Biochemical characterization of mRNA capping enzyme from Faustovirus

Abstract

The mammalian mRNA 5′ cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5′-mediated mRNA turnover. Hence, installation of the proper 5′ cap is crucial in therapeutic applications of synthetic mRNA. The core 5′ cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5′ triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity towards RNA containing secondary structures, and broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.

Streamlined DNA template preparation and co-transcriptional 5′ capped RNA synthesis enabled by solid-phase catalysis

“…Fig.4b (and Supplementary Fig.7) shows the activity of FCE at various temperatures. Consistent with previous results, 27 FCE is highly active across a broad range of temperatures from 25 to 55°C. Immobilization on BG-PEG 750 -coated magnetic beads (FCE@PEG) improved FCE activity at the higher end of the temperature range (55-65°C).…”

“…Faustovirus capping enzyme (FCE) is a single-subunit RNA capping enzyme that exhibits high specific activity, broad temperature range, and possesses a three-domain fold similar to the larger subunit of VCE ( Fig.4a ). 27 To incorporate RNA capping in an immobilized enzyme workflow, we produced SNAP-tagged versions of FCE and vaccinia RNA cap 2′-O-methyltransferase (2′OMTase) and immobilized them on BG- and PEG 750 -coated magnetic beads. Both SNAP-tagged enzymes were immobilized to the beads at high efficiency and exhibited higher enzyme activity on PEG 750 -coated beads (FCE@PEG and 2′OMTase@PEG) ( Supplementary Fig.2, Supplementary Table 1 ).…”

“…4a). 27 To incorporate RNA capping in an immobilized enzyme workflow, we produced SNAP-tagged versions of FCE and vaccinia RNA cap 2′-Omethyltransferase (2′OMTase) and immobilized them on BG-and PEG750-coated magnetic beads. Both SNAP-tagged enzymes were immobilized to the beads at high efficiency and exhibited higher enzyme activity on PEG750-coated beads (FCE@PEG and 2′OMTase@PEG) (Supplementary Fig.…”