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International audience; Peptide-based hydrogels are physical gels formed through specific supramolecular self-assembling processes, leading to ordered nanostructures which constitutes the water entrapping scaffold of the soft material. Thanks to the inherent properties of peptides, these hydrogels are highly considered in the biomedical domain and open new horizons in terms of application in advanced therapies and biotechnologies. The use of one, and only one, native peptide to formulate a gel is by far the most reported approach to design such materials, but suffers from several limitations, including in terms of mechanical properties. To improve peptide-based hydrogels interest and give rise to innovative properties, several strategies have been proposed in the recent years, and the development of multicomponent peptide-based hydrogels appears as a promising and relevant strategy. Indeed, mixing two or more compounds to develop new materials is a much-used approach that has proven its effectiveness in a wide variety of domains, including polymers, composites and alloys. While still limited to a handful of examples, we would like to report herein on the formulation and the comprehensive study of multicomponent hybrid DNA-nucleobase/peptide-based hydrogels using a multiscale approach based on a large panel of analytical techniques (i.e., rheometry, proton relaxometry, SAXS, electronic microscopy, infrared, circular dichroism, fluorescence, Thioflavin T assays). Among the six multicomponent systems studied, the results highlight the synergistic role of the presence of the two complementary DNA-nucleobases (i.e., adenine/thymine and guanine/cytosine) on the coassembling process from structural (e.g., morphology of the nanoobjects) to physicochemical (e.g., kinetics of formation, fluorescence properties) and mechanical properties (e.g., stiffness, resistance to external stress). All the data confirm the relevance of the multicomponent peptide-based approach in the design of innovative hydrogels and bring another brick in the wall of the understanding of these complex and promising systems.

Over the last twenty years, low-molecular weight gelators and, in particular, peptide-based hydrogels, have drawn great attention from scientists thanks to both their inherent advantages in terms of properties and their high modularity (

Soy and pea proteins are two rich sources of essential amino acids. The hydrolysis of these proteins reveals functional and bioactive properties of the produced small peptide mixtures. In our study, we employed the hydrolysis of soy and pea protein isolates with the endopeptidases Alcalase® and Protamex®, used alone or followed by the exopeptidase Flavourzyme®. The sequential enzyme treatments were the most efficient regarding the degree of hydrolysis. Then, soy and pea protein hydrolysates (SPHs and PPHs, respectively) were ultrafiltrated in order to select peptides of molecular weight ≤ 1 kDa. Whatever the protein source or the hydrolysis treatment, the hydrolysates showed similar molecular weight distributions and amino acid compositions. In addition, all the ultrafiltrated hydrolysates possess metal-chelating activities, as determined by UV-spectrophotometry and Surface Plasmon Resonance (SPR). However, the SPR data revealed better chelating affinities in SPHs and PPHs when produced by sequential enzymatic treatment.

International audience; Molecular self-assembly is a fascinating process which has become an area of great interest in supramolecular chemistry, as it leads in certain cases to molecular gels. Organogels formulated from low molecular weight compounds (LMWOGs) have attracted much interest in the past decades due to their applications as new soft materials. Herein, we report on the ability of the cyclic pseudopeptide cyclo-[-(D-Phe-azaPhe-Ala)2-] (2) to self-assemble in some aromatic solvents and to form organogels driven by non-covalent forces, mainly hydrogen bonding and π-stacking interactions. Comprehensive FTIR and NMR studies emphasized that this cyclic aza-peptide adopts a β-turn conformation at low concentration in toluene, while an equilibrium between the monomeric states (intramolecular forces) and the supramolecular structures (intra- and intermolecular forces) is established at high concentration (gel state). Rheological investigations of the organogels highlight the dependence of their stiffness (up to ∼4 kPa) and sol/gel transition temperatures (up to 100 °C) as a function of the solvent and concentration of gelator used. The formulation of fibrous structures confirmed the phenomenon of self-assembly. Finally, we found that cyclo-[-(D-Phe-azaPhe-Ala)2-] is an effective organogelator for application in phase selective gelation (PSG) of organic solvents from aqueous/organic mixtures with recovery percents up to 96%.

