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.

What tunes guanine ionization potential in a nucleosome ? An all-in-one systematic QM/MM assessment.

Guanine radical cations are precursors to oxidatively-induced DNA lesions and the determination of oxidative DNA hot spots beyond oligonucleotides remains a current challenge. In order to rationalize the finetuned ionization properties of the ∼60 guanines in a nucleosome core particle (NCP), we report a robust MD-then-FO-DFTB/MM simulation protocol spanning 20 microseconds. Our work allows to identify several factors governing guanine ionization potential and to map oxidative hotspots. Our results highlight the predominant role of the proximity of positively-charged histone residues in the modulation of the guanine ionization potential up to 0.6 eV. Consequently, fast long range hole transfer in nucleosomal DNA might vary on the proximity of histone tails, so, on a biological point of view, on the chromatin state.

Influence of Chemical Modifications of the Crystallophore on Protein Nucleating Properties and Supramolecular Interactions Network.

Crystallophores are lanthanide complexes that have demonstrated outstanding induction of crystallization for various proteins. This article explores the effect of tailored modifications of the crystallophore first generation and their impact on the nucleating properties and protein crystal structures. Through high-throughput crystallization experiments and dataset analysis, we evaluated the effectiveness of these variants, in comparison to the first crystallophore generation G1. In particular, the V1 variant, featuring a propanol pendant arm, demonstrated the ability to produce new crystallization conditions for the proteins tested (hen-egg white lysozyme, proteinase K and thaumatin). Structural analysis performed in the case of hen egg-white lysozyme along with Molecular Dynamics simulations, highlights V1's unique behavior, taking advantage of the flexibility of its propanol arm to explore different protein surfaces and form versatile supramolecular interactions.

One touch is all it takes: the supramolecular interaction between ubiquitin and lanthanide complexes revisited by paramagnetic NMR and molecular dynamics.

The supramolecular interaction between lanthanide complexes and proteins is at the heart of numerous chemical and biological studies. Some of these complexes have demonstrated remarkable interaction properties with proteins or peptides in solution and in the crystalline state. Here we have used the paramagnetism of lanthanide ions to characterize the affinity of two lanthanide complexes for ubiquitin. As the interaction process is dynamic, the acquired NMR data only reflect the time average of the different steps. We have used molecular dynamics (MD) simulations to get a deeper insight into the detailed interaction scenario at the microsecond scale. This NMR/MD approach enabled us to establish that the tris-dipicolinate complex interacts specifically with arginines and lysines, while the crystallophore explores the protein surface through weak interactions with carboxylates. These observations shed new light on the dynamic interaction properties of these complexes, which will ultimately enable us to propose a crystallization mechanism.

Situating the phosphonated calixarene-cytochrome C association by molecular dynamics simulations.

Protein-calixarenes binding plays an increasingly central role in many applications, spanning from molecular recognition to drug delivery strategies and protein inhibition. These ligands obey a specific bio-supramolecular chemistry, which can be revealed by computational approaches, such as molecular dynamics simulations. In this paper, we rely on all-atom, explicit-solvent molecular dynamics simulations to capture the electrostatically driven association of a phosphonated calix-[4]-arene with cytochome-C, which critically relies on surface-exposed paired lysines. Beyond two binding sites identified in direct agreement with the x-ray structure, the association has a larger structural impact on the protein dynamics. Then, our simulations allow a direct comparison to analogous calixarenes, namely, sulfonato, similarly reported as "molecular glue." Our work can contribute to a robust in silico predictive tool to assess binding sites for any given protein of interest for crystallization, with the specificity of a macromolecular cage whose endo/exo orientation plays a role in the binding.

DNA-Histone Cross-Link Formation via Hole Trapping in Nucleosome Core Particles.

Radical cations (holes) produced in DNA by ionizing radiation and other oxidants yield DNA-protein cross-links (DPCs). Detailed studies of DPC formation in chromatin via this process are lacking. We describe here a comprehensive examination of DPC formation within nucleosome core particles (NCPs), which are the monomeric component of chromatin. DNA holes are introduced at defined sites within NCPs that are constructed from the bottom-up. DPCs form at DNA holes in yields comparable to those of alkali-labile DNA lesions that result from water trapping. DPC-forming efficiency and site preference within the NCP are dependent on translational and rotational positioning. Mass spectrometry and the use of mutant histones reveal that lysine residues in histone N-terminal tails and amino termini are responsible for the DPC formation. These studies are corroborated by computational simulation at the microsecond time scale, showing a wide range of interactions that can precede DPC formation. Three consecutive dGs, which are pervasive in the human genome, including G-quadruplex-forming sequences, are sufficient to produce DPCs that could impact gene expression.

