DNA duplex structure for embedding in hemin, which generated strong ECL signal in the presence of coreactors for ultrasensitive detection of exosomes. Under the optimal conditions, it exhibited a good linearly of exosomes ranging from 10 to 104 exosomes particle μL-1 with limit of detection down to 5.01 exosome particle μL-1. Furthermore, the high ratio of ECL signal and minor change of ECL intensity indicated the good specificity, stability, and repeatability of this ECL biosensor. Given the good performance for exosome analysis, this ultrasensitive ECL biosensor has a promising application in the clinical diagnosis of early cancers.

Structural basis of water-mediated cis Watson-Crick/Hoogsteen base-pair formation in non-CpG methylation.

Non-CpG methylation is associated with several cellular processes, especially neuronal development and cancer, while its effect on DNA structure remains unclear. We have determined the crystal structures of DNA duplexes containing -CGCCG- regions as CCG repeat motifs that comprise a non-CpG site with or without cytosine methylation. Crystal structure analyses have revealed that the mC:G base-pair can simultaneously form two alternative conformations arising from non-CpG methylation, including a unique water-mediated cis Watson-Crick/Hoogsteen, (w)cWH, and Watson-Crick (WC) geometries, with partial occupancies of 0.1 and 0.9, respectively. NMR studies showed that an alternative conformation of methylated mC:G base-pair at non-CpG step exhibits characteristics of cWH with a syn-guanosine conformation in solution. DNA duplexes complexed with the DNA binding drug echinomycin result in increased occupancy of the (w)cWH geometry in the methylated base-pair (from 0.1 to 0.3). Our structural results demonstrated that cytosine methylation at a non-CpG step leads to an anti→syntransition of its complementary guanosine residue toward the (w)cWH geometry as a partial population of WC, in both drug-bound and naked mC:G base pairs. This particular geometry is specific to non-CpG methylated dinucleotide sites in B-form DNA. Overall, the current study provides new insights into DNA conformation during epigenetic regulation.

Accelerated Screening of Alternative DNA Base‐Organic Molecule‐Base Architectures via Integrated Theory and Experiment

AbstractOrganic molecule‐mediated noncanonical DNA self‐assembly expands the standard DNA base‐pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base‐pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub‐stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule‐incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA‐molecule architectures beyond Watson–Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA‐based delivery system and probe design for sensitive detection of certain molecules.

Accelerated Screening of Alternative DNA Base-Organic Molecule-Base Architectures via Integrated Theory and Experiment.

Organic molecule-mediated noncanonical DNA self-assembly expands the standard DNA base-pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base-pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub-stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule-incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA-molecule architectures beyond Watson-Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA-based delivery system and probe design for sensitive detection of certain molecules.

Liposome‐Based Delivery of a Bis‐benzimidazole Derivative to Duplex DNA: A Spectroscopic Study

AbstractDue to the immense pharmacological significance, the interaction of small molecules with liposome and the exact knowledge of their binding location and distribution within the lipid bilayer of liposome is an active field of biophysical research. Here, we have studied the binding interaction of H33258 with DPPC liposome, a model biological membrane. From the steady‐state fluorescence spectroscopy it was revealed that H33258 bounded to DPPC liposome and the estimated binding constant value was 2.5×102 M−1. It was also revealed that the H33258 essentially partitioned within the lipid bilayer of DPPC liposome. Moreover, from steady‐state fluorescence, time‐resolved lifetime, time‐resolved anisotropy and ITC studies it was established that the probe molecules were sequestrated from the lipid bilayer and bounded to a duplex DNA structure. Our results on the binding consequences of H33258 with liposome will aid development of a simple and safe strategy to deliver drugs to the target sites using liposome, endogenously.

Securing LYTAC with Logic-Identification System for Cancer Cell-Selective Membrane Protein Degradation.