International audience; Antioxidant compounds are chemicals of primary importance, especially for their applications in nutrition and healthcare, thanks to their abilities to prevent oxidation processes and to limit and/or rebalance the oxidative stress, well-known for its impact on a wide variety of diseases. While several biomolecules are well-known for their antioxidant properties (e.g., ascorbic acid, carotenoids, phenolic derivatives), bio-sourced antioxidants have drawn considerable attention in the last decades, especially bioactive peptides, mainly obtained by the hydrolysis process. Antioxidant peptide sequences are mainly identified a posteriori, thanks to fastidious and time-consuming approaches and techniques, limiting the discovery of new efficient peptides. In this context and taking inspiration from nature, we report herein on a new series of three bio-inspired antioxidant peptides derived from the milk protein casein. These phosphopeptides, designed to chelate the redox-active iron(III) and forming highly soluble complexes up to pH 9, act both as indirect (i.e., inhibition of the metal redox activity) and direct (i.e., radical scavenging) antioxidants

International audience; Metal-chelating peptides (MCP) are considered as indirect antioxidants due to their capacity to inhibit radical chain reaction and oxidation. Here, we propose a new proof of concept for the screening of MCPs present in protein hydrolysates for valorizing their antioxidant properties by using the emerging time-resolved molecular dynamics technology, switchSENSE. This method unveils possible interactions between MCPs and immobilized nickel ions using fluorescence and electro-switchable DNA chips. The switchSENSE method was first set up on synthetic peptides known for their metal-chelating properties. Then, it was applied to soy and tilapia viscera protein hydrolysates. Their Cu2+-chelation capacity was, in addition, determined by UV-visible spectrophotometry as a reference method. The switchSENSE method has displayed a high sensitivity to evidence the presence of MCPs in both hydrolysates. Hence, we demonstrate for the first time that this newly introduced technology is a convenient methodology to screen protein hydrolysates in order to determine the presence of MCPs before launching time-consuming separations.

International audience; Minerals are essential to different biological processes in living beings. Likewise, free transition metals such as copper and iron promote the formation of reactive oxygen species (ROSs). However, metal-binding peptides obtained by enzymatic hydrolysis of proteins or synthesized by solid-phase peptide synthesis avoid ROS formation catalyzed by free transition metals. Interactions between metals and mineral-binding peptides (MBPs) are usually studied using spectroscopic techniques, isothermal titration calorimetry, and more recently by surface plasmon resonance and switchSENSE. Through electrospray ionization-mass spectrometry, the changes in the binding affinities can be studied. MBPs can be purified using immobilized metal-ion affinity chromatography, and its process simulation. Various cellular models are used to investigate the health effects of MBPs.

International audience; Production of iron-chelating peptides from protein hydrolysates requires robust and adequate screening methods to optimize their purification and subsequently valorize their potential antioxidant properties. An original methodology was developed for direct and sensitive screening of iron(II)-chelating peptides based on ion-pair reverse phase liquid chromatography (IP-RPLC) coupled to high-resolution mass spectrometry (HRMS). Peptide mixture was first added to iron(II) solution to form iron(II)-peptide complexes. Then IP-RPLC-HRMS analysis was conducted on this iron-peptide mixture and on the iron-free peptide solution for comparative mass spectra analysis. This protocol, initially applied to a range of low molecular weight standard peptides, allowed detection of [(Peptide-H)+ Fe ] complex ion for iron(II)-chelating peptides (GGH, EAH, DAH, βAH, DMH, DTH, DSH). GGH was added in complex peptide mixtures and targeted analysis of [(GGH-H)+ Fe ] complex showed a limit of detection (LOD) below 0.77 mg L of GGH. This protocol was finally tested in combination with metabolomics software and additional digital processing for non-targeted search for iron(II)-chelating peptides. Applicability of this new screening methodology has been validated by detection of GGH as iron(II)-chelating peptide when added at 0.77 mg L in caseinhydrolysate.

