Biology subjects

Whipple, J. M.

Publications and source records attributed to Whipple, J. M..

3 recordsLinked to original sources

bioRxiv2023

Biochemical characterization of mRNA capping enzyme from Faustovirus

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.

biochemistry↗

bioRxiv2022

RNase H-based analysis of synthetic mRNA 5-prime cap incorporation

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.

biochemistry↗

bioRxiv2019

In vitro characterization of the yeast DcpS, a scavenger mRNA decapping enzyme

Eukaryotic mRNAs are modified at their 5 end early during transcription by the addition of N7-methylguanosine (m7G), which forms the \"cap\" on the first 5 nucleotide. Identification of the 5 nucleotide on mRNA is necessary for determination of the Transcription Start Site (TSS). We explored the effect of various reaction conditions on the activity of the yeast scavenger mRNA decapping enzyme DcpS (yDcpS) and examined decapping of 30 chemically distinct cap structures varying the state of methylation, sugar, phosphate linkage, and base composition on 25mer RNA oligonucleotides. Contrary to the generally accepted belief that DcpS enzymes only decap short oligonucleotides, we found that yDcpS efficiently decaps RNA transcripts as long as 1400 nucleotides. Further, we validated the application of yDcpS for enriching capped RNA using a strategy of specifically tagging the 5 end of capped RNA by first decapping and then recapping it with an affinity-tagged guanosine nucleotide.

molecular biology↗