Lysosome-targeting chimera (LYTAC) links proteins of interest (POIs) with lysosome-targeting receptors (LTRs) to achieve membrane protein degradation, which is becoming a promising therapeutic modality. However, cancer cell-selective membrane protein degradation remains a big challenge considering expressions of POIs in both cancer cells and normal cells, as well as broad tissue distribution of LTRs. Here a logic-identification system is designed, termed Logic-TAC, based on cell membrane-guided DNA calculations to secure LYTAC selectively for cancer cells. Logic-TAC is designed as a duplex DNA structure, with both POI and LTR recognition regions sealed to avoid systematic toxicity during administration. MCF-7 and MCF-10A are chosen as sample cancer cell and normal cell respectively. As input 1 for logic-identification, membrane proteins EpCAM, which is highly expressed by MCF-7 but barely by MCF-10A, reacts with Logic-TAC to expose POI recognition region. As input 2 for logic-identification, Logic-TAC binds to POI, membrane protein MUC1, to expose LTR recognition region. As output, MUC1 is connected to LTR and degraded via lysosome pathway selectively for cancer cell MCF-7 with little side effect on normal cell MCF-10A. The logic-identification system also demonstrated satisfactory in vivo therapeutic results, indicating its promising potential in precise targeted therapy.

Native Mass Spectrometric Insights into the Formation and Stability of DNA Triplexes.

Deoxyribonucleic acid is a genetic biomacromolecule that contains the inherited information required to build and maintain a living organism. While the canonical duplex DNA structure is rigorously characterized, the structure and function of higher order DNA structures such as DNA triplexes are comparatively poorly understood. Previous literature has shown that these triplexes can form under physiological conditions, and native mass spectrometry offers a useful platform to study their formation and stability. However, experimental conditions including buffer salt concentration, pH, and instrumentation parameters such as ion mode can confound analysis by impacting the amount of triplex observed by mass spectrometry. Using model 30mer Y-type triplex sequences, we demonstrate the influence a range of experimental variables have on the detection of DNA triplex structures, informing suitable conditions for the study. When carefully considered conditions are used, mass spectrometry offers a powerful complementary tool for the analysis of higher order DNA assemblies.

DNA Cryogels with Anisotropic Mechanical and Responsive Properties for Specific Cell Capture and Release.

Due to their programmable stimuli-responsiveness, excellent biocompatibility, and water-rich and soft structures similar to biological tissues, smart DNA hydrogels hold great promise for biosensing and biomedical applications. However, most DNA hydrogels developed to date are composed of randomly oriented and isotropic polymer networks, and the resulting slow response to biotargets and lack of anisotropic properties similar to those of biological tissues have limited their extensive applications. Herein, anisotropic DNA hydrogels consisting of unidirectional void channels internally oriented up to macroscopic length scales were constructed by a directional cryopolymerization method, as exemplified by a DNA-incorporated covalently cross-linked DNA cryogel and a DNA duplex structure noncovalently cross-linked DNA cryogel. Results showed that the formation of unidirectional channels significantly improved the responsiveness of the gel matrix to biomacromolecular substances and further endowed the DNA cryogels with anisotropic properties, including anisotropic mechanical properties, anisotropic swelling/shrinking behaviors, and anisotropic responsiveness to specific biotargets. Moreover, the abundant oriented and long macroporous channels in the gel matrix facilitated the migration of cells, and through the introduction of aptamer structures and thermosensitive polymers, an anisotropic DNA cryogel-based platform was further constructed to achieve the highly efficient capture and release of specific cells. These anisotropic DNA hydrogels may provide new opportunities for the development of anisotropic separation and biosensing systems.

Purification and biochemical characterization of the G4 resolvase and DNA helicase FANCJ.

G-quadruplex (G4) DNA or RNA poses a unique nucleic acid structure in genomic transactions. Because of the unique topology presented by G4, cells have exquisite mechanisms and pathways to metabolize G4 that arise in guanine-rich regions of the genome such as telomeres, promoter regions, ribosomal DNA, and other chromosomal elements. G4 resolvases are often represented by a class of molecular motors known as helicases that disrupt the Hoogsteen hydrogen bonds in G4 by harnessing the chemical energy of nucleoside triphosphate hydrolysis. Of special interest to researchers in the field, including us, is the human FANCJ DNA helicase that efficiently resolves G4 DNA structures. Notably, FANCJ mutations are linked to Fanconi Anemia and are prominent in breast and ovarian cancer. Since our discovery that FANCJ efficiently resolves G4 DNA structures 15 years ago, we and other labs have characterized mechanistic aspects of FANCJ-catalyzed G4 resolution and its biological importance in genomic integrity and cellular DNA replication. In addition to its G4 resolvase function, FANCJ is also a classic DNA helicase that acts on conventional duplex DNA structures, which are relevant to the enzyme's role in interstrand cross link repair, double-strand break repair via homologous recombination, and response to replication stress. Here, we describe detailed procedures for the purification of recombinant FANCJ protein and characterization of its G4 resolvase and duplex DNA helicase activity.

