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Optimizing mRNA Capping and Analysis

Posted on Monday, May 13, 2024

By Nicole Kelesoglu

Topic: Tips for the lab

As mRNA has translated from research into therapeutics, 5´ capping and characterization workflows have become even more critical. NEB provides a range of solutions for mRNA capping – but have you heard about the higher activity of our new capping enzyme and our improved approach for LC-MS RNA cap analysis?

Capping mRNA is necessary for RNA integrity and function. The Cap-1 structure is a critical quality attribute for mRNA therapeutic biopharmaceuticals. NEB supported multiple mRNA vaccine makers for global scale roll-out using our reagents and expertise during the pandemic. If you’re seeking guidance for mRNA therapeutics development, we invite you to download our new eBook, From Template to Transcript, Overcoming challenges in mRNA manufacturing.
Our RNA capping portfolio includes both enzymatic and co-transcriptional solutions at research grade and GMP-grade*. Our research has contributed to advancing mRNA techniques, as demonstrated by two recent studies, one characterizing a new capping enzyme with properties advantageous to mRNA manufacturing, and another describing an improved approach for LC-MS RNA cap analyses.


Solutions for enzymatic and co-transcriptional mRNA capping

NEB’s toolbox for mRNA capping includes capping enzymes, synthetic cap analogs, and kits for efficient RNA capping and poly(A) tailing workflows. We recommend co-transcriptional capping when a wide variety of transcripts need to be generated quickly and with minimal optimization. We recommend enzymatic capping for large-scale studies and mRNA manufacturing. The HiScribe® T7 mRNA Kit with CleanCap® Reagent AG (NEB #E2080) is optimized for high-yield, co-transcriptional capping of mRNA in a single, simple reaction. Post-transcriptional enzymatic capping can enable higher yields for mRNA manufacturing in some cases.  Vaccinia Capping Enzyme (VCE) (NEB #M2080) has been a stalwart of enzymatic capping and when combined with mRNA Cap 2´-O-methyltransferase (NEB #M0366) can generate the Cap-1 structure. Faustovirus Capping Enzyme (FCE) is a new capping enzyme developed at NEB that offers properties to improve mRNA manufacturing workflows.


Higher activity of Faustovirus Capping Enzyme for mRNA manufacturing

Faustovirus Capping Enzyme (NEB #M2081), colloquially referred to as “FCE”, is a novel capping enzyme offering several advantages for mRNA manufacturing workflows. FCE has higher capping activity in general compared to VCE, making it a more cost-effective option for scaling up to mRNA manufacturing. FCE is a single-subunit RNA capping enzyme derived from an amoeba giant virus that can be used with mRNA Cap 2´-O-methyltransferase in a one-step reaction, in the same way that Vaccinia Capping Enzyme is used to generate an mRNA Cap-1 structure. While Faustovirus Capping Enzyme’s overall higher capping efficiency is exciting, it’s worth noting that it can also be useful with certain challenging mRNA substrates. NEB researchers recently shared data on FCE’s nuanced properties in the study: Biochemical characterization of mRNA capping enzyme from Faustovirus. The capabilities of FCE make it our primary recommendation for enzymatic RNA capping reactions, and certainly for any cases when the design of synthetic mRNA, including its 5′ sequence, impacts the effectiveness of VCE capping.  Another advantage of FCE is its robust activity across a broader temperature range. It can enable low-temperature capping to help prevent the degradation of long synthetic RNA molecules and higher-temperature capping to improve the capping efficiency of RNA molecules with more complex secondary structures. Even if you’re familiar with VCE, given the desirable properties of FCE, it can be worthwhile to test both capping enzymes.

mRNA manufacturing Faustovirus capping enzyme Vaccinia Capping Enzyme infographic 


RNase 4 simplifies mRNA cap LC-MS analysis design

Liquid chromatography-mass spectrometry (LC-MS) analysis is the gold standard for verifying 5´ cap structures of synthetic mRNA - but the method is not without its limitations. NEB has designed a simplified, improved assay that utilizes RNase 4 within the workflow.

LC-MS analysis of mRNA 5´cap structures is a multi-step workflow. A pre-defined 5´ fragment is first cleaved from the synthetic mRNA to a size amenable to LC-MS analysis. RNase H (NEB #M0297), Thermostable RNase H  (NEB #M0523), ribozymes and deoxyribozymes (DNAzymes) have been used for this purpose. However, substantial method development on cleavage site selection is needed to achieve efficient and precise RNA cleavage. Scientists at NEB recently developed a method using RNase 4 that can sidestep the tedious cleavage site selection and simplify the method development process. In addition, we found that RNase 4 tolerates many nucleobase modifications, making it a more robust option for RNA containing modified nucleotides. You can explore using RNase 4 in 5´ cap analysis in this report: Selective Characterization of mRNA 5' End-Capping by DNA Probe-Directed Enrichment with Site-Specific Endoribonucleases.

We recently released RNase 4 (NEB #M1284) as well as a protocol for DNA probe-directed analysis of mRNA 5′ cap structures. We hope that deploying RNase 4 for RNA cap analyses will benefit mRNA research and development.

LC-MS Capping Analysis RNase 4 RNase H probes

Figure 1: The analysis of 5'-end mRNA capping by LC-MS/MS is streamlined using RNase 4 and a simple DNA probe as opposed to the chimeric DNA-RNA probe required by RNase H.



LC-MS RNA capping analysis RNase 4

Figure 2:  mRNA capping can be analyzed by LC-MS/MS following hybridization with a DNA probe and digestion with RNase 4. RNase 4 offers cut site flexibility and specificity.



Expertise in enzymatic RNA capping and quality analysis

If you are looking for a partner with enzyme expertise, pressure-tested production capacity, and regulatory experience in mRNA-based therapeutics, please get in touch with NEB’s OEM & Customized Solutions Team.


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