A critical quality attribute (CQA) of any synthetic messenger RNA (mRNA) construct is the 5' cap structure. This modification, typically a 7-methylguanosine (m7G) linked to the first transcribed nucleotide via a 5'-5' triphosphate bridge, is essential for protecting mRNA from exonuclease-mediated degradation and recruiting eukaryotic translation initiation factor 4E (eIF4E) for protein synthesis. CD BioGlyco provides a specialized mRNA capping efficiency analysis service designed to quantify these species with unparalleled precision. By determining the exact ratio of capped to uncapped molecules, we empower researchers to optimize their manufacturing processes, ensure therapeutic potency, and fulfill stringent regulatory requirements for structural characterization.
At CD BioGlyco, our service scope is meticulously aligned with the therapeutic nucleic acid development platform, specifically targeting mRNA-based vaccine development through comprehensive mRNA structural characterization. Our analysis services encompass the detailed profiling of various cap structures, including the distinction between Cap-0 and the more biologically relevant Cap-1, which features a 2'-O-methylation on the first nucleotide.
We provide a one-stop analytical solution that includes the development of sequence-specific cleavage assays for any mRNA candidate, regardless of length or complexity. Our experts handle the purification of 5' cleavage products and utilize ion-pair reversed-phase high-performance LC (IP-RP-HPLC) coupled with MS to resolve species that differ by only a single methyl group or phosphate. This service applies to mRNAs produced via various technologies. By integrating these high-end analytical methods, CD BioGlyco ensures that your mRNA product meets the highest standards of identity and purity, facilitating a smoother transition from discovery to preclinical research.
We design custom DNA/RNA chimera probes or ribozymes optimized for the specific 5' untranslated region (UTR) to ensure efficient and site-specific cleavage.
The target mRNA is hybridized with the designed probes under precisely controlled thermal conditions. This step ensures that the cleavage enzymes access the target site with high specificity, protecting the 5' terminal fragment from non-specific degradation.
Using RNase H or specialized ribozyme assays, the mRNA is cleaved to release a short 5' terminal oligonucleotide. This reduction in molecular weight is essential for achieving the mass resolution required to distinguish between various capping states.
To minimize background noise and remove the large 3' mRNA body, we perform high-efficiency purification. This step ensures that only the relevant 5' terminal fragments are introduced into the LC-MS system, enhancing sensitivity.
The purified 5' fragments are analyzed using IP-RP-HPLC coupled with HRMS. This allows for the separation of Cap-0, Cap-1, and uncapped species based on both their retention time and their highly accurate mass-to-charge (m/z) ratios.
Our data scientists process the resulting mass spectra, integrating peak areas to calculate the percentage of each capping species. A comprehensive report is generated, including raw data, quantified results, and an expert interpretation of the capping efficiency.
DoI: 10.1093/nar/gkaa955
Journal: Nucleic Acids Research
IF: 13.1
Published: 2020
Results: This study describes a high-resolution biosensor based on the chimeric protein B4E (fusing murine eIF4E and β-lactamase) for simultaneous detection of mRNA capping levels and integrity. The biosensor binds mRNA via poly-deoxythymidine-functionalized beads and recognizes m7G caps through eIF4E, with colorimetric readout via β-lactamase. It works for mRNAs of 250-2700 nt, detects ≥20% capping variations (LOD: 2.4 pmol), and requires minimal instrumentation. Validated on in vitro transcripts, mRNA vaccines (up to 6507 nt), and in vivo mRNAs from CHO cells, it avoids mRNA modification or amplification. This simple, versatile tool bridges the gap between basic and advanced techniques, enabling applications in mRNA vaccine quality control and in vitro capping optimization.
Fig.1 High resolution B4E biosensor to test the capping level. (Moya-Ramírez, et al., 2020)
mRNA Vaccine Development
Essential for ensuring that vaccine candidates possess the Cap-1 structure necessary to maximize antigen expression while minimizing unwanted inflammatory responses.
Protein Replacement Therapy
Crucial for quantifying the stability and translational efficiency of mRNAs designed to treat genetic disorders, where consistent protein production levels are vital for therapeutic efficacy.
Gene Editing
Used to characterize the guide RNA (gRNA) or mRNA components, ensuring that the capping status does not interfere with the precise assembly of the editing machinery.
Cancer Immunotherapy
Facilitates the development of personalized mRNA-based neoantigen vaccines by providing rigorous quality control of the capping process during rapid-response manufacturing cycles.
Unrivaled Mass Resolution
Our high-resolution LC-MS distinguishes between species with mass differences as small as a single methyl group, ensuring accurate Cap-0 vs. Cap-1 quantification.
Sensitivity for Trace Impurities
We detect and quantify uncapped or partially processed mRNA species even at levels below 1%, providing a detailed impurity profile for data submissions.
Customizable Probe Engineering
Our team excels at designing cleavage assays for challenging sequences, including those with secondary structures or extensive chemical modifications in the 5' UTR.
Accelerated Turnaround Times
By utilizing optimized workflows and automated data analysis, CD BioGlyco delivers comprehensive capping efficiency results rapidly to keep your development timelines on track.
"Their ability to resolve Cap-1 from Cap-0 in our modified mRNA construct gave us the confidence needed to proceed to the next experiments."
– D.R., Senior Scientist
"CD BioGlyco's analysis revealed a significant percentage of uncapped species that our standard gel electrophoresis missed. Their technical support in interpreting the MS data was exceptional."
– D.S., Principal Investigator
"The customized ribozyme cleavage assay they developed for our proprietary 5' UTR worked perfectly. We will certainly be using their characterization services for our future vaccine candidates."
– E.T., Director of R&D
At CD BioGlyco, we understand that the success of mRNA therapeutics hinges on the precision of their structural modifications. Our mRNA capping efficiency analysis service provides the high-resolution data necessary to ensure your constructs are stable, potent, and safe. Please feel free to contact us for detailed information on our services or to request a formal quotation.
Reference