Double-enzyme active MnO2@BSA mediated lab-on-paper dual-modality aptasensor for di(2-ethylhexyl)phthalate

Di(2-ethylhexyl)phthalate (DEHP), as an environmental endocrine disruptor, has adverse effects on eco-environments and health. Thus, it is crucial to highly sensitive on-site detect DEHP. Herein, a double-enzyme active MnO2@BSA mediated dual-modality photoelectrochemical (PEC)/colorimetric aptasensing platform with the cascaded sensitization structures of ZnIn2S4 and TiO2 as signal generators was engineered for rapid and ultrasensitive detection of DEHP using an all-in-one lab-on-paper analytical device. Benefitting from cascaded sensitization effect, the ZnIn2S4/TiO2 photosensitive structures-assembled polypyrrole paper electrode gave an enhanced photocurrent signal. The MnO2@BSA nanoparticles (NPs) with peroxidase-mimic and oxidase-mimic double-enzymatic activity induced multiple signal quenching effects and catalyzed color development. Specifically, the MnO2@BSA NPs acted as peroxidase mimetics to generate catalytic precipitates, which not only obstructed interfacial electron transfer but also served as electron acceptors to accept photogenerated electrons. Besides, the steric hindrance effect from MnO2@BSA NPs-loaded branchy polymeric DNA duplex structures further decreased photocurrent signal. The target recycling reaction caused the detachment of MnO2@BSA NPs to increase PEC signal, realizing the ultrasensitive detection of DEHP with a low detection limit of 27 fM. Ingeniously, the freed MnO2@BSA NPs flowed to colorimetric zone with the aid of fluid channels and acted as oxidase mimetics to induce color intensity enhancement, resulting in the rapid visual detection of DEHP. This work provided a prospective paradigm to develop field-based paper analytical tool for DEHP detection in aqueous environment.

Study on reproductive endocrine disturbance and DNA damage mechanism of female Ruditapes philippinarum under Benzo[a]pyrene stress

The reproductive toxicity of polycyclic aromatic hydrocarbons (PAHs) in aquatic organisms has attracted increasing attention from scholars. Currently, research in this field primarily focuses on vertebrates such as zebrafish and other model species. However, there is still a significant knowledge gap in the toxicity of PAHs to invertebrates and its potential mechanisms. Benzo[a]pyrene (B[a]P) is one of the most representative PAHs. In this study, female Ruditapes philippinarum (R. philippinarum) was treated with B[a]P concentrations of 0, 0.8, 4, and 20 μg/L to investigate reproductive indicators in the proliferative, growth, mature, and spawn stages. Transcriptomics was used to investigate the expression of genes associated with the reproductive endocrine system, DNA repair, autophagy, apoptosis, and ovarian development at different reproductive stages. Our results suggested that B[a]P disrupted the endocrine system by interfering with the production of steroid hormones and the transmission of estrogen signals in female R. philippinarum. The structure of the ovarian DNA duplex is severely damaged under the stress of B[a]P, and a series of cellular responses caused by DNA damage are also interfered. Additionally, we observed a reduction in the gonadosomatic index (GSI) and mature oocytes numbers after B[a]P exposed. Tissue section indicated that severe damage to the ovarian structure at mature and spawn stages. In conclusion, this study combined transcriptomic and toxicological to explore the negative effects on ovarian development induced by B[a]P, focusing on reproductive endocrine disturbance and DNA damage.

Evolution of Organic Solvent-Resistant DNA Polymerases.