International audience; Reactive oxygen species (ROS) are major sources of oxidative stress playing prominent roles in the development of several pathologies including cardiovascular and neurodegenerative diseases or cancers. The presence of transition biometal ions, specifically copper and iron, induces ROS formation by catalyzing the reduction of molecular oxygen to superoxide anion (O 2 À), hydrogen peroxide (H 2 O 2) and hydroxyl (HO) radical. To limit ROS production and their detrimental effects, we report on the synthesis, physicochemical studies and antioxidant assays of an innovative series of synthetic pentapeptides exhibiting a dual direct/indirect mode of action, both as iron(III)-chelators and as radical scavengers. These combined effects lead to a drastic reduction of in vitro reactive oxygen species production up to 95% for the more reactive hydroxyl radical. Significance to metallomics We report herein on a new series of peptides exhibiting iron(III)-chelation abilities from acidic to neutral pH corresponding to conditions in which redox-active iron(III) is found in vivo, such as in both the gastrointestinal tract and the lysosomal iron labile pool, and in the blood plasma, respectively. Thanks to their chelating properties and their inherent design, this series of peptides act as effective antioxidants based on a dual indirect (i.e., inhibition of the metal redox activity) and direct (i.e., hydroxyl radical scavenging) mode of action, drastically limiting the production of reactive oxygen species up to À99% for the hydroxyl radical.

Hybrid molecular/macromolecular gels were formulated by mixing amino-acid based gelators with dextran, a nonionic bacterial polysaccharide, in mixtures of water and dimethylsulfoxide. Gelators were hydrophobic derivatives of L -lysine in which the Nα was acylated with dodecanoic acid and the Ne was acylated by benzoyloxycarbonyl or o-chloro-benzoyloxycarbonyl group. Increasing dextran concentration led to a decrease of the stationary values of storage and loss modulus of the gels. In addition, gel formation was slowed down upon increasing dextran concentration. Nevertheless, the characteristics of aggregates as well as their kinetics of growth, as deduced from rheological data, remained identical to those observed with gelator alone (mainly linear fractal aggregates and unidirectional growing mechanism) whatever dextran concentration. Raman microscopy confirmed conclusions drawn from rheological data. It was concluded that dextran macromolecules were mainly at the origin of steric hindrance with supramolecular aggregates.

International audience

Guanine and related nucleobases such as guanosine, deoxyguanosine and isoguanosine are notable molecular tools for designing functional supramolecular assemblies. This popularity originates in their ability to self-assemble via a unique topological pluralism — as isolated nucleobases, discrete macrocyclic quartets and virtually infinite linear ribbons — that endows them with a considerable functional versatility. Many programmes have been launched to fine-tune the chemical properties of guanine derivatives, to make them usable under different experimental conditions, such as in organic or aqueous environments, and responsive to external stimuli, such as ionic strength, pH, light or temperature. These strategies aim to translate the chemical information encoded in a basic guanine unit into programmable, higher-order supramolecular architectures. Spectacular results have been recently obtained in various chemical fields, from supramolecular chemistry to chemical biology, from soft matter to catalysis. In this Review, we detail these advances and demonstrate how these multidisciplinary investigations cast a bright light on the diversity that guanines, synthetic guanines and related nucleobases uniquely offer in terms of both structure and function. The propensity of guanine and its derivatives to assemble into guanine quartets makes them a privileged tool in the design of functional supramolecular assemblies. This Review describes the production of such functional suprastructures and their applications in nanotechnology, soft matter and chemical biology.

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International audience

Three new low-molecular-weight gelators (LMWGs) based on L-Leucine derivatives bearing different chain lengths (i.e. 9, 12 or 16) were designed and synthesized (designated as 1-3). The N-terminal is carrying the various alkyl chain lengths where the C-terminal is modified with hydrazide moiety. The prepared gelators 1-3 were fully characterized using 1H, 13C NMR, FTIR and mass spectroscopy. The gelation behavior has also been investigated using different organic solvents and oils with determination of critical gelation concentration (CGC). The self-assembly process was investigated by recording FTIR spectra gel states to investigate the driving forces for gelation process. The morphology of the prepared xerogels was studied using SEM.