Rationalizing the environment-dependent photophysical behavior of a DNA luminescent probe by classical and non-adiabatic molecular dynamics simulations.

Environment-sensitive fluorescent nucleoside analogs are of utmost importance to investigate the structure of nucleic acids, their intrinsic flexibility, and sequence-specific DNA- and RNA-binding proteins. The latter play indeed a key role in transcription, translation as well as in the regulation of RNA stability, localization and turnover, and many other cellular processes. The sensitivity of the embedded fluorophore to polarity, hydration, and base stacking is clearly dependent on the specific excited-state relaxation mechanism and can be rationalized combining experimental and computational techniques. In this work, we elucidate the mechanisms leading to the population of the triplet state manifold for a versatile nucleobase surrogate, namely the 2-thienyl-3-hydroxychromone in gas phase, owing to non-adiabatic molecular dynamics simulations. Furthermore, we analyze its behavior in the B-DNA environment via classical molecular dynamics simulations, which evidence a rapid extrusion of the adenine facing the 2-thienyl-3-hydroxychromone nucleobase surrogate. Our simulations provide new insights into the dynamics of this family of chromophores, which could give rise to an integrated view and a fine tuning of their photochemistry, and namely the role of excited-state intramolecular proton transfer for the rational design of the next generation of fluorescent nucleoside analogs.

Probing the dynamical interaction of the para-sulfonato-calix[4]arene with an antifungal protein.

Calixarenes are hallmark molecules in supramolecular chemistry as hosts for small ligands. They have also conversely proved their interest as ligands toward assisted co-crystallization of proteins. These functionalized macrocycles target positively-charged residues, and notably surface-exposed lysines, with a site-selectivity finely characterized experimentally, but that remains to be assessed. Relying on a tailored molecular dynamics simulations protocol, we explore the association of para-sulfonato-calix[4]arenes with an antifungal protein, as a small yet most competitive system with 13 surface-exposed lysines. Our computational approach probes de novo the electrostatically-driven interaction, ruled out by a competition with salt bridges, corroborating the presence of two main binding sites probed by X-ray. The attach-pull-release (APR) method provides a very good assessment of the overall binding free energy measured experimentally (-6.42 ± 0.5 vs. -5.45 kcal mol-1 by isothermal titration calorimetry). This work also probes dynamic modifications upon ligand binding, and our computational protocol could be generalized to situate the supramolecular forces ruling out the calixarene-assisted co-crystallization of proteins.

Ni-Centered Coordination-Induced Spin-State Switching Triggered by Electrical Stimulation.

We herein report the synthesis and magnetic properties of a Ni(II)-porphyrin tethered to an imidazole ligand through a flexible electron-responsive mechanical hinge. The latter is capable of undergoing a large amplitude and fully reversible folding motion under the effect of electrical stimulation. This redox-triggered movement is exploited to force the axial coordination of the appended imidazole ligand onto the square-planar Ni(II) center, resulting in a change in its spin state from low spin (S = 0) to high spin (S = 1) proceeding with an 80% switching efficiency. The driving force of this reversible folding motion is the π-dimerization between two electrogenerated viologen cation radicals. The folding motion and the associated spin state switching are demonstrated on the grounds of NMR, (spectro)electrochemical, and magnetic data supported by quantum calculations.

Capturing the Recognition Dynamics of para-Sulfonato-calix[4]arenes by Cytochrome c: Toward a Quantitative Free Energy Assessment.

Calix[n]arenes' selective recognition of protein surfaces covers a broad range of timely applications, from controlling protein assembly and crystallization to trapping partially disordered proteins. Here, the interaction of para-sulfonated calix-[4]-arenes with cytochrome c is investigated through all-atom, explicit water molecular dynamics simulations which allow characterization of two binding sites in quantitative agreement with experimental evidence. Free energy calculations based on the MM-PBSA and the attach-pull-release (APR) methods highlight key residues implicated in the recognition process and provide binding free energy results in quantitative agreement with isothermal titration calorimetry. Our study emphasizes the role of MD simulations to capture and describe the "walk" of sulfonated calix-[4]-arenes on the cytochrome c surface, with the arginine R13 as a pivotal interacting residue. Our MD investigation allows, through the quasi-harmonic multibasin (QHMB) method, probing an allosteric reinforcement of several per-residue interactions upon calixarene binding, which suggests a more complex mode of action of these supramolecular auxiliaries.

How Fragile We Are: Influence of Stimulator of Interferon Genes (STING) Variants on Pathogen Recognition and Immune Response Efficiency.

