mRNA Capping Efficiency Assay

mRNA Capping Efficiency Assay

In the field of molecular biology, the emergence of mRNA capping efficiency measurement is a key advancement in understanding and manipulating genetic material. BOC Sciences is a leading provider of RNA solutions, and we deeply recognize the importance of mRNA modification in various biomedical applications in ensuring the production of high-quality and stable mRNA products, especially in the development of mRNA vaccines and nucleic acid drugs. Here, BOC Sciences offers comprehensive mRNA in vitro transcription (IVT) services, with mRNA capping efficiency measurement playing a crucial role in assessing the quality and stability of mRNA.

What is mRNA Capping?

mRNA capping is a fundamental post-transcriptional modification process essential for the maturation and stability of mRNA. It involves the addition of a 7-methylguanosine (m7G) cap structure to the 5' end of the mRNA molecule. This modification occurs co-transcriptionally and serves as a crucial determinant of mRNA fate, influencing processes such as RNA splicing, transport, translation efficiency, and mRNA turnover.

What is the Significance of the Capping of mRNA?

The capping of mRNA confers several critical advantages to the transcript. Firstly, it protects the mRNA from exonuclease degradation by providing a stable chemical structure at the 5' end. Additionally, the cap structure facilitates the recruitment of translation initiation factors, promoting efficient translation initiation. Moreover, mRNA capping plays a pivotal role in mRNA export from the nucleus to the cytoplasm, thereby regulating gene expression dynamics.

What is mRNA Capping Efficiency?

mRNA capping efficiency refers to the percentage of mRNA transcripts within a cellular population that possess a functional m7G cap structure at their 5' ends. This parameter serves as a crucial indicator of the cellular machinery's proficiency in mRNA capping and reflects the overall translational competence and gene expression dynamics within the system. The measurement of mRNA capping efficiency holds immense significance in elucidating the molecular mechanisms underlying gene expression regulation and cellular homeostasis. mRNA capping efficiency assays provide valuable insights into the efficiency of mRNA capping processes, enabling researchers to discern aberrations associated with various pathological conditions such as viral infections, cancer, and neurodegenerative disorders.

BOC Sciences' mRNA Capping Efficiency Assay Service

At BOC Sciences, we offer comprehensive mRNA capping efficiency detection services, providing a comprehensive solution for assessing the efficiency of mRNA capping processes, aiming to meet the diverse needs of researchers and pharmaceutical companies. Our mRNA capping efficiency measurement service utilizes proprietary methods and equipment, delivering precise and reproducible measurements of mRNA capping efficiency. We are committed to inspiring valuable insights into gene expression regulation and cellular homeostasis for customers in the global biopharmaceutical field.

mRNA Capping Efficiency Assay Methods Available at BOC Sicences

Options for Assay MethodsDescription
ElectrophoresisLong-chain mRNA typically consists of thousands of nucleotides (nt), and the only difference between capped and uncapped mRNA is a single methylated guanosine (m7G) at the 5' end, making it difficult to distinguish within the full-length mRNA. The current approach to measuring capping efficiency involves enzymatic cleavage to obtain 5' end RNA fragments (approximately tens of nucleotides long), followed by separation of different lengths of 5' end oligonucleotide fragments using methods such as polyacrylamide gel electrophoresis, high-performance liquid chromatography (HPLC), or liquid chromatography-mass spectrometry (LC-MS), allowing for quantitative evaluation of mRNA capping efficiency.
Fluorescent Cap AnalogsA very direct method for measuring capping efficiency is to directly label the 5' Cap with a fluorescent marker, and the number of capped molecules can be directly determined by the fluorescence intensity. This can be achieved using chemically synthesized modified guanosine nucleotides or cap analogs with a fluorescent label located in a part of the molecule that does not interfere with its biological function.
qRT-PCRThe capping status of mRNA can also be assessed using qRT-PCR. qSL-RT-PCR was originally developed for measuring mRNA decapping and analysis of therapeutic RNA samples. qSL-RT-PCR uses bridging or primers oligonucleotides to anneal to the 5' end of RNA, forming a single-stranded extension upstream. Subsequently, complementary anchor oligonucleotides are annealed, allowing for the extension of a unique sequence detectable by RT-qPCR from un-capped RNA molecules anchored to the primer. Capped RNAs are blocked from ligation and thus remain undetected. Therefore, the amount of uncapped RNA can be quantified and correlated with the total sample concentration to determine the capping efficiency.
Liquid Chromatography-Mass Spectrometry (LC-MS)LC-MS is a powerful analytical tool for the separation, identification, and quantification of molecules, with high sensitivity and resolution, making it particularly suitable for quality control in the pharmaceutical industry. However, the small size of the cap structure compared to mRNA fragments makes direct analysis of the 5' Cap structure on full-length mRNA challenging. An approach to overcome this limitation is to use enzymes or ribonucleases to cleave mRNA near the 5' end, generating smaller mRNA fragments, which can then be used to determine the presence of the cap structure by LC-MS. Enzyme pre-treatment combined with LC-MS has now evolved into an efficient method for mRNA capping detection analysis and has become the industry standard for release testing of critical batches.
Nanopore Sequencing MethodNanopore sequencing is a platform technology that enables direct sequencing of modified/unmodified mRNA. Individual RNA molecules are driven through nanopore protein channels embedded in a synthetic membrane. The sequence of RNA is determined by measuring changes in current as molecules pass through the pore. Engineered nanopores can be used to directly identify various RNA modifications, including m7G, ψ, m6A, m5C, m1A, etc.