Among all the materials used in industry, gels play an increasingly important role. These so-called soft-matter materials are defined by their ability to fix a large amount of solvent, either organic (organogels) or aqueous (hydrogels). The large majority of hydrogels are made of natural or synthetic polymers, or natural proteins. However, a new kind of hydrogel has appeared: the peptide-based hydrogels, developed from short amino acids sequences (

Recent and unambiguous evidences of the formation of DNA and RNA G-quadruplexes in cells has provided solid support for these structures to be considered as valuable targets in oncology. Beyond this, they have lent further credence to the anticancer strategies relying on small molecules that selectively target these higher-order DNA/RNA architectures, referred to as G-quadruplex ligands. They have also shed bright light on the necessity of designing multitasking ligands, displaying not only enticing quadruplex interacting properties (affinity, structural selectivity) but also additional features that make them usable for detecting quadruplexes in living cells, notably for determining whether, when, and where these structures fold and unfold during the cell cycle and also for better assessing the consequences of their stabilization by external agents. Herein, we report a brand new design of such multitasking ligands, whose structure experiences a quadruplex-promoted conformational switch that triggers not only its quadruplex affinity (i.e., smart ligands, which display high affinity and selectivity for DNA/RNA quadruplexes) but also its fluorescence (i.e., smart probes, which behave as selective light-up fluorescent reporters on the basis of a fluorogenic electron redistribution). The first prototype of such multifunctional ligands, termed PyroTASQ, represents a brand new generation of quadruplex ligands that can be referred to as "twice-as-smart" quadruplex ligands.

Secondary nucleic acid structures, such as DNA and RNA quadruplexes, are potential targets for cancer therapies. Ligands that interact with these targets could thus find application as anticancer agents. Synthetic G-quartets have recently found numerous applications, including use as bioinspired G-quadruplex ligands. Herein, the design, synthesis and preliminary biophysical evaluation of a new prototype multitarget G-quadruplex ligand, (PNA)PorphySQ, are reported, where peptidic nucleic acid guanine ((PNA)G) was incorporated in the porphyrin-templated synthetic G-quartet (PorphySQ). Using fluorescence resonance energy transfer (FRET)-melting experiments, PorphySQ was shown to possess enhanced quadruplex-interacting properties thanks to the presence of four positively charged (PNA)G residues that improve its electrostatic interactions with the binding site of both DNA and RNA quadruplexes (i.e., their negatively charged and accessible G-quartets), thereby making (PNA)PorphySQ an interesting prototype of a multitarget ligand. Both the chemical stability and water solubility of (PNA)PorphySQ are improved over the non-PNA derivative (PorphySQ), which are desirable properties for drug development, and while improvements remain to be made, this ligand is a promising lead for the further development of multitarget G-quadruplex ligands.

A new series of gold(I) N-heterocyclic carbene (NHC) complexes based on xanthine ligands have been synthesized and characterized by mass spectrometry, NMR, and X-ray diffraction. The compounds have been tested for their antiproliferative properties in human cancer cells and nontumorigenic cells in vitro, as well as for their toxicity in healthy tissues ex vivo. The bis-carbene complex [Au(caffein-2-ylidene)(2)] [BF4] (complex 4) appeared to be selective for human ovarian cancer cell lines and poorly toxic in healthy organs. To gain preliminary insights into their actual mechanism of action, two biologically relevant in cellulo targets were studied, namely, DNA (more precisely a higher-order DNA structure termed G-quadruplex DNA that plays key roles in oncogenetic regulation) and a pivotal enzyme of the DNA damage response (DDR) machinery (poly-(adenosine diphosphate (ADP)-ribose) polymerase 1 (PARP-1), strongly involved in the cancer resistance mechanism). Our results indicate that complex 4 acts as an efficient and selective G-quadruplex ligand while being a modest PARP-1 inhibitor (i.e., poor DDR impairing agent) and thus provide preliminary insights into the molecular mechanism that underlies its antiproliferative behavior.

