Access to High-Purity 7m G-cap RNA in Substantial Quantities by a Convenient All-Chemical Solid-Phase Method.

Given the importance of mRNA with 5'-cap, easy access to RNA substrates with different 7m G caps, of high quality and in large quantities is essential to elucidate the roles of RNA and the regulation of underlying processes. In addition to existing synthetic routes to 5'-cap RNA based on enzymatic, chemical or chemo-enzymatic methods, we present here an all-chemical method for synthetic RNA capping. The novelty of this study lies in the fact that the capping reaction is performed on solid-support after automated RNA assembly using commercial 2'-O-propionyloxymethyl ribonucleoside phosphoramidites, which enable final RNA deprotection under mild conditions while preserving both 7m G-cap and RNA integrity. The capping reaction is efficiently carried out between a 5'-phosphoroimidazolide RNA anchored on the support and 7m GDP in DMF in the presence of zinc chloride. Substantial amounts of 7m G-cap RNA (from 1 to 28 nucleotides in length and of any sequence with or without internal methylations) containing various cap structures (7m GpppA, 7m GpppAm , 7m Gpppm6 A, 7m Gpppm6 Am , 7m GpppG, 7m GpppGm ) were obtained with high purity after IEX-HPLC purification. This capping method using solid-phase chemistry is convenient to perform and provides access to valuable RNA substrates as useful research tools to unravel specific issues regarding cap-related processes.

AT-752 targets multiple sites and activities on the Dengue virus replication enzyme NS5.

AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2'-methyl-2'-fluoro guanosine 5'-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2'-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 Å resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2'-O but not N7-methylation activity. AT-9010 is discriminated ∼10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibition through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC50 ≈ 0.50 µM), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses.

The methyltransferase domain of the Respiratory Syncytial Virus L protein catalyzes cap N7 and 2'-O-methylation.

Respiratory syncytial virus (RSV) is a negative sense single-stranded RNA virus and one of the main causes of severe lower respiratory tract infections in infants and young children. RSV RNA replication/transcription and capping are ensured by the viral Large (L) protein. The L protein contains a polymerase domain associated with a polyribonucleotidyl transferase domain in its N-terminus, and a methyltransferase (MTase) domain followed by the C-terminal domain (CTD) enriched in basic amino acids at its C-terminus. The MTase-CTD of Mononegavirales forms a clamp to accommodate RNA that is subsequently methylated on the cap structure and depending on the virus, on internal positions. These enzymatic activities are essential for efficient viral mRNA translation into proteins, and to prevent the recognition of uncapped viral RNA by innate immunity sensors. In this work, we demonstrated that the MTase-CTD of RSV, as well as the full-length L protein in complex with phosphoprotein (P), catalyzes the N7- and 2'-O-methylation of the cap structure of a short RNA sequence that corresponds to the 5' end of viral mRNA. Using different experimental systems, we showed that the RSV MTase-CTD methylates the cap structure with a preference for N7-methylation as first reaction. However, we did not observe cap-independent internal methylation, as recently evidenced for the Ebola virus MTase. We also found that at µM concentrations, sinefungin, a S-adenosylmethionine analogue, inhibits the MTase activity of the RSV L protein and of the MTase-CTD domain. Altogether, these results suggest that the RSV MTase domain specifically recognizes viral RNA decorated by a cap structure and catalyzes its methylation, which is required for translation and innate immune system subversion.

Modified Galacto- or Fuco-Clusters Exploiting the Siderophore Pathway to Inhibit the LecA- or LecB-Associated Virulence of Pseudomonas aeruginosa.

Galacto- and fuco-clusters conjugated with one to three catechol or hydroxamate motifs were synthesised to target LecA and LecB lectins of Pseudomonas aeruginosa (PA) localised in the outer membrane and inside the bacterium. The resulting glycocluster-pseudosiderophore conjugates were evaluated as Trojan horses to cross the outer membrane of PA by iron transport. The data suggest that glycoclusters with catechol moieties are able to hijack the iron transport, whereas those with hydroxamates showed strong nonspecific interactions. Mono- and tricatechol galactoclusters (G1C and G3C) were evaluated as inhibitors of infection by PA in comparison with the free galactocluster (G0). All of them exhibited an inhibitory effect between 46 to 75 % at 100 µM, with a higher potency than G0. This result shows that LecA localised in the outer membrane of PA is involved in the infection mechanism.

