Françoise Debart

Respiratory syncytial virus (RSV) causes severe respiratory infections, with viral replication occurring in cytoplasmic membraneless viral factories formed by liquid-liquid phase separation (LLPS). The interactions among the RSV nucleoprotein N, phosphoprotein P, transcription factor M2-1, and RNA drive these condensates. Here, we used a microfluidic PhaseScan platform, together with biochemical and cellular assays, to systematically characterize LLPS involving RSV proteins and RNA. We identified optimal concentrations of oligomeric N and P tetramers for condensate formation in vitro without crowding agents, demonstrated that monomeric N inhibits LLPS and revealed that M2-1 enhances condensate formation by increasing multivalency. Notably, we found that M2-1 preferentially binds 5′ capped RNA, distinguishing it from N, which binds uncapped RNA. These findings elucidate molecular determinants of RSV viral factory assembly and subcompartmentalization, providing insights into viral replication mechanisms and informing potential antiviral strategies targeting LLPS processes.

Respiratory syncytial virus (RSV) causes severe respiratory infections, with viral replication occurring in cytoplasmic membraneless viral factories formed by liquid-liquid phase separation (LLPS). The interactions between the RSV nucleoprotein N, phosphoprotein P, transcription factor M2-1, and RNA drive these condensates. Here we employed a microfluidic Phase Scan platform, together with biochemical and cellular assays to systematically characterise LLPS involving RSV proteins and RNA. We identified optimal stoichiometric ratios of N oligomers and P tetramers for condensate formation, demonstrated that monomeric N inhibits LLPS, and revealed that M2-1 enhances condensate formation by increasing multivalency. Notably, we discovered that M2-1 preferentially binds 5’ capped RNA, distinguishing it from N, which binds uncapped RNA. These findings elucidate molecular determinants of RSV viral factory assembly and subcompartmentalisation, providing insights into viral replication mechanisms and informing potential antiviral strategies targeting LLPS processes.

By applying the Liebeskind-Srogl cross-coupling reaction to pyrimidine nucleosides, a Pd(0)-catalyzed, Cu(I)-mediated process that effectively couples thioesters and boronic acids to form ketones, we previously described an efficient and easy access to 5'-C-acyl nucleosides. By reduction of the 5'-carbonyl group into a secondary alcohol, we report herein the synthesis of new 5'-C-aryl-5'-O-dimethoxytrityl-3'-O-phosphoramidite building blocks ready to be incorporated into oligonucleotides by automated synthesis. We describe the preparation of two sets of (R)- and (S)-diastereoisomers of 5'-C-phenylthymidine (^PhT) and 5'-C-pyrenylthymidine (^PyT), their conversion to phosphoramidites and incorporation into oligodeoxynucleotides (ODN). We also discuss the challenges faced by the synthesis of ^PyT-modified ODN, which required to modify the automated cycle to prevent backbone degradation in subsequent steps. The affinity and specificity of several 5'-C-aryl modified-ODN toward their complementary sequence were studied by UV-melting experiments. While thermal melting analysis failed to differentiate between matched and mismatched pairs, fluorescence measurements demonstrated that pyrenyl-modified ODN can effectively distinguish correct from incorrect base pairs. This highlights their potential use in the detection of genetic mutations through structural changes in DNA duplexes.

Original N -arylsulfonamide acyclic analogues of adenosine as bisubstrates of RNA cap guanine N 7-methyltransferase nsp14 of SARS-CoV-2 were prepared and some of them were barely active at 50 μM against this RNA cap methylation enzyme.

Nucleic acid photolithography is the only microarray fabrication process that has demonstrated chemical versatility accommodating any type of nucleic acid. The current approach to RNA microarray synthesis requires long coupling and photolysis times and suffers from unavoidable degradation postsynthesis. In this study, we developed a series of RNA phosphoramidites with improved chemical and photochemical protection of the 2′- and 5′-OH functions. In so doing, we reduced the coupling time by more than half and the photolysis time by a factor of 4. Sequence libraries that would otherwise take over 6 hours to synthesize can now be prepared in half the time. Degradation is substantially lowered, and concomitantly, hybridization signals can reach over seven times those of the previous state of the art. Under those conditions, high-density RNA microarrays and RNA libraries can now be synthesized at greatly accelerated rates. We also synthesized fluorogenic RNA Mango aptamers on microarrays and investigated the effect of sequence mutations on their fluorogenic properties.

SAH-derived bisubstrates of SARS-CoV-2 cap RNA N 7-methyltransferase were synthesized, and two adenosines with an N -arylsulfonamide core attached by an N -ethylthioether linker proved to be effective inhibitors in the submicromolar range.

Given the importance of mRNA with 5’‐cap, easy access to RNA substrates with different ^7mG 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 ^7mG‐cap and RNA integrity. The capping reaction is efficiently carried out between a 5’‐phosphoroimidazolide RNA anchored on the support and ^7mGDP in DMF in the presence of zinc chloride. Substantial amounts of ^7mG‐cap RNA (from 1 to 28 nucleotides in length and of any sequence with or without internal methylations) containing various cap structures (^7mGpppA, ^7mGpppA_m, ^7mGppp^m6A, ^7mGppp^m6A_m, ^7mGpppG, ^7mGpppG_m) 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.

Whereas BAIB-TEMPO oxidation of 2′,3′-O-TBDMS-N4-acetylcytidine results in the expected 5′-carboxylic acid nucleoside, its 2′,3′-O-isopropylidene analogue reacts in a radically different way. We have demonstrated here that hypervalent iodine(III) in water triggers an unprecedented oxidative cyclization leading to a mixture of C5-substituted O6,5′-cyclo-5,6-dihydrouridines. This mixture of cyclouridines can be opened under basic conditions and, after deprotection, yields 5-hydroxyuridine, an important post-transcriptional modification of uridine at the wobble position (U34) of bacterial tRNA. NMR experimental values and calculations were performed to provide further insight on the specific reactivity of 2′,3′-O-isopropylidene N4-acetylcytidine.