Trinucleotide mRNA Cap Analogue N6-Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo.

Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am─a common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap-m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of m6Am in mRNA.

A New Way of Belonging: Active-Site Investigation of L-DOPA Dioxygenase, a VOC Family Enzyme from Lincomycin Biosynthesis.

Extradiol dioxygenase chemistry is essential for catechol breakdown. The largest natural reservoir of catechols, or 1,2-dihydroxybenzenes, is the plant woody-tissue polymer lignin. Vicinal-oxygen-chelate (VOC) dioxygenases make up the largest group of characterized extradiol dioxygenases, and while most are found as part of catabolic pathways degrading a variety of natural and human-made aromatic rings, L-DOPA (l-3,4-dihydroxyphenylalanine) dioxygenase is a VOC enzyme that participates in the biosynthesis of a natural product. All VOC superfamily members shared conserved elements of catalysis, yet despite decades of investigation of VOC enzymes, the relationships between VOC domain architecture and enzymatic function remain complex and poorly understood. Herein, we present evidence that L-DOPA dioxygenase is the representative member of a new topological class of VOC extradiol dioxygenases. Guided by its evolutionary similarity to glyoxylase enzymes, we performed a careful investigation of the Streptomyces lincolnensis L-DOPA dioxygenase (LmbB1) active site through mutagenesis, kinetic, and pH studies. Our results demonstrate that the L-DOPA dioxygenase reaction depends upon an active-site tyrosine and histidine and is remarkably resilient to mutation, even at the iron-ligating residues. Evaluation of the cleavage reaction as a function of pH supports the role of a histidine in acid-base catalysis. The active-site architecture is functionally consistent with the existing knowledge of VOC extradiol dioxygenase catalysis.