Solid Supports for the Synthesis of 3'-Aminooxy Deoxy- or Ribo-oligonucleotides and Their 3'-Conjugation by Oxime Ligation.

Mono- and triethylene glycol aminooxy derivatives were reacted with levulinic acid, protected with dimethoxytrityl, and immobilized on solid support. The resulting solid supports were used for elongation of oligonucleotides. Then, a mild ammonia treatment was applied to remove the oligonucleotide protecting groups, followed by a treatment with 50 mM methoxyamine at pH 4.2, releasing the 3'-aminooxy oligonucleotides by an oxime exchange reaction. The resulting 3'-aminooxy deoxy- or ribo-oligonucleotides were conjugated to various ketones and aldehydes with high efficiency by oxime ligation.

Screening of a Library of Oligosaccharides Targeting Lectin LecB of Pseudomonas Aeruginosa and Synthesis of High Affinity Oligoglycoclusters.

The Gram negative bacterium Pseudomonas aeruginosa (PA) is an opportunistic bacterium that causes severe and chronic infection of immune-depressed patients. It has the ability to form a biofilm that gives a selective advantage to the bacteria with respect to antibiotherapy and host defenses. Herein, we have focused on the tetrameric soluble lectin which is involved in bacterium adherence to host cells, biofilm formation, and cytotoxicity. It binds to l-fucose, d-mannose and glycan exposing terminal fucose or mannose. Using a competitive assay on microarray, 156 oligosaccharides and polysaccharides issued from fermentation or from the biomass were screened toward their affinity to LecB. Next, the five best ligands (Lewisa, Lewisb, Lewisx, siayl-Lewisx and 3-fucosyllactose) were derivatized with a propargyl aglycon allowing the synthesis of 25 trivalent, 25 tetravalent and 5 monovalent constructions thanks to copper catalyzed azide alkyne cycloaddition. The 55 clusters were immobilized by DNA Directed immobilization leading to the fabrication of a glycocluster microarray. Their binding to LecB was studied. Multivalency improved the binding to LecB. The binding structure relationship of the clusters is mainly influenced by the carbohydrate residues. Molecular simulations indicated that the simultaneous contact of both binding sites of monomer A and D seems to be energetically possible.

Design and Synthesis of Galactosylated Bifurcated Ligands with Nanomolar Affinity for Lectin LecA from Pseudomonas aeruginosa.

Lectin A (LecA) from Pseudomonas aeruginosa is an established virulence factor. Glycoclusters that target LecA and are able to compete with human glycoconjugates present on epithelial cells are promising candidates to treat P. aeruginosa infection. A family of 32 glycodendrimers of generation 0 and 1 based on a bifurcated bis-galactoside motif have been designed to interact with LecA. The influences both of the central multivalent core and of the aglycon of these glycodendrimers on their affinity toward LecA have been evaluated by use of a microarray technique, both qualitatively for rapid screening of the binding properties and also quantitatively (Kd ). This has led to high-affinity LecA ligands with Kd values in the low nanomolar range (Kd =22 nm for the best one).

Toward the Rational Design of Galactosylated Glycoclusters That Target Pseudomonas aeruginosa Lectin A (LecA): Influence of Linker Arms That Lead to Low-Nanomolar Multivalent Ligands.

Anti-infectious strategies against pathogen infections can be achieved through antiadhesive strategies by using multivalent ligands of bacterial virulence factors. LecA and LecB are lectins of Pseudomonas aeruginosa implicated in biofilm formation. A series of 27 LecA-targeting glycoclusters have been synthesized. Nine aromatic galactose aglycons were investigated with three different linker arms that connect the central mannopyranoside core. A low-nanomolar (Kd =19 nm, microarray) ligand with a tyrosine-based linker arm could be identified in a structure-activity relationship study. Molecular modeling of the glycoclusters bound to the lectin tetramer was also used to rationalize the binding properties observed.

Membrane interactions of synthetic peptides with antimicrobial potential: effect of electrostatic interactions and amphiphilicity.