Highlights of Our mRNA Capping Efficiency Assay Service

Customizable Assay Protocols

We understand that different research objectives may require customized detection protocols. Therefore, our team collaborates closely with clients to develop tailored detection workflows to meet specific experimental needs and sample types. Faced with various types of modified RNA, we ensure that our detection methods are optimized for accuracy and sensitivity.

Selectivity, High Sensitivity & Specificity

Our mRNA capping efficiency assay possesses high sensitivity and specificity, enabling precise detection and quantification of both capped and uncapped RNA species. By selectively employing advanced molecular biology techniques and cap-specific detection methods, we minimize background noise and maximize signal intensity, ensuring reliable results.

Expert Technical Support

At BOC Sciences, we take pride in our professional team of experienced scientists who provide expert technical support throughout the detection process. From experimental design and sample preparation to data analysis and interpretation, our team is dedicated to delivering excellent service and guidance at every step.

Accelerate Your Research Discoveries

By entrusting mRNA capping efficiency detection to BOC Sciences, you gain access to cutting-edge technology and specialized expertise, accelerating the pace of your research and discoveries. Our comprehensive services enable you to uncover new insights into the molecular mechanisms underlying gene expression dynamics and disease pathogenesis.

Contact BOC Sciences today to learn more about our mRNA capping efficiency assay service and embark on a journey towards groundbreaking discoveries.

Case Study

Case study 1 Assessing the Capping Efficiency of In Vitro-Transcribed mRNA.

Flowing diagram to quantify the capping efficiency of in vitro transcribed mRNAs by ribozyme-mediated cleavage.Flowing diagram to quantify the capping efficiency of in vitro transcribed mRNAs by ribozyme-mediated cleavage. (Vlatkovic, I.; et al, 2022)

In the dynamic field of mRNA therapeutics, ensuring the quality and efficacy of in vitro-transcribed mRNA (IVT mRNA) is paramount. A crucial aspect of IVT mRNA stability and translational efficiency lies in the presence of the cap structure at its 5'-end. However, variations in capping efficiency due to different synthesis methods and mRNA features present a challenge for quality control. In response, researchers have developed a pioneering approach using ribozyme cleavage assays to specifically target and assess the capping efficiency of IVT mRNA. By designing ribozymes to cleave mRNA at predetermined positions, followed by purification and analysis of capped and uncapped fragments, this method offers a robust solution for optimizing synthesis methods and ensuring the quality of mRNA-based therapeutics across diverse applications, including vaccine development and cancer immunotherapies.

FAQ

1. How to measure capping efficiency?

Several methodologies have been developed to assess mRNA capping efficiency, including enzymatic assays, cap-specific antibody-based techniques, and high-throughput sequencing approaches. These methods typically involve the isolation of mRNA transcripts followed by the detection and quantification of capped and uncapped RNA species, allowing for the precise determination of capping efficiency.

2. What is 5 prime capping in transcription?

5' prime capping in transcription is a process where a modified guanine nucleotide is added to the 5' end of the newly synthesized mRNA molecule. This cap serves to protect the mRNA from degradation and aids in its recognition and processing during translation.

3. What is capping and tailing of mRNA?

Capping adds a protective structure to one end of the mRNA, while tailing attaches a string of nucleotides to the other end. These modifications stabilize the mRNA and facilitate its function in the cell.

4. Why is 5 cap added to mRNA?

5' cap is added to mRNA for several reasons. It helps in stabilizing the mRNA molecule, protecting it from degradation by cellular enzymes. Additionally, it aids in the initiation of translation, the process where mRNA is used as a template to build proteins. The cap also assists in transporting the mRNA out of the nucleus and into the cytoplasm where translation occurs. Overall, the 5' cap plays crucial roles in mRNA stability, translation initiation, and cellular localization.

5. What is the purpose of capping?

The addition of a 5' cap to mRNA is crucial for its stability and functionality. This modification not only enhances mRNA processing but also aids in its export from the nucleus to the cytoplasm. Moreover, the 5' cap plays a pivotal role in the translation process by facilitating the recruitment of ribosomes. Following capping, a phosphorylation event occurs, which triggers the recruitment of machinery required for RNA splicing. This splicing process is essential for removing non-coding regions (introns) from the mRNA molecule, ultimately resulting in the production of a mature mRNA transcript.

6. What is the Difference Between Capped and Uncapped RNA?

The distinction between capped and uncapped RNA lies in their susceptibility to degradation and translational efficiency. Capped RNA, with its protective m7G cap structure, exhibits enhanced stability against exonucleases and increased translational competence compared to uncapped RNA molecules. Conversely, uncapped RNA is prone to rapid degradation and inefficient translation initiation, compromising gene expression regulation.

Reference

  1. Vlatkovic, I.; et al. Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA. Pharmaceutics. 2022, 14(2): 328.
* Only for research. Not suitable for any diagnostic or therapeutic use.
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