The introduction of thermostable polymerases revolutionized the polymerase chain reaction (PCR) and biotechnology. However, many GC-rich genes cannot be PCR-amplified with high efficiency in water, irrespective of temperature. Although polar organic cosolvents can enhance nucleic acid polymerization and amplification by destabilizing duplex DNA and secondary structures, nature has not selected for the evolution of solvent-tolerant polymerase enzymes. Here, we used ultrahigh-throughput droplet-based selection and deep sequencing along with computational free-energy and binding affinity calculations to evolve Taq polymerase to generate enzymes that are both stable and highly active in the presence of organic cosolvents, resulting in up to 10% solvent resistance and over 100-fold increase in stability at 97.5 °C in the presence of 1,4-butanediol, as well as tolerance to up to 10 times higher concentrations of the potent cosolvents sulfolane and 2-pyrrolidone. Using these polymerases, we successfully amplified a broad spectrum of GC-rich templates containing regions with over 90% GC content, including templates recalcitrant to amplification with existing polymerases, even in the presence of cosolvents. We also demonstrated dramatically reduced GC bias in the amplification of genes with widely varying GC content in quantitative polymerase chain reaction (qPCR). By expanding the scope of solvent systems compatible with nucleic acid polymerization, these organic solvent-resistant polymerases enable a dramatic reduction of sequence bias not achievable through thermal resistance alone, with significant implications for a wide range of applications including sequencing and synthetic biology in mixed aqueous-organic media.

Biochemical properties of naturally occurring human bloom helicase variants.

Bloom syndrome helicase (BLM) is a RecQ-family helicase implicated in a variety of cellular processes, including DNA replication, DNA repair, and telomere maintenance. Mutations in human BLM cause Bloom syndrome (BS), an autosomal recessive disorder that leads to myriad negative health impacts including a predisposition to cancer. BS-causing mutations in BLM often negatively impact BLM ATPase and helicase activity. While BLM mutations that cause BS have been well characterized both in vitro and in vivo, there are other less studied BLM mutations that exist in the human population that do not lead to BS. Two of these non-BS mutations, encoding BLM P868L and BLM G1120R, when homozygous, increase sister chromatid exchanges in human cells. To characterize these naturally occurring BLM mutant proteins in vitro, we purified the BLM catalytic core (BLMcore, residues 636-1298) with either the P868L or G1120R substitution. We also purified a BLMcore K869A K870A mutant protein, which alters a lysine-rich loop proximal to the P868 residue. We found that BLMcore P868L and G1120R proteins were both able to hydrolyze ATP, bind diverse DNA substrates, and unwind G-quadruplex and duplex DNA structures. Molecular dynamics simulations suggest that the P868L substitution weakens the DNA interaction with the winged-helix domain of BLM and alters the orientation of one lobe of the ATPase domain. Because BLMcore P868L and G1120R retain helicase function in vitro, it is likely that the increased genome instability is caused by specific impacts of the mutant proteins in vivo. Interestingly, we found that BLMcore K869A K870A has diminished ATPase activity, weakened binding to duplex DNA structures, and less robust helicase activity compared to wild-type BLMcore. Thus, the lysine-rich loop may have an important role in ATPase activity and specific binding and DNA unwinding functions in BLM.

Promoter G-Quadruplex Binding Styryl Benzothiazolium Derivative for Applications in Ligand Affinity Ranking and as Ethidium Bromide Substitute in Gel Staining.

Fluorescent compounds that can preferentially interact with certain nucleic acids are of great importance in new drug discovery in a multitude of functions including fluorescence-based displacement assays and gel staining. Here, we report the discovery of an orange emissive styryl-benzothiazolium derivative (compound 4) which interacts preferentially with Pu22 G-quadruplex DNA among a pool of nucleic acid structures containing G-quadruplex, duplex, and single-stranded DNA structures as well as RNA structures. Fluorescence-based binding analysis revealed that compound 4 interacts with Pu22 G-quadruplex DNA in a 1:1 DNA to ligand binding stoichiometry. The association constant (Ka) for this interaction was found to be 1.12 (±0.15) × 106 M-1. Circular dichroism studies showed that the binding of the probe does not cause changes in the overall parallel G-quadruplex conformation; however, signs of higher-order complex formation were seen in the form of exciton splitting in the chromophore absorption region. UV-visible spectroscopy studies confirmed the stacking nature of the interaction of the fluorescent probe with the G-quadruplex which was further complemented by heat capacity measurement studies. Finally, we have shown that this fluorescent probe can be used toward G-quadruplex-based fluorescence displacement assays for ligand affinity ranking and as a substitute for ethidium bromide in gel staining.

