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Recent years have led to the identification of a number of enzymes responsible for RNA decapping. This has provided a basis for further research to identify their role, dependency and substrate specificity. However, the multiplicity of these enzymes and the complexity of their functions require advanced tools to study them. Here, we report a high-throughput fluorescence intensity assay based on RNA aptamers designed as substrates for decapping enzymes. Using a library of differently capped RNA probes we generated a decapping susceptibility heat map, which confirms previously reported substrate specificities of seven tested hydrolases and uncovers novel. We have also demonstrated the utility of our assay for evaluating inhibitors of viral decapping enzymes and performed kinetic studies of the decapping process. The assay may accelerate the characterization of new decapping enzymes, enable high-throughput screening of inhibitors and facilitate the development of molecular tools for a better understanding of RNA degradation pathways., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Polymeric bags are a widely applied, simple, and cost-effective method for the storage and offline analysis of gaseous samples. Various materials have been used as sampling bags, all known to contain impurities and differing in their cost, durability, and storage capabilities. Herein, we present a comparative study of several well-known bag materials, Tedlar (PVF), Kynar (PVDF), Teflon (PTFE), and Nalophan (PET), as well as a new material, ethylene vinyl copolymer (EVOH), commonly used for storing food. We investigated the influences of storage conditions, humidity, bag cleaning, and light exposure on volatile organic compound concentration (acetone, acetic acid, isoprene, benzene, limonene, among others) in samples of exhaled human breath stored in bags for up to 48 h. Specifically, we show high losses of short-chain fatty acids (SCFAs) in bags of all materials (for most SCFAs, less than 50% after 8 h of storage). We found that samples in Tedlar, Nalophan, and EVOH bags undergo changes in composition when exposed to UV radiation over a period of 48 h. We report high initial impurity levels in all the bags and their doubling after a period of 48 h. We compare secondary electrospray ionization and proton transfer reaction mass spectrometry in the context of offline analysis after storage in sampling bags. We provide an analytical perspective on the temporal evolution of bag contents by presenting the intensity changes of all significant m / z features. We also present a simple, automated, and cost-effective offline sample introduction system, which enables controlled delivery of collected gaseous samples from polymeric bags into the mass spectrometer. Overall, our findings suggest that sampling bags exhibit high levels of impurities, are sensitive to several environmental factors (e.g. light exposure), and provide low recoveries for some classes of compounds, e.g. SCFAs., (Creative Commons Attribution license.)

In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5' cap biosynthesis, involving RNA cap guanine-N7 methyltransferase (N7-MTase). N7-MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7-MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7-MTase activity assay based on small-molecule fluorescent probes. We synthesized 12 fluorescent substrate analogues (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3'-O position of adenosine acted as an artificial substrate with the properties of a turn-off probe for all three tested N7-MTases (human, parasite, and viral). Using this compound, a N7-MTase inhibitor assay adaptable to high-throughput screening was developed and used to screen synthetic substrate analogues and a commercial library. Several inhibitors with nanomolar activities were identified., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lectin microarray technology was applied to compare the glycosylation pattern of the monoclonal antibody MB311 expressed in SP2.0 cells to an antibody-dependent cellular cytotoxic effector function (ADCC)-optimized variant (MB314). MB314 was generated by a plant expression system that uses genetically modified moss protoplasts ( Physcomitrella patens ) to generate a de-fucosylated version of MB311. In contrast to MB311, no or very low interactions of MB314 with lectins Aspergillus oryzae l-fucose (AOL), Pisum sativum agglutinin (PSA), Lens culinaris agglutinin (LCA), and Aleuria aurantia lectin (AAL) were observed. These lectins are specific for mono-/biantennary N-glycans containing a core fucose residue. Importantly, this fucose indicative lectin-binding pattern correlated with increased MB314 binding to CD16 (FcγRIII; receptor for the constant region of an antibody)-whose affinity is mediated through core fucosylation-and stronger ADCC. In summary, these results demonstrate that lectin microarrays are useful orthogonal methods during antibody development and for characterization.

During the analytical characterization of the humanized Lewis-Y specific monoclonal antibody IGN311 (IgG1/kappa) used for passive anti-cancer therapy in humans, isoelectric focusing (IEF) experiments revealed that IGN311 batches produced in serum-containing and serum-free medium, respectively, displayed different banding patterns. The additional bands in the IEF pattern correlated with additional peaks observed by subsequent cation exchange (CEX)-HPLC analysis. Since the IEF pattern is one of the specification criteria in the quality control of monoclonal antibodies and a non-matching pattern may be indicative for lot-to-lot inconsistency, this phenomenon was investigated in detail. First, we investigated whether a difference in antibody glycosylation was the cause for the observed charge heterogeneity. De-N-glycosylation experiments demonstrated that charge heterogeneity observed in the IEF pattern is not a consequence of glycosylation. In contrast, sample treatment by carboxypeptidase B, removing the carboxy-terminal lysine residues from the two heavy chains of the antibody, resulted in reduced charge heterogeneity eliminating the two most basic bands observed in IEF. These data were supported by reversed phase HPLC-MALDI-TOF-MS analysis of enzymatically cleaved peptides of the antibody as well as by carboxy-terminal sequencing of the heavy chains. It was demonstrated that the differences in the IEF banding pattern were due to lysine clipping occurring during the production of the antibody. The antibody batch produced under serum-free conditions was less affected by lysine clipping. Both antibody variants--clipped and unclipped--elicited the same potency in a complement dependent cytotoxicity (CDC) assay demonstrating that lysine clipping of IGN311 does not impair Fc-mediated effector functions.