Cationic antimicrobial peptides are considered promising candidates to complement currently used antibiotics, which are less effective against increasingly resistant pathogens. To determine the mechanism of action of these peptides, a better understanding of each molecular determinant involved in their membrane interactions is of great importance. In this study, we have focused on the role of electrostatic interactions and amphiphilicity on the membrane interactions since the large majority of natural antimicrobial peptides are cationic. Therefore, cationic and anionic peptides have been prepared based on a model 14-mer peptide. The latter is a synthetic peptide composed of ten leucines and four phenylalanines, which are modified by the addition of the crown ether. Infrared spectroscopy results indicate that the position of substitution is the main determinant involved in the secondary structure adopted by the peptides, and not the charge of the substituted residues. Fluorescence vesicle leakage assays indicate, however, differences between the ability of cationic and anionic peptides to induce calcein release in zwitterionic and anionic lipid vesicles, suggesting an importance of electrostatic interactions and repulsions. Finally, (31)P NMR results indicate that the vesicle morphologies is not significantly affected by the interactions with both cationic and anionic peptides but that their effect on lipid bilayers is mainly determined by their secondary structure. This study therefore indicates that the membrane interactions of model 14-mer peptides are mainly governed by their secondary structure, which depends on the position of substitution, and not the charge of the residues.

Investigation of the mechanism of action of novel amphipathic peptides: insights from solid-state NMR studies of oriented lipid bilayers.

We have investigated in the present study the effect of both non-selective and selective cationic 14-mer peptides on the lipid orientation of DMPC bilayers by (31)P solid-state nuclear magnetic resonance (NMR) spectroscopy. Depending on the position of substitution, these peptides adopt mainly either an α-helical structure able to permeabilize DMPC and DMPG vesicles (non-selective peptides) or an intermolecular ß-sheet structure only able to permeabilize DMPG vesicles (selective peptides). Several systems have been investigated, namely bilayers mechanically oriented between glass plates as well as bicelles oriented with their normal perpendicular or parallel to the external magnetic field. The results have been compared with spectral simulations with the goal of elucidating the difference in the interaction of these two types of peptides with zwitterionic lipid bilayers. The results indicate that the perturbation induced by selective peptides is much greater than that induced by non-selective peptides in all the lipid systems investigated, and this perturbation has been associated to the aggregation of the selective ß-sheet peptides in these systems. On the other hand, the oriented lipid spectra obtained in the presence of non-selective peptides suggest the presence of toroidal pores. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Determining the mode of action involved in the antimicrobial activity of synthetic peptides: a solid-state NMR and FTIR study.

We have previously shown that leucine to lysine substitution(s) in neutral synthetic crown ether containing 14-mer peptide affect the peptide structure and its ability to permeabilize bilayers. Depending on the substitution position, the peptides adopt mainly either a α-helical structure able to permeabilize dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) vesicles (nonselective peptides) or an intermolecular ß-sheet structure only able to permeabilize DMPG vesicles (selective peptides). In this study, we have used a combination of solid-state NMR and Fourier transform infrared spectroscopy to investigate the effects of nonselective α-helical and selective intermolecular ß-sheet peptides on both types of bilayers. (31)P NMR results indicate that both types of peptides interact with the headgroups of DMPC and DMPG bilayers. (2)H NMR and Fourier transform infrared results reveal an ordering of the hydrophobic core of bilayers when leakage is noted, i.e., for DMPG vesicles in the presence of both types of peptides and DMPC vesicles in the presence of nonselective peptides. However, selective peptides have no significant effect on the ordering of DMPC acyl chains. The ability of these 14-mer peptides to permeabilize lipid vesicles therefore appears to be related to their ability to increase the order of the bilayer hydrophobic core.

Revisiting peptide amphiphilicity for membrane pore formation.

It has previously been shown that an amphipathic de novo designed peptide made of 10 leucines and four phenylalanines substituted with crown ethers induces vesicle leakage without selectivity. To gain selectivity against negatively charged dimyristoylphosphatidylglycerol (DMPG) bilayers, one or two leucines of the peptide were substituted with positively charged residues at each position. All peptides induce significant calcein leakage of DMPG vesicles. However, some peptides do not induce significant leakage of zwitterionic dimyristoylphosphatidylcholine vesicles and are thus active against only bacterial model membranes. The intravesicular leakage is induced by pore formation instead of membrane micellization. Nonselective peptides are mostly helical, while selective peptides mainly adopt an intermolecular ß-sheet structure. This study therefore demonstrates that the position of the lysine residues significantly influences the secondary structure and bilayer selectivity of an amphipathic 14-mer peptide, with ß-sheet peptides being more selective than helical peptides.