Catalysis of chemical reactions is crucial for both chemical industry and research. However, scientists are not the first ones to use catalysts in their laboratory. In fact, they are also essential for nature which designs plenty of biocatalysts, playing a pivotal role in living systems. For a long time, it was thought that only enzymes had this property. However, since the beginning of the 1980s, it is known that ribonucleic acids (also termed RNA) can acquire this ability, making them compulsory for key reactions (e.g., for the translation of messenger RNA in the ribosome). Based on that, chemists designed several synthetic DNA catalysts (termed DNAzymes) for a large variety of reactions and applications. Among the DNA structures used, G-quadruplexes are guanine-rich noncanonical DNA structures (i.e., differing from duplex DNA) composed of native G-quartets and particularly interesting for their ability to catalyze reactions of peroxidation. This peroxidase-mimicking system found plenty of applications detailed in this chapter. Moreover, optimizations of experimental conditions are also discussed and highlight the versatility and easy-to-use characteristics of G-quadruplexes DNA. Also, synthetic G-quartets, mainly TASQ (for template-assembled synthetic G-quartets), developed by chemists showed their ability to mimic G-quadruplexes, thanks to the presence of a G-quartet. Thus, synthetic G-quartets proved their capability to catalyze peroxidase-mimicking reactions, and these new exciting nature-mimicking catalytic systems are presented in detail in this chapter.

The targeting of higher-order DNA structures has been thoroughly developed with G-quadruplex DNA but not with other structures like branched DNA (also known as DNA junctions). Because these alternative higher-order DNA architectures might be of high biological relevance, we implemented a high-throughput version of the FRET melting assay that enabled us to map the interactions of a candidate with four different DNA structures (duplex- and quadruplex DNA, three- and four-way junctions) in a rapid and reliable manner. We also introduce a novel index, the BONDS (branched and other noncanonical DNA selectivity) index, to conveniently quantify this differential affinity.

Since the initial discovery of the catalytic capability of short DNA fragments, this peculiar enzyme-like property (termed DNAzyme) has continued to garner much interest in the scientific community because of the virtually unlimited applications in developing new molecular devices. Alongside the exponential rise in the number of DNAzyme applications in the last past years, the search for convenient ways to improve its overall efficiency has only started to emerge. Credence has been lent to this strategy by the recent demonstration that the quadruplex-based DNAzyme proficiency can be enhanced by ATP supplements. Herein, we have made a further leap along this path, trying first of all to decipher the actual DNAzyme catalytic cycle (to gain insights into the steps ATP may influence), and subsequently investigating in detail the influence of all the parameters that govern the catalytic efficiency. We have extended this study to other nucleotides and quadruplexes, thus demonstrating the versatility and broad applicability of such an approach. The defined exquisitely efficient DNAzyme protocols were exploited to highlight the enticing advantages of this method via a 96-well plate experiment that enables the detection of nanomolar DNA concentrations in real-time with the naked-eye (see movie as Supplementary Data).

Recent years have witnessed a tremendous increase in the biotechnological applications of nucleic acid-based nanotools. Beyond their biological relevance, nucleobases have indeed found new scopes of applications in bionanotechnology, which are expanding nowadays at an accelerated pace. Among the four canonical nucleobases (adenine, guanine, cytosine and thymine), guanine is certainly the most useful and used base, thanks to its versatile H-bond donating/accepting properties that make it suitable for being involved in various assemblies ranging from base-pairs to base-quartets. Here, we would like to report on an innovative guanine-based molecular tool named Tb. Pyro-DOTASQ: this metal complex has a sophisticated chemical structure that allows formation of an intramolecular G-quartet upon interaction with alternative secondary structures known as G-quadruplexes. This target-promoted molecular switch triggers a luminescence response that would permit the use of Tb. Pyro-DOTASQ to search and detect quadruplex-forming DNA and RNA sequences: its unique design indeed allows it i) to create specific interaction with quadruplexes, ii) to provide an easily readable luminescent output to monitor this association and iii) to be readily immobilized on graphene surface, thus making Tb. Pyro-DOTASQ a high-value molecular device. Results obtained in the course of in-depth biophysical analyses raise questions about the actual supramolecular structure of Tb. Pyro-DOTASQ: these results thus shed a bright light on the care that must be exercised when using intricate molecular architectures to construct elaborated supramolecular metal complexes.

