Matthieu Sainlos

Self-assembled lamellar complexes of siRNA with lipidic aminoglycoside derivatives promote efficient siRNA delivery and interference

RNA interference requires efficient delivery of small double-stranded RNA molecules into the target cells and their subsequent incorporation into RNA-induced silencing complexes. Although current cationic lipids commonly used for DNA transfection have also been used for siRNA transfection, a clear need still exists for better siRNA delivery to improve the gene silencing efficiency. We synthesized a series of cationic lipids characterized by head groups bearing various aminoglycosides for specific interaction with RNA. siRNA complexation with such lipidic aminoglycoside derivatives exhibited three lipid/siRNA ratio-dependent domains of colloidal stability. Fluorescence and dynamic light-scattering experiments showed that cationic lipid/siRNA complexes were formed at lower charge ratios, exhibited a reduced zone of colloidal instability, and had smaller mean diameters compared with our previously described guanidinium-based cationic lipids. Cryo-transmission electron microscopy and x-ray-scattering experiments showed that, although the final in toto morphology of the lipid/siRNA complexes depended on the aminoglycoside type, there was a general supramolecular arrangement consisting of ordered lamellar domains with an even spacing of 67 Å. The most active cationic lipid/siRNA complexes for gene silencing were obtained with 4,5-disubstituted 2-deoxystreptamine aminoglycoside derivatives and were characterized by the siRNA being entrapped in small particles exhibiting lamellar microdomains corresponding to siRNA molecules sandwiched between the lipid bilayers. These results clearly show that lipidic aminoglycoside derivatives constitute a versatile class of siRNA nanocarriers allowing efficient gene silencing.

Dynamic and specific interaction between synaptic NR2-NMDA receptor and PDZ proteins

The relative content of NR2 subunits in the NMDA receptor confers specific signaling properties and plasticity to synapses. However, the mechanisms that dynamically govern the retention of synaptic NMDARs, in particular 2A-NMDARs, remain poorly understood. Here, we investigate the dynamic interaction between NR2 C termini and proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) scaffold proteins at the single molecule level by using high-resolution imaging. We report that a biomimetic divalent competing ligand, mimicking the last 15 amino acids of NR2A C terminus, specifically and efficiently disrupts the interaction between 2A-NMDARs, but not 2B-NMDARs, and PDZ proteins on the time scale of minutes. Furthermore, displacing 2A-NMDARs out of synapses lead to a compensatory increase in synaptic NR2B-NMDARs, providing functional evidence that the anchoring mechanism of 2A- or 2B-NMDARs is different. These data reveal an unexpected role of the NR2 subunit divalent arrangement in providing specific anchoring within synapses, highlighting the need to study such dynamic interactions in native conditions.

A general screening strategy for peptide-based fluorogenic ligands: Probes for dynamic studies of PDZ domain-mediated interactions

A systematic and general approach for identifying efficient probes for class I PDZ domains based on environment-sensitive chromophores is presented. A series of peptides derived from the C-terminal sequence of Stargazin was first used with PDZ domains of PSD-95 and Shank3 to identify the optimal position and linker length for the 4-DMAP chromophore. The results were applied to well-characterized ligand sequences for each set of domains to generate high affinity probes that retain their native sequence specificity and yield remarkable fluorescence increases upon binding. These probes constitute efficient tools to study the dynamics and regulatory mechanisms of PDZ domain-mediated interactions.

Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin

The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures.

Caged Mono- and Divalent Ligands for Light-Assisted Disruption of PDZ Domain-Mediated Interactions

We report a general method for light-assisted control of interactions of PDZ domain binding motifs with their cognate domains by the incorporation of a photolabile caging group onto the essential C-terminal carboxylate binding determinant of the motif. The strategy was implemented and validated for both simple monovalent and biomimetic divalent ligands, which have recently been established as powerful tools for acute perturbation of native PDZ domain-dependent interactions in live cells.

Tools for investigating peptide–protein interactions: peptide incorporation of environment-sensitive fluorophores through SPPS-based 'building block' approach

This protocol presents the synthesis and peptide incorporation of environment-sensitive fluorescent amino acids derived from the dimethylamino-phthalimide family. The procedure uses anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol by these authors. In this study, the corresponding fluorescent amino acids can be readily obtained in Fmoc-protected form for convenient use as building blocks in solid phase peptide synthesis (SPPS). The time required to complete the procedure depends on the size and the number of peptides targeted. Alternatively, the chromophores can be incorporated directly after SPPS via on-resin derivatization of peptides, which is an option described in a related protocol by these authors.

Tools for investigating peptide–protein interactions: peptide incorporation of environment-sensitive fluorophores via on-resin derivatization

This protocol presents the peptide incorporation of environment-sensitive fluorophores derived from the dimethylaminophthalimide family. The procedure utilizes anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol from these authors. In this protocol, the fluorophores are directly incorporated after solid-phase peptide synthesis (SPPS) via on-resin derivatization of peptides prepared using commercially available diamino acids, which are Alloc-protected on the side-chain amino group. The time required to complete the procedure depends on the size and number of peptides targeted. As an alternative to this approach, the corresponding fluorescent amino acids can be obtained in an Fmoc-protected form for convenient use as building blocks in SPPS. This option is described in a related protocol by these authors.

Inhibition of PDZ domain-mediated interactions.

Modulating protein-protein interactions constitutes a promising strategy both for the investigation of biological mechanisms and for developing new therapeutic approaches. Among the many types of inter-actions, PDZ domain-mediated interactions (PDMIs) have emerged over the last decade as attractive targets in the drug discovery field. Indeed, these small domains are involved in the regulation of many signaling pathways and possess structural properties which are favorable for the design of competing ligands. Herein, we describe the recent approaches developed to inhibit this class of protein-protein interactions.

Synthesis of anhydride precursors of the environment-sensitive fluorophores 4-DMAP and 6-DMN

This protocol describes the synthesis of cyclic anhydride precursors of the environment-sensitive fluorophores 4-dimethylaminophthalimide (4-DMAP) and 6-dimethylaminonaphthalimide (6-DMN). The condensation of these anhydrides with a primary amino group confers on molecules of interest solvatochromic properties. In particular, two strategies for the insertion of the chromophores into peptides are presented in two companion protocols. The anhydride syntheses can be completed on the gram scale in 2 d for the 4-DMAP precursor and 10–15 d for the 6-DMN precursor.

Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin

Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM)). This method is complementary to the previously described anti-GFP-nanobody/SNAP-tag strategies, with the main advantage being that it requires only a short 15-amino-acid tag, and can thus be used with proteins resistant to fusion with large tags and for multicolor imaging. The protocol requires standard molecular biology/biochemistry equipment, making it easily accessible for laboratories with only basic skills in cell biology and biochemistry. The production/purification/conjugation steps take ∼5 d, and labeling takes a few minutes to an hour.