In addition to their role in protein synthesis by the ribosomes, aminoacyl-tRNAs participate in various metabolic pathways as a source of ester-activated amino acids. Among the tRNA-dependent aminoacyl transferases, enzymes of the Fem family catalyze an essential step of peptidoglycan synthesis in pathogenic bacteria and are considered as attractive targets for the development of novel antibiotics. FemX, the model enzyme of the family, transfers L-Ala from Ala-tRNA to the epsilon-amino group of L-Lys in the peptidoglycan precursor UDP-MurNAc-pentapeptide (UM5K). The crystal structures of the apo-enzyme and of a UM5K-FemX complex have been determined but co-crystallization with Ala-tRNA has not been obtained. We propose to develop the semi-synthesis of highly modified aminoacyl-tRNAs and bi-substrates to explore the catalytic mechanism of FemX. We will synthesize chemical probes that will specifically interact with FemX and its substrates. Azides and alkynes will be introduced into the tRNA and in UM5K, respectively. The Huisgen-Sharpless Cu(I)-catalyzed cycloaddition reaction will afford bi-substrates containing the tRNA covalently linked to the peptidoglycan precursor. In parallel, the active center of FemX will be used to catalyze the same reaction. By this approach, we will obtain molecules suitable for co-crystallization with FemX. Because the in situ generated reaction products are likely to trap a single conformational state of FemX corresponding to the catalytically active form of the enzyme, this approach is likely to be more powerful than the conventional crystallogenesis screens made with the substrates or products of the reaction. Phospho-derivatives of the tRNA will be synthesized to mimic the putative tetrahedral intermediate resulting from the intramolecular nucleophilic attack of the carbonyl of Ala-tRNA by the vicinal ribose hydroxyl. These phospho-derivatives will also be used to trap a relevant conformation of the enzyme that allows the trans-acylation reaction of the amino acid between the 2’ and 3’ positions of Ala-tRNA to occur within the active site. The enzyme-catalyzed cycloaddition reaction will be further investigated both to identify inhibitors of FemX and to decipher the mechanism of the enzyme-assisted catalyzed cycloaddition reaction, which is poorly understood. We will assess and compare the contributions of substrate binding and substrate activation (i) in the CuI- and FemX-catalyzed cycloaddition reactions using the functionalized substrates, and (ii) in the amino acid transfer reaction catalyzed by FemX with the “natural” substrates. The information gathered on the catalytic mechanism of FemX and on the structure of its active site should provide the critical information for the rationale design of drugs active on Fem transferases from pathogenic bacteria such as methicillin-resistant staphylococci. The approach will be of broad application in RNA biology.

Genome editing mediated by CRISPR-Cas9 has shown great promise for the treatment of retinal dystrophies (RD). Currently, adeno-associated viruses are the most widely used vectors for retinal gene therapies but their small packaging capacity and permanent transgene expression makes them suboptimal for CRISPR-Cas delivery. Transient delivery of Cas9 protein and its guide RNA as ribonucleoprotein (RNP) complexes have been reported in the retinal pigment epithelium (RPE) and into the inner ear cells in vivo. Members of our consortium investigated transient delivery of either Cas9 mRNA or Cas9 RNP into the retinal pigment epithelium (RPE) and photoreceptors as these are the target cells for most prevalent inherited retinal degenerations. Cas9 mRNA complexed with different lipid or peptide vectors led to low rates of indels at the target sequence in vivo and mostly in the RPE. Major changes to the delivery system are needed to increase the efficiency of gene editing, and finally, safety and cost of the therapeutic approach need to be taken into account when designing such vector systems. Our objectives are to address some of these challenges in this project by developing a novel polymer based non-viral carrier for in vivo targeted delivery of CRISPR-based RNP complexes; and to assess the efficacy and safety of our novel delivery approach in a mouse model of retinal dystrophy.