PH-Responsive and Reversible A-Motif-Based DNA Hydrogel: Synthesis and Biosensing Application.

Functional DNA hydrogels with various motifs and functional groups require perfect sequence design to avoid cross-bonding interference with themselves or other structural sequences. This work reports an A-motif functional DNA hydrogel that does not require any sequence design. A-motif DNA is a noncanonical parallel DNA duplex structure containing homopolymeric deoxyadenosines (poly-dA) strands that undergo conformation changes from single strands at neutral pH to a parallel duplex DNA helix at acidic pH. Despite this and other advantages over other DNA motifs like no cross-bonding interference with other structural sequences, the A-motif has not been explored much. We successfully synthesized a DNA hydrogel by using an A-motif as a reversible handle to polymerize a DNA three-way junction. The A-motif hydrogel was initially characterized by electrophoretic mobility shift assay, and dynamic light scattering, which showed the formation of higher-order structures. Further, we used imaging techniques like atomic force microscopy and scanning electron microscope to validating its hydrogel like highly branched morphology. pH-induced conformation transformation from monomers to gel is quick and reversible, and was analysed for multiple acid-base cycles. The sol-to-gel transitions and gelation properties were further examined in rheological studies. The use of the A-motif hydrogel in the visual detection of pathogenic target nucleic acid sequence was demonstrated for the first time in a capillary assay. Moreover, pH-induced hydrogel formation was observed in situ as a layer over the mammalian cells. The proposed A-motif DNA scaffold has enormous potential in designing stimuli-responsive nanostructures that can be used for many biological applications.

Interaction of an anticancer benzopyrane derivative with DNA: Biophysical, biochemical, and molecular modeling studies

BackgroundSIMR1281 is a potent anticancer lead candidate with multi- target activity against several proteins; however, its mechanism of action at the molecular level is not fully understood. Revealing the mechanism and the origin of multitarget activity is important for the rational identification and optimization of multitarget drugs. MethodsWe have used a variety of biophysical (circular dichroism, isothermal titration calorimetry, viscosity, and UV DNA melting), biochemical (topoisomerase I & II assays) and computational (molecular docking and MD simulations) methods to study the interaction of SIMR1281 with duplex DNA structures. ResultsThe biophysical results revealed that SIMR1281 binds to dsDNA via an intercalation-binding mode with an average binding constant of 3.1 × 106 M−1. This binding mode was confirmed by the topoisomerases' inhibition assays and molecular modeling simulations, which showed the intercalation of the benzopyrane moiety between DNA base pairs, while the remaining moieties (thiazole and phenyl rings) sit in the minor groove and interact with the flanking base pairs adjacent to the intercalation site. ConclusionsThe DNA binding characteristics of SIMR1281, which can disrupt/inhibit DNA function as confirmed by the topoisomerases' inhibition assays, indicate that the observed multi-target activity might originate from ligand intervention at nucleic acids level rather than due to direct interactions with multiple biological targets at the protein level. General significanceThe findings of this study could be helpful to guide future optimization of benzopyrane-based ligands for therapeutic purposes.

Front Cover: Orientational Control of Circularly Polarized Luminescence from Pyrene Clusters by Using a DNA Scaffold (Chem. Eur. J. 22/2023)

Circularly polarized luminescent (CPL) depends on orientation. The DNA helix is depicted as a spiral staircase on which chromophore people stand. The CPL intensity, which is shown as helical light, varies depending not only on the distance between chromophores but also on their relative orientation. Helical structures of DNA duplexes are also illustrated. More information can be found in the Research Article by H. Kashida, H. Asanuma, and co-workers (DOI: 10.1002/chem.202300182).