The stimulator of interferon genes (STING) protein is a cornerstone of the human immune response. Its activation by cGAMP in the presence of cytosolic DNA stimulates the production of type I interferons and inflammatory cytokines. In the human population, several STING variants exist and exhibit dramatic differences in their activity, impacting the efficiency of the host defense against infections. Understanding the molecular mechanisms of these variants opens perspectives for personalized medicine treatments against diseases such as viral infections, cancers, or autoinflammatory diseases. Through microsecond-scale molecular modeling simulations, contact analyses, and machine learning techniques, we reveal the dynamic behavior of four STING variants (wild type, G230A, R293Q, and G230A/R293Q) and rationalize the variability of efficiency observed experimentally. Our results show that the decrease in STING activity is linked to a stiffening of key structural elements of the binding cavity together with changes in the interaction patterns within the protein.

Organo-Polyoxometalate-Based Hydrogen-Bond Catalysis.

Several urea-inserted organo-polyoxometalates (POMs) derived from polyoxotungstovanadate [P2 V3 W15 O61 ]9- were prepared. The insertion of the carbonyl into the polyoxometallic framework activates the urea toward Hydrogen-bond catalysis. This was shown on the Friedel-Crafts arylation of trans-ß-nitrostyrene. Modelling shows that the most stable form of the organo-POMs features a cis-trans arrangement of the two N-H bonds, but that the likely catalytically active trans-trans form is accessible at room temperature. Finally, it is possible that the oxo substituents next to the vanadium atoms may help the approach of the nucleophile via H-bonding.

Synthesis and Properties of Higher Nuclearity Polyazanes.

Polyazanes (i.e., higher nuclearity homologues of hydrazines) with increasing numbers of bound nitrogen atoms (from 3 to 5), including the first pentazane ever described, were prepared by the addition of lower-order polyazanes to diazo reagents. A structure was obtained. It was shown that the polynitrogen chains adopt a helical conformation. DFT modeling shows that the arrangement persists in solution. Although the polyazanes are all reducing agents, they become less so as the number of nitrogens increases.

Electron-Triggered Metamorphism in Palladium-Driven Self-Assembled Architectures.

A metal-induced self-assembly strategy is used to promote the π-dimerization of viologen-based radicals at room temperature and in standard concentration ranges. Discrete box-shaped 2:2 (M:L) macrocycles or coordination polymers are formed in solution by self-assembly of a viologen-based ditopic ligand with cis-[Pd(en)(NO3)2], trans-[Pd(CH3CN)2(Cl)2], or [Pd(CH3CN)4(BF4)2]. Changing the redox state of the bipyridium units involved in the tectons, from their dicationic state to their radical cation state, results in a reversible "inflation/deflation" of the discrete 2:2 (M:L) macrocyclic assemblies associated to a large modification in the size of their inner cavity. Viologen-centered electron transfer is also used to trigger a dissociation of the coordination polymers formed with tetrakis(acetonitrile)Pd(II), the driving force of the disassembling process being the formation of discrete box-shaped 2:2 (M:L) assemblies stabilized by π-dimerization of both viologen cation radicals.

Influence of Divalent Cations in the Protein Crystallization Process Assisted by Lanthanide-Based Additives.

The use of lanthanide complexes as powerful auxiliaries for biocrystallography prompted us to systematically analyze the influence of the commercial crystallization kit composition on the efficiency of two lanthanide additives: [Eu(DPA)3]3- and Tb-Xo4. This study reveals that the tris(dipicolinate) complex presents a lower chemical stability and a strong tendency toward false positives, which are detrimental for its use in a high-throughput robotized crystallization platform. In particular, the crystal structures of (Mg(H2O)6)3[Eu(DPA)3]2·7H2O (1), {(Ca(H2O)4)3[Eu(DPA)3]2}n·10nH2O (2), and {Cu(DPA)(H2O)2}n (3), resulting from spontaneous crystallization in the presence of a divalent alkaline-earth cation and transmetalation, are reported. On the other hand, Tb-Xo4 is perfectly soluble in the crystallization media, stable in the presence of alkaline-earth dications, and slowly decomposes (within days) by transmetalation with transition metals. The original structure of [Tb4L4(H2O)4]Cl4·15H2O (4) is also described, where L represents a bis(pinacolato)triazacyclononane ligand. This paper also highlights a potential synergy of interactions between Tb-Xo4 and components of the crystallization mixtures, leading to the formation of complex adducts like {AdkA/Tb-Xo4/Mg2+/glycerol} in the protein binding sites. The observation of such multicomponent adducts illustrated the complexity and versatility of the supramolecular chemistry occurring at the surface of the proteins.

Capturing the dynamic association between a tris-dipicolinate lanthanide complex and a decapeptide: a combined paramagnetic NMR and molecular dynamics exploration.

