Production Overview

Capping efficiency is a critical quality attribute of mRNA vaccines. The 5' cap structure protects mRNA from degradation and facilitates protein translation. The mRNA capping efficiency sample pretreatment kit enables efficient and reproducible sample preparation for 5'-terminal sequence analysis via LC-MS. This kit contains pre-formulated 4× RNase H Mix, streptavidin magnetic beads and optimized protocols, and the samples ready for mass spectrometry detection can be prepared within 1.5 hours.

Key Features

Easy to operate and User-friendly

Breakthrough one-step workflow from probe combination to RNase H digestion, the whole process only takes 1.5 hours.

Reliable results

100% mRNA cutting efficiency and high recovery rate of 5' end sequence. Combined with LC-MS analysis, it can achieve accurate analysis of the cap structure at the 5' end of mRNA.

High stability of RNase H Mix

Tolerant to 37°C thermal acceleration for 45 days, tolerant to open tubes for 10 times with no effect on the enzyme digestion activity.

Ready-to-use Positive Control Template

The kit contains a positive control mRNA which is capped by co-transcriptional capping.

Applications

Specially designed for sample pre-treatment and preparation in mRNA capping efficiency detection; widely applicable to IVT mRNA raw materials, modified mRNA samples, mRNA vaccine and therapeutic mRNA research & development, as well as process quality control of mRNA capping reaction.

Performance Data

Probe design and synthesis

This kit does not contain any specific probes but is needed. The user needs to design a 14-30 nt probe complementary to the 5' end sequence of the target mRNA with 4-6 DNA bases at the 5' end or middle and the 2'-O-methyl modified RNA base, and the 3' end should carry Biotin TEG tag as shown in Fig 1.

Sample processing procedure

Fig 2. Hzymes’ Fast Sample treatment process within 1.5h.

LC-MS analysis

EGFP mRNA positive control within the kit Capped by co-transcriptional capping was processed by Sample Preparation Kit for mRNA Capping Efficiency Detection (Cat No. HBP005001) and analyzed by LC-MS to check the capping profile of EGFP control mRNA. The 2.71 min peak is the target fragment and 3.37 min peak is the probe.

Fig 3. HPLC-MS TIC profile of EGFP mRNA Capped by co-transcriptional capping

Fig 4. Target peak deconvolved molecular weight map (left) and probe peak deconvolved molecular weight map (right) of EGFP mRNA Capped by co-transcriptional capping.

Table 1. Target deconvolution profile

Data Analysis

The Calculation formula for Co-transcriptional capping process is as follow:

Capping rate = Cap1 / (mon_unCap + di_unCap + tri_unCap + Cap1)

The capping rate of the EGFP mRNA Capped by co-transcriptional capping is 99.46%.

FAQ

Is the kit compatible with all types of mRNA cap structures?

Yes, it is adapted to mainstream Cap 0, Cap 1 and Cap 2 structure mRNA, and supports capping efficiency detection of conventional enzymatic capping and co-transcriptional capping mRNA products.

Are the probes and RNase H enzyme provided in the kit?

All core components except 3’ biotin-modified specific probe, RNase H enzyme, reaction buffer and streptavidin magnetic beads are provided as ready-to-use reagents.

Is the kit for research use only?

This kit is for research use only, and it has been applied to sample pretreatment work in multiple mRNA drug IND declaration projects with stable and validated performance.

What is the applicable mRNA length range?

It is suitable for conventional IVT mRNA of common lengths, and can stably complete specific cleavage and magnetic bead purification for both short-sequence (500 nt) and long-chain mRNA samples (12000 nt).