Revealing intrinsic changes of DNA induced by spore photoproduct lesion through computer simulation

In bacterial endospores, a cross-linked thymine dimer, 5-thyminyl-5,6-dihydrothymine, commonly referred to as the spore photoproduct (SP), is found as the dominant DNA photo lesion under UV radiation. During spore germination, SP is faithfully repaired by the spore photoproduct lyase (SPL) for normal DNA replication to resume. Despite this general mechanism, the exact way in which SP modifies the duplex DNA structure so that the damaged site can be recognized by SPL to initiate the repair process is still unclear. A previous X-ray crystallographic study, which used a reverse transcriptase as a DNA host template, captured a protein-bound duplex oligonucleotide containing two SP lesions; the study showed shortened hydrogen bonds between the AT base pairs involved in the lesions and widened minor grooves near the damaged sites. However, it remains to be determined whether the results accurately reflect the conformation of SP-containing DNA (SP-DNA) in its fully hydrated pre-repair form. To uncover the intrinsic changes in DNA conformation caused by SP lesions, we performed molecular dynamics (MD) simulations of SP-DNA duplexes in aqueous solution, using the nucleic acid portion of the previously determined crystal structure as a template. After MD relaxation, our simulated SP-DNAs showed weakened hydrogen bonds at the damaged sites compared to those in the undamaged DNA. Our analyses of the MD trajectories revealed a range of local and global structural distortions of DNA induced by SP. Specifically, the SP region displays a greater tendency to adopt an A-like-DNA conformation, and curvature analysis revealed an increase in the global bending compared to the canonical B-DNA. Although these SP-induced DNA conformational changes are relatively minor, they may provide a sufficient structural basis for SP to be recognized by SPL during the lesion repair process.

From single-molecule to synchrotron: New tools for investigating the molecular mechanisms of aberrant DNA diseases.

Damage in DNA can interrupt and alter its conformational flexibility—which drives crucial site-specific functions—leading to aberrant DNA diseases. It is not fully understood exactly what role these conformations play in the processes undertaken by DNA, nor how they might be affected by various damage. Here we present an overview and comparison of two novel and emergent techniques to study such conformations; single molecule Förster resonance energy transfer (smFRET) and X-ray scattering interferometry (XSI). We are focusing on the RNaseH2 complex, mutations that are responsible for Aicardi-Goutières syndrome (AGS), to establish how the search space is minimised as it finds and cleaves RNA/DNA hybrids, and whether this is driven by conformational changes. We also introduce O6-methylguanine (O6-MeG), ribonucleotide, nicks and gaps; all prevalent modifications to DNA which without repair would lead to toxic intermediates. It is hypothesised that when the damage is present the increase in flexibility of DNA signals proteins to promote repair. smFRET was used to measure the conformational changes when the different types of damage, were present in a DNA duplex. A change in FRET efficiency is seen for all the types of damage, with the nick and gap damage having a greater impact on the mean and spread of the FRET efficiency. These results provide initial support for the hypothesis that damage alters the overall structure and dynamics of DNA duplexes and that the presence of these lesions and their respective repair mechanisms binds the DNA in varying levels of flexibility.

Selective, Disruptive Luminescent Ru(II) Polypyridyl Probes of G-Quadruplex.

Sensors capable of transducing G-quadruplex DNA binding are important both in solution and for imaging and interrogation in cellulo. Ru(II)-based light switches incorporating dipyridylphenazine (dppz) ligands are effective probes for recognition and imaging of DNA and its polymorphs including G-quadruplex, although selectivity is a limitation. While the majority of Ru(II)-based light switches reported to date, stabilize the quadruplex, imaging/theranostic probes that can disrupt G4s are of potentially enormous value in study and therapy for a range of disease states. We report here, on a Ru(II) complex (Ru-PDC3) that assembles the light switch capability of a Ru(II) dipyridylphenazine complex with the well-known G4-selective ligand Phen-DC3, into a single structure. The complex shows the anticipated light switch effect and strong affinity for G4 structures. Affinity depended on the G4 topology and sequence, but across all structures bar one, it was roughly an order of magnitude greater than for duplex or single-stranded DNA. Moreover, photophysical and Raman spectral data showed clear discrimination between duplex DNA and G4-bound structures offering the prospect of discrimination in imaging as well as in solution. Crucially, unlike the constituent components of the probe, Ru-PDC3 is a powerful G4 disrupter. From circular dichroism (CD), a reduction of ellipticity of the G4 between 70 and 95% was observed depending on topology and in many cases was accompanied by an induced CD signal for the metal complex. The extent of change in ellipticity is amongst the largest reported for small-molecule ligand G4 binding. While a promising G4 probe, without modification, the complex is fully water-soluble and readily permeable to live cells.