MRNA process development and production – mRNA process development – mRNA synthesis and modification

MRNA (messenger RNA) is an important molecular biology messenger that can carry genetic information within cells, converting genetic information in DNA into instructions for protein synthesis. In recent years, mRNA technology has received increasing attention in the medical field, especially in vaccine development and gene therapy.

With the rapid development of genomics and proteomics, people’s understanding of gene expression regulation and protein function is constantly deepening. Traditional gene therapy methods usually use gene transfer vectors to integrate target genes into the host genome, but this method has many limitations, such as poor stability and immune response. MRNA technology, which synthesizes mRNA molecules to directly provide genetic information, can bypass these limitations and become a gene therapy method with great potential.

The development and production of mRNA technology have received widespread attention and investment. At present, mRNA technology is mainly applied in the following fields:

Vaccine development: mRNA vaccines are a novel vaccine technology that converts target pathogen protein coding information into mRNA, guiding host cells to synthesize pathogen proteins themselves and inducing immune responses. In the development and application of COVID-19 vaccines, mRNA technology has achieved remarkable success, providing new ideas and methods for vaccine development for other diseases.

Gene therapy: mRNA technology is also widely used in the field of gene therapy. By synthesizing mRNA molecules, genetic information required for damaged cells can be provided, promoting cell repair and regeneration. For example, using mRNA to encode appropriate proteins can treat various diseases such as rare genetic diseases, cancer, and cardiovascular diseases.

Cell engineering: mRNA technology can also be used in the field of cell engineering. By introducing specific mRNA into cells, the functions and characteristics of cells can be altered, such as increasing protein production and improving cell activity, making them widely used in fields such as regenerative medicine, tissue engineering, and biopharmaceuticals.

It is worth noting that mRNA technology still faces many challenges and challenges, such as mRNA stability, delivery efficiency, toxicity, etc. In addition, the production and large-scale production of mRNA technology also need to address many process and economic issues, such as optimizing synthesis processes, increasing production, and reducing costs.

Despite facing some challenges, the breakthrough progress in mRNA technology is highly anticipated. I believe that with the continuous development and improvement of technology, more breakthroughs and applications will be made in the development and production of mRNA technology, bringing more benefits to human health.

Starting today, we will introduce in detail the development and production of mRNA processes. The following is a directory about mRNA process development and production. We welcome everyone to continue to follow:

overview

MRNA synthesis is a complex process that involves DNA transcription and subsequent RNA processing and modification.

Initial transcription: Transcription is the process by which DNA produces mRNA. It is carried out in the nucleus, using RNA polymerase to hydrolyze DNA and synthesize RNA strands using one strand of DNA as a template. This RNA strand is the precursor of mRNA, known as primary transcript or precursor mRNA (pre mRNA).

Precursor mRNA processing: Primary transcripts require a series of splicing, splicing, and modification processes to generate mature mRNA. These processing processes include:

Splicing: Splicing is the process of removing introns (i.e. non coding regions) from precursor mRNA and connecting encoded exons (i.e. coding regions). Splicing is achieved through a complex, which is composed of snRNPs (small nuclear ribonucleoproteins) and other proteins.

Capping: Capping is the addition of a special chemical structure called 7-methylguanosine (cap) to the 5 ‘end of mRNA. The addition of cap helps to enhance mRNA stability, promote transcription termination, and improve translation efficiency.

Tail modification: Add a string of adenosine monophosphate (poly A tail) to the 3 ‘end of mRNA. This poly-A tail helps to stabilize mRNA and promote the initiation and termination of transcription and translation.

MRNA transport: Mature mRNA needs to be transported to the cytoplasm to participate in protein synthesis. This is achieved through nuclear protein conformations and nuclear pore complexes. The nuclear pore complex is a complex composed of proteins that allows mRNA and other nucleic acid and protein molecules to enter the cytoplasm from the nucleus.

MRNA translation: In the cytoplasm, mRNA participates in the translation process along with tRNA and rRNA by binding to ribosomes. Ribosomes match the codons on mRNA with the anti codons on tRNA, and sequentially connect amino acids to synthesize proteins.

The synthesis and modification of mRNA is a complex process that includes transcription, splicing, capping, tail modification, as well as mRNA transport and translation. These processes are highly regulated in cells to ensure proper synthesis and regulation of protein expression. Below, we will provide a detailed introduction to these main processes.

Synthetic mRNA

The synthesis process of mRNA, also known as transcription, can be divided into three stages: initiation, extension, and termination.

Initiation: The initiation process of transcription involves the unwinding of DNA and the formation of transcription initiation complexes.

Transcription factor binding: During the initiation process, transcription factors bind ligands to the promoter region of DNA. Transcription factors help to locate and recruit RNA polymerase.

RNA polymerase hydrolyzes DNA: Once a transcription factor binds to the promoter region, RNA polymerase binds to DNA, hydrolyzes DNA strands, and forms open complexes.

Elongation: During the elongation stage, RNA polymerase moves along the DNA template chain to synthesize mRNA strands.

Transcription: RNA polymerase synthesizes mRNA strands based on the coding information of