Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5’ cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5’ cap incorporation is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of pre-defined short fragments from the 5’ end of the kilobase-long synthetic mRNA molecules using RNase H, a ribozyme or a DNAzyme, enrichment of the 5’ cleavage products, and LC-MS intact mass analysis. In this communication, we describe 1) a framework to design site-specific RNA cleavage using RNase H; 2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; 3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5’ cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community.

Salt stress impedes plant growth and development, and leads to yield loss. Recently, a halophyte species Mesembryanthemum crystallinum has become a model to study plant photosynthetic responses to salt stress. It has an adaptive mechanism of shifting from C 3 photosynthesis to crassulacean acid metabolism (CAM) photosynthesis under stresses, which greatly enhances water usage efficiency and stress tolerance. In this study, we focused on investigating the morphological and physiological changes [e.g., leaf area, stomatal movement behavior, gas exchange, leaf succulence, and relative water content (RWC)] of M. crystallinum during the C 3 to CAM photosynthetic transition under salt stress. Our results showed that in M. crystallinum seedlings, CAM photosynthesis was initiated after 6 days of salt treatment, the transition takes place within a 3-day period, and plants became mostly CAM in 2 weeks. This result defined the transition period of a facultative CAM plant, laid a foundation for future studies on identifying the molecular switches responsible for the transition from C 3 to CAM, and contributed to the ultimate goal of engineering CAM characteristics into C 3 crops.

Crassulacean acid metabolism (CAM) is a specialized type of photosynthesis: stomata close during the day, enhancing water conservation, and open at night, allowing CO 2 uptake. Mesembryanthemum crystallinum (common ice plant) is a facultative CAM species that can shift from C 3 photosynthesis to CAM under salt or drought stresses. However, the molecular mechanisms underlying the stress induced transition from C 3 to CAM remain unknown. Here we determined the transition time from C 3 to CAM in M. crystallinum under salt stress. In parallel, single-cell-type transcriptomic profiling by 3'-mRNA sequencing was conducted in guard cells to determine the molecular changes in this key cell type during the transition. In total, 495 transcripts showed differential expression between control and salt-treated samples during the transition, including 285 known guard cell genes, seven CAM-related genes, 18 transcription factors, and 185 other genes previously not found to be expressed in guard cells. PEPC1 and PPCK1, which encode key enzymes of CAM photosynthesis, were up-regulated in guard cells after seven days of salt treatment, indicating that guard cells themselves can transition from C 3 to CAM. This study provides important information towards introducing CAM stomatal behavior into C 3 crops to enhance water use efficiency.

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Deoxy sugars are ubiquitous in nature and contribute to diverse biological activities. Attempts to design systems to control or to mimic their functions are hampered, however, by the lack of biosynthetic knowledge of these unique sugars. To elucidate the mechanism by which the sugar deoxygenation is effected, we have initiated a study to explore the biosynthesis of CDP-ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharides of Yersinia pseudotuberculosis, and our initial focus centered on C-3 deoxygenation catalyzed by E 1 andEj. We have now purified the wild-type enzymes, cloned the corresponding genes (ascC for.B, and ascDfor~), and overexpressed the gene products in Escherichia coli. The purified E] is aflavoprotein comprising an iron-sulfur Center and E] is an iron-sulfur containing, pyridoxamine 5'-phosphate-dependent enzyme. Since these iron-sulfur clusters are well known one-electron carriers, reactions mediated by E I and E 3 must proceed via a radical mechanism. Recently, EPR analysis of El~catalysis indicated a potential new redox role for pyridoxamine as a cofactor. These findings make this system unique from two perspectives: E 1 is the only coenzyme B 6 -dependent catalyst that interacts with a sugar and not with an amino acid, and it is the first example in which coenzyme B 6 may facilitate one-electron redox chemistry.Thus, the unprecedented mechanisms of E 1 and E 3 distinguish this system asa novel radical deoxygenation with potentially interesting future developments. 627