In the realm of biomolecules, peptides can present a large diversity of structures. Our study sheds new light on the structural interplay between a tris-dipicolinate lanthanide probe and a decapeptide SASYKTLPRG. Although a rather trivial, electrostatically driven interaction was expected, the combination of paramagnetic NMR and molecular dynamics simulations reveals a highly dynamic association process and allows for providing extensive insights into the interaction sites and their occupancy. This study highlights the importance of a large conformational sampling to reconcile characteristic time in NMR with molecular dynamics simulations, where sampling in the microsecond range is needed. This study opens the door for a detailed mechanistic elucidation of the early steps of lanthanide complex-peptide or lanthanide complex-protein interaction or self-assembly processes.

Assessing the sequence dependence of pyrimidine-pyrimidone (6-4) photoproduct in a duplex double-stranded DNA: A pitfall for microsecond range simulation.

Sequence dependence of the (6-4) photoproduct conformational landscape when embedded in six 25-bp duplexes is evaluated along extensive unbiased and enhanced (replica exchange with solute tempering, REST2) molecular dynamics simulations. The structural reorganization as the central pyrimidines become covalently tethered is traced back in terms of non-covalent interactions, DNA bending, and extrusion of adenines of the opposite strands. The close sequence pattern impacts the conformational landscape around the lesion, inducing different upstream and downstream flexibilities. Moreover, REST2 simulations allow us to probe structures possibly important for damaged DNA recognition.

Molecular Mechanisms Associated with Clustered Lesion-Induced Impairment of 8-oxoG Recognition by the Human Glycosylase OGG1.

The 8-oxo-7,8-dihydroguanine, referred to as 8-oxoG, is a highly mutagenic DNA lesion that can provoke the appearance of mismatches if it escapes the DNA Damage Response. The specific recognition of its structural signature by the hOGG1 glycosylase is the first step along the Base Excision Repair pathway, which ensures the integrity of the genome by preventing the emergence of mutations. 8-oxoG formation, structural features, and repair have been matters of extensive research; more recently, this active field of research expended to the more complicated case of 8-oxoG within clustered lesions. Indeed, the presence of a second lesion within 1 or 2 helix turns can dramatically impact the repair yields of 8-oxoG by glycosylases. In this work, we use µs-range molecular dynamics simulations and machine-learning-based postanalysis to explore the molecular mechanisms associated with the recognition of 8-oxoG by hOGG1 when embedded in a multiple-lesion site with a mismatch in 5' or 3'. We delineate the stiffening of the DNA-protein interactions upon the presence of the mismatches, and rationalize the much lower repair yields reported with a 5' mismatch by describing the perturbation of 8-oxoG structural features upon addition of an adjacent lesion.

Dynamic Molecular Metamorphism Involving Palladium-Assisted Dimerization of π-Cation Radicals.

A dynamic supramolecular approach is developed to promote the π-dimerization of viologen radicals at room temperature and in standard concentration ranges. The approach involves cis- or trans-protected palladium centers serving as inorganic hinges linking two functionalized viologens endowed with metal-ion coordinating properties. Based on detailed spectroscopic, electrochemical and computational data, we show that the one-electron electrochemical reduction of the viologen units in different dynamic metal/ligand mixtures leads to the formation of the same intramolecular π-dimer, regardless of the initial environment around the metallic precursor and of the relative ratio between metal and ligand initially introduced in solution. The large-scale electron-triggered reorganization of the building blocks introduced in solution thus involves drastic changes in the stoichiometry and stereochemistry of the palladium/viologen complexes proceeding in some cases through a palladium centered trans→cis isomerization of the coordinated ligands.

Probing interaction of a trilysine peptide with DNA underlying formation of guanine-lysine cross-links: insights from molecular dynamics.

DNA-protein cross-links constitute bulky DNA lesions that interfere with the cellular machinery. Amongst these stable covalently tethered adducts, the efficient nucleophilic addition of the free amino group of lysines onto the guanine radical cation has been evidenced. In vitro addition of a trilysine peptide onto a guanine radical cation generated in a TGT oligonucleotide is so efficient that competitive addition of a water molecule, giving rise to 8-oxo-7,8-dihydroguanine, is not observed. This suggests a spatial proximity between guanine and lysine for the stabilization of the prereactive complex. We report all-atom microsecond scale molecular dynamics simulations that probe the structure and interactions of the trilysine peptide (KKK) with two oligonucleotides. Our simulations reveal a strong, electrostatically driven yet dynamic interaction, spanning several association modes. Furthermore, the presence of neighbouring cytosines has been identified as a factor favoring KKK binding. Relying on ab initio molecular dynamics on a model system constituted of guanine and methylammonium, we also corroborate a mechanistic pathway involving fast deprotonation of the guanine radical cation followed by hydrogen transfer from ammonium leaving as a result a nitrogen reactive species that can subsequently cross-link with guanine. Our study sheds new light on a ubiquitous mechanism for DNA-protein cross-links also stressing out possible sequence dependences.