International audience

Amyloid supramolecular assemblies have found widespread exploitation as ordered nanomaterials in a range of applications from materials science to biotechnology. New strategies are, however, required for understanding and promoting mature fibril formation from simple monomer motifs through easy and scalable processes. Noncovalent interactions are key to forming and holding the amyloid structure together. On the other hand, the halogen bond has never been used purposefully to achieve control over amyloid self-assembly. Here we show that single atom replacement of hydrogen with iodine, a halogen-bond donor, in the human calcitonin-derived amyloidogenic fragment DFNKF results in a super-gelator peptide, which forms a strong and shape-persistent hydrogel at 30-fold lower concentration than the wild-type pentapeptide. This is remarkable for such a modest perturbation in structure. Iodination of aromatic amino acids may thus develop as a general strategy for the design of new hydrogels from unprotected peptides and without using organic solvents., Amyloid assemblies lie at the heart of many physiological functions, as well as being the cause of numerous diseases. Here, the authors subtly modify wild-type pentapeptides with halides, and discover that the new halogen bonding interactions have a remarkable influence on their physical properties.

The structure of the double helix of deoxyribonucleic acid (DNA, also called duplex-DNA) was elucidated sixty years ago by Watson, Crick, Wilkins and Franklin. Since then, DNA has continued to hold a fascination for researchers in diverse fields including medicine and nanobiotechnology. Nature has indeed excelled in diversifying the use of DNA: beyond its canonical role of repository of genetic information, DNA could also act as a nanofactory able to perform some complex catalytic tasks in an enzyme-mimicking manner. The catalytic capability of DNA was termed DNAzyme; in this context, a peculiar DNA structure, a quadruple helix also named quadruplex-DNA, has recently garnered considerable interest since its autonomous catalytic proficiency relies on its higher-order folding that makes it suitable to interact efficiently with hemin, a natural cofactor of many enzymes. Quadruplexes have thus been widely studied for their hemoprotein-like properties, chiefly peroxidase-like activity, i.e., their ability to perform hemin-mediated catalytic oxidation reactions. Recent literature is replete with applications of quadruplex-based peroxidase-mimicking DNAzyme systems. Herein, we take a further leap along the road to biochemical applications, assessing the actual efficiency of catalytic quadruplexes for the detection of picomolar levels of surface-bound analytes in an enzyme-linked immunosorbent (ELISA)-type assay. To this end, we exploit an innovative strategy based on the functionalization of DNA by a multitasking platform named RAFT (for regioselectivity addressable functionalized template), whose versatility enables the grafting of DNA whatever its nature (duplex-DNA, quadruplex-DNA, etc.). We demonstrate that the resulting biotinylated RAFT/quadruplex systems indeed acquire catalytic properties that allow for efficient luminescent detection of picomoles of surface-bound streptavidin. We also highlight some of the pitfalls that have to be faced during optimization, notably demonstrating that highly optimized experimental conditions can make DNA pre-catalysts catalytically competent whatever their secondary structures.

Natural G-quartets, a cyclic and coplanar array of four guanine res- idues held together through a Watson- Crick/Hoogsteen hydrogen-bond net- work, have received recently much at- tention due to their involvement in G- quadruplex DNA, an alternative higher-order DNA structure strongly suspected to play important roles in key cellular events. Besides this, syn- thetic G-quartets (SQ), which artificial- ly mimic native G-quartets, have also been widely studied for their involve- ment in nanotechnological applications (i.e., nanowires, artificial ion channels, etc.). In contrast, intramolecular syn- thetic G-quartets (iSQ), also named template-assembled synthetic G-quar- tets (TASQ), have been more sparingly investigated, despite a technological potential just as interesting. Herein, we report on a particular iSQ named PNA DOTASQ, which demonstrates very interesting properties in terms of DNA and RNA interaction (notably its selec- tive recognition of quadruplexes ac- cording to a bioinspired process) and catalytic activities, through its ability to perform peroxidase-like hemin-mediat- ed oxidations either in an autonomous fashion (i.e., as pre-catalyst for TASQ- zyme reactions) or in conjunction with quadruplex DNA (i.e., as enhancing agents for DNAzyme processes). These results provide a solid scientific basis for TASQ to be used as multitasking tools for bionanotechnological applica- tions.

