Automated glycan assembly of ¹⁹F labelled glycan probes enables high‐throughput NMR studies of protein‐glycan interactions

Protein‐glycan interactions mediate important biological processes, including pathogen host invasion and cellular communication. Major challenges to monitoring these low affinity interactions are the required high sensitivity of a biophysical assay and to cover a breath of synthetic well‐defined structures. Here, we showcase an expedite approach that integrates automated glycan assembly (AGA) of 19 F labelled probes and high‐throughput NMR methods, enabling the study of protein‐glycan interactions. Synthetic Lewis type 2 antigens were screened against seven glycan binding proteins (GBPs), including DC‐SIGN and BambL, respectively involved in HIV‐1 and lung infections in immunocompromised patients, confirming the preference for fucosylated glycans (Le x , H type 2, Le y ). Previously unknown glycan‐lectin weak interactions were detected, and thermodynamic data were obtained. Enzymatic reactions were monitored in real‐time, delivering kinetic parameters. These results demonstrate the utility of AGA combined with 19 F NMR for the discovery and characterization of glycan‐protein interactions, opening up new perspectives for 19 F labelled complex glycans

Targeting the central pocket of the Pseudomonas aeruginosa lectin LecA

Pseudomonas aeruginosa is an opportunistic ESKAPE pathogen that produces two lectins, LecA and LecB, as part of its large arsenal of virulence factors. Both carbohydrate-binding proteins are central to the initial and later persistent infection processes, i. e. bacterial adhesion and biofilm formation. The biofilm matrix is a major resistance determinant and protects the bacteria against external threats such as the host immune system or antibiotic treatment. Therefore, the development of drugs against the P. aeruginosa biofilm is of particular interest to restore efficacy of antimicrobials. Carbohydrate-based inhibitors for LecA and LecB were previously shown to efficiently reduce biofilm formations. Here, we report a new approach for inhibiting LecA with synthetic molecules bridging the established carbohydrate-binding site and a central cavity located between two LecA protomers of the lectin tetramer. Inspired by in silico design, we synthesized various galactosidic LecA inhibitors with aromatic moieties targeting this central pocket. These compounds reached low micromolar affinities, validated in different biophysical assays. Finally, X-ray diffraction analysis revealed the interactions of this compound class with LecA. This new mode of action paves the way to a novel route towards inhibition of P. aeruginosa biofilms

An integrated approach to the registration and preservation of a cultivar gene pool in the VIR genebank exemplified in cultivars bred by the Ural Federal Agrarian Research Center of the Ural Branch of the Russian Academy of Sciences

As part of a comprehensive program for registering and preserving the gene pool of Russian varieties in the VIR genebank, initiated at the N.I. Vavilov All-Russian Institute of Plant Genetic Resources, nomenclatural standards were assigned to potato cultivars ‘Alâska’, ‘Argo’, ‘Bravo’, ‘Irbitskij’, ‘Legenda’, ‘Lûks’, ‘Terra’, and ‘Šah’, and a voucher specimen was prepared for pre-cultivar ‘Bagira’. All of them were bred at the Ural Federal Agrarian Research Centre (Ural Branch of the Russian Academy of Sciences). Nomenclatural standards of these cultivars are preserved in the Nomenclatural standard collection at the Herbarium of cultivated plants and their wild relatives and weeds (VIR Herbarium, WIR). For each of them, a genetic passport was developed using DNA preparations isolated from plant material donated by the cultivar author to the VIR herbarium. The genetic passport, which includes information on the allelic composition of eight chromosome-specific microsatellite loci, is supplemented by the molecular screening data with markers of 11 R-genes for resistance to pests, as well as the data on the types of cytoplasm. The author of cultivars also supplied VIR with samples of virus-free in vitro plants. Microplants of eight Ural cultivars and one pre-cultivar were genotyped using the same SSR markers and included in the VIR in vitro collection, and then in the cryopreservation program. Most of the accessions were characterized by a relatively high, above 39%, level of post-cryogenic regeneration. Frozen explants of the genotyped Ural cultivars (‘Alâska’, ‘Argo’, ‘Legenda’, ‘Terra’, and ‘Šah’) and pre-cultivar ‘Bagira’ were placed for long-term storage in the VIR Cryobank

Multiplexed set of 10 microsatellite markers for identification of potato varieties

Genetic identification of potato varieties is a demanded instrument for development of new cultivars registration system, protection of plant breeders’ rights, and variety homogeneity control. The most perspective approach for distinction and identification of varieties continues to remain the use of short tandem repeats. STR amplification with the subsequent high resolution electrophoresis allows such a unique characteristic of a variety to be obtained as the DNA profile. A large scale of samples requires the creation of a robust and time-saving technique based on fragment sizing. We selected 10 polymorphic STR loci of potato: STI0032, STG0016, STI0001, STI0004, STM1104, STM5127, STI0030, STI0033, STI0014, STM5114 and designed a multiplex panel for potato DNA profiling. Fluorescent labelling of primers and size distinction of amplicones allowed us to use one tube for PCR and capillary electrophoresis. We also modified the CTAB-protocol for DNA extraction from tubers and other parts of potato plants, the PCR mix recipe and the amplification protocol for good results. Using Genetic Analyzer allows the length of alleles to be defined with an accuracy of one nucleotide and digitized genetic profiles to be developed. We created a unique DNA profile for each of 40 varieties and 23 breeding lines from Russia and other countries and evaluated the homogeneity of 8 varieties. The proposed technique оf potato DNA profiling allows a large number of samples to be rapidly analyzed in the 96-well plate format