MARCEL 2.0 proposes an original concept where metallic nanocatalysts (Au, Ag, Cu nanoparticles) are functionalized with molecular hosting cavities bearing metallic complexes in order to direct the reactivity in ORR and CO2RR electrocatalysis. Both of these processes are complex and require efficient and highly selective catalysts as the metalloenzymes. Inspired form such biological systems whose functioning is based on confinement and supramolecular effects, MARCEL seeks the rational control of forming and stabilizing intermediates to guide specific reaction pathways. This innovative design that relies on surface supramolecular effects will be further combined to plasmonic effect in order to enhance the electrocatalytic performance. The reactivity and interfacial phenomena will be thoroughly investigated by combining experimental (electrochemistry, in situ spectroscopies) and computational analyses.

The structure of RNA molecules and their complexes are crucial for understanding biology. Notorious examples of large RNAs include the genomes of RNA viruses (Influenza, HIV, Chikungunya, SARS-CoV2...), whose lengths exceed the current capabilities of predictive computational methods, as well as high-res experimental structural techniques. In the INSSANE project, we will develop integrated experimental protocols, together with efficient computational methods for the structural modeling of large RNAs. We will accurately probe and predict the genomic RNA architectures of, bio-medically relevant, viruses. The scope of applicability of our methodologies in bioinformatics will extend beyond viruses, and could be used to model the structure of other large RNAs (lncRNAS, Introns). Towards that goal, we will introduce a novel protocol, named SHAPE-Cut, to streamline the probing of large RNAs. SHAPE-Cut will measure position-specific solvent accessibility by combining novel chemistry and long-read sequencing. In comparison to existing protocols, we expect SHAPE-Cut to avoid typical biases, be easier to implement, and provide increased accuracy, when coupled with specific data analyses and computational methods. We will combine the complementary data of crosslinking and probing experiments: the former reveals long-range interactions, while the latter, through accessibility profiles, has been shown to greatly improve the prediction of local structures. We will implement a recent crosslinking protocol and use its data in index-based genome-wide search of thermodynamically stable RNA-RNA interactions. Then, we devise an integrative structure prediction method that combines SHAPE reactivity, long-range interactions, homology, and thermodynamic stability. Finally, a novel visualization tool will represent genome-scale RNAs and streamline the interdisciplinary dialogue. Algorithmic hurdles will be overcome to improve the processing of sequencing data produced by RNA structure-targeting experiments. All modern RNA probing protocols are based on sequencing technologies, and reveal structural information indirectly, through an alteration that is observable at the RNA sequence level (mutations, stops/cut). However, the crucial mapping of primary sequencing data has received relatively scarce attention in the context of probing techniques, despite specific challenges (chimeric reads, informative errors/stops) having been identified at the root of biases and technical artifacts. We will tailor mapping to our protocols, and develop data structures and indexing techniques to fully exploit sequencing data to its fullest extent. We will also inform mapping by predicted accessibility, e.g. to disambiguate the mapping of erroneous (but probably informative) reads. Beyond increasing mappability, we will deconvolute isoforms/subgenomes, which are known to occur in viral genomes. Our final integrative structure modeling method will consider evolutionary information, and will be formulated as a Maximum-Independent-Set (MIS) graph problem for a conflict graph including both alternative local structure and long-range interactions. We will implement a Fixed Parameter Tractable algorithm based on the treewidth to produce a model with maximal support and thermodynamic stability. By including experts in bioinformatics of RNA structure, sequence analysis, biochemistry, and organic chemistry, our consortium is uniquely positioned to address the timely challenges tackled in the project. Its implementation requires a combination of expertise from traditionally distinct areas of bioinformatics, namely combinatorial structure prediction and high-throughput sequencing analysis. Its synergies will build on existing pairwise collaborations and will streamline the communication between partners representing complementary perspectives on RNA as an object of study.

The loss of functional ? cells is the root cause for the development of diabetes. Therapies aimed at the replenishment of the pancreatic ? cell are among the most promising strategies to treat diabetes. In this project, we will investigate the effects of new selective Dyrk1A inhibitors developed by a French Biotech, on the proliferation of human ? cells, and the regeneration of ? cell in relevant preclinical models of type 2 diabetes. This translational research program will pave the way for the rapid clinical evaluation of these highly advanced Dyrk1A inhibitors in diabetic patients.