How to Detect mRNA Capping Efficiency?

Messenger RNA (mRNA) is a ribonucleotide sequence that encodes a protein, primarily consisting of five main parts: the 5' end is a cap structure (Cap), the 5' untranslated region (UTR) related to translation efficiency, the open reading frame (ORF) area that encodes a protein, the 3' UTR related to translation efficiency, and the polyA tail synthesized by multiple adenosine monophosphates, which is related to mRNA stability and translation efficiency.

 

The cap structure is formed by the methylation modification of the first guanosine at the 5' end of mRNA (7-methyl guanosine, abbreviated as m7G), which has functions such as protecting RNA from degradation, recruiting related proteins to participate in intron splicing, polyA tailing, nucleation, and translation. The proportion of mRNA with cap structure can affect the immunogenicity and translation efficiency of mRNA, so the capping rate is a key quality indicator of mRNA vaccines or drugs.

 

The molecular weight of mRNA is generally hundreds to thousands of KDa, while the cap structure is only about 500 Da. The tiny cap structure appears insignificant in the face of the mRNA molecule, making it difficult to distinguish between capped and uncapped mRNA structurally. Therefore, it is necessary to use highly sensitive and high-resolution technical means to accurately detect and distinguish them.

 

The Commonly Used Methods for Detecting mRNA Capping Rates

1. Immunoprecipitation-PCR (IP-PCR)

(1) Principles

Use specific antibodies to capture mRNA with an m7G cap, and then use PCR to amplify the target sequence. The capping rate of mRNA is analyzed by detecting the amplified product.

 

(2) Characteristics

The method is simple and easy to operate, suitable for large-scale sample detection; but it may be affected by non-specific binding and amplification, resulting in false negatives or false positives.

 

2. Enzyme Digestion Method

(1) Principles

Precise cutting of the 5' end of mRNA is carried out by specific enzymes to produce oligonucleotide fragments of suitable length. Subsequently, different 5' end structures are identified by liquid chromatography-mass spectrometry (LC-MS), and the capping rate is calculated.

 

(2) Characteristics

High resolution and accuracy; but this method requires high quality and purity of the sample, and is easily affected by enzyme activity. It is not suitable for some complex and easily degradable samples.

 

3. Mass Spectrometry

(1) Principles

The presence and degree of capping are determined by measuring the mass of the mRNA molecule and the specific characteristics of the cap structure.

 

(2) Characteristics

High sensitivity and accuracy; but the operation is relatively complex and requires professional equipment and technical personnel.

 

Different methods of mRNA capping rate detection have their advantages and disadvantages. Researchers can choose the appropriate method according to experimental requirements, sample type, operation difficulty and other factors. MtoZ Biolabs uses Thermo's Orbitrap Fusion Lumos mass spectrometer combined with Nano-LC nanoscale chromatography technology to establish an mRNA 5' capping rate analysis platform, which can provide one-stop mRNA 5' capping rate analysis services. You only need to tell us the purpose of the experiment and send the samples, and MtoZ Biolabs is responsible for all subsequent projects. For more details, feel free to consult!