Abstract With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of long, complex RNAs has become essential. Tandem liquid chromatography–mass spectrometry (LC–MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence analysis. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a larger population of uniquely mappable cleavage products. We deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine–two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5′ cap incorporation into in vitro transcribed mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC–MS/MS.

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Human RNase 4 improves mRNA sequence characterization by LC–MS/MS

https://doi.org/10.1016/j.jpba.2022.115174

Challenges and emerging trends in liquid chromatography-based analyses of mRNA pharmaceuticals

Auto-inducible promoter systems have been reported to increase soluble product formation in the periplasm of E. coli compared to inducer-dependent systems. In this study, we investigated the phosphate (PO4)-sensitive phoA expression system (pAT) for the production of a recombinant model antigen-binding fragment (Fab) in the periplasm of E. coli in detail. We explored the impact of non-limiting and limiting PO4 conditions on strain physiology as well as Fab productivity. We compared different methods for extracellular PO4 detection, identifying automated colorimetric measurement to be most suitable for at-line PO4 monitoring. We showed that PO4 limitation boosts phoA-based gene expression, however, the product was already formed at non-limiting PO4 conditions, indicating leaky expression. Furthermore, cultivation under PO4 limitation caused physiological changes ultimately resulting in a metabolic breakdown at PO4 starvation. Finally, we give recommendations for process optimization with the phoA expression system. In summary, our study provides very detailed information on the E. coli phoA expression system, thus extending the existing knowledge of this system, and underlines its high potential for the successful production of periplasmic products in E. coli.

/pdf/recombinant-protein-production-in-e-coli-using-the-phoa-34wirntu.pdf

Recombinant Protein Production in E. coli Using the phoA Expression System

Knowledge of the cleavage specificity of ribonucleases is critical for their application in RNA modification mapping or RNA-protein binding studies. Here, we detail the cleavage specificity and efficiency of ribonuclease MC1 and cusativin using a customized RNA sequence that contained all dinucleotide combinations and homopolymer sequences. The sequencing of the oligonucleotide digestion products by a semi-quantitative liquid chromatography coupled with mass spectrometry (LC-MS) analysis documented as little as 0.5–1% cleavage levels for a given dinucleotide sequence combination. While RNase MC1 efficiently cleaved the [A/U/C]pU dinucleotide bond, no cleavage was observed for the GpU bond. Similarly, cusativin efficiently cleaved Cp[U/A/G] dinucleotide combinations along with UpA and [A/U]pU, suggesting a broader specificity of dinucleotide preferences. The molecular interactions between the substrate and active site as determined by the dinucleotide docking studies of protein models offered additional evidence and support for the observed substrate specificity. Targeted alteration of the key amino acid residues in the nucleotide-binding site confirms the utility of this in silico approach for the identification of key interactions. Taken together, the use of bioanalytical and computational approaches, involving LC-MS and ligand docking of tertiary structural models, can form a powerful combination to help explain the RNA cleavage behavior of RNases.

/pdf/rna-cleavage-properties-of-nucleobase-specific-rnase-mc1-and-2extyas2.pdf

RNA Cleavage Properties of Nucleobase-Specific RNase MC1 and Cusativin Are Determined by the Dinucleotide-Binding Interactions in the Enzyme-Active Site

Kelly Becker

Papers

Enhanced expression and purification of nucleotide-specific ribonucleases MC1 and Cusativin.