The capability of DNA to acquire enzyme-like properties has led to the emergence of the so-called DNAzyme field; herein, we take a further leap along this nature-inspired road, demonstrating that a template assembled synthetic G-quartet (TASQ) can act as a pre-catalyst for catalytic peroxidase-mimicking oxidation reactions, whatever its nature (guanine or guanosine-based G-quartets), in an ATP-dependent manner, thereby bringing this bioinspired TASQzyme process even closer to nature.

An unprecedented strategy to control the quadruplex- vs duplex-DNA selectivity of a ligand is reported. We designed a compound whose structure can rearrange when it interacts with a G-quadruplex, thereby controlling its affinity. Thus, the first "smart G-quadruplex ligand" is reported, since this ligand experiences a structural change in the presence of quadruplexes but not in the presence of duplexes, ensuring a high level of quadruplex selectivity.

Template-assembled synthetic G-quartet (TASQ) has been reported recently as a G-quadruplex ligand interacting with DNA according to an unprecedented, nature-inspired ‘like likes like’ approach, based on the association between two G-quartets, one being native (quadruplex) and the other one artificial (ligand). Herein, a novel TASQ-based ligand is designed, synthesized and its quadruplex-recognition properties are evaluated in vitro: PorphySQ (for porphyrin-templated synthetic G-quartet) displays enhanced quadruplex recognition properties as compared to the very first reported prototype (DOTASQ, for DOTA-templated synthetic G-quartet), since the porphyrin template insures a more stable intramolecular G-quartet fold due to self-stabilizing interactions that may take place intramolecularly between the porphyrin ring and the formed G-quartet.

Three-way junction DNA (TWJ-DNA, also known as 3WJ-DNA) is an alternative secondary DNA structure comprised of three duplex-DNAs that converge towards a single point, termed the branch point. This point is characterized by unique geometrical properties that make its specific targeting by synthetic small-molecules possible. Such a targeting has already been demonstrated in the solid state but not thoroughly biophysically investigated in solution. Herein, a set of simple biophysical assays has been developed to identify TWJ-specific small-molecule ligands; these assays, inspired by the considerable body of work that has been reported to characterize the interactions between small-molecules and other higher-order DNA (notably quadruplex-DNA), have been calibrated with a known non-specific DNA binder (the porphyrin TMPyP4) and validated via the study of a small series of triazacyclononane (TACN) derivatives (metal-free or not) and the identification of a fairly-affinic and exquisitely TWJ-selective candidate (a TACN-quinoline construct named TACN-Q).

The end of human telomeres is comprised of a long G-rich single-stranded DNA (known as 3'-overhang) able to adopt an unusual three-dimensional "beads-on-the-string" organization made of consecutively stacked G-quadruplex units (so-called quadruplex multimers). It has been widely demonstrated that, upon interaction with hemin, discrete quadruplexes acquire peroxidase-mimicking properties, oxidizing several organic probes in H(2)O(2)-rich conditions; this property, known as DNAzyme, has found tens of applications in the last two decades. However, little is known about the DNAzyme activity of multimeric quadruplexes; this is an important question to address, especially in light of recent reports that exploit the DNAzyme process to optically assess the activity of an enzyme that elongates the telomeric overhang, the telomerase. Herein, we thoroughly investigate the DNAzyme activity of long telomeric fragments, with a particular focus on both the nature of the hemin/multimeric quadruplex interactions and the putative higher-order fold of the studied fragments; in light of our results, we also propose possible ways that may be followed to improve the use of DNAzyme to evaluate the telomerase activity.

International audience

DOTASQ (for DOTA-templated Synthetic G-quartet) is the first prototype of nature-inspired G-quadruplex ligand: its design, founded on a possible intramolecular G-quartet formation, enables it to interact with G-quadruplex DNA via an unprecedented nature-mimicking binding mode, based on the association between two G-quartets, one being native (quadruplex) and the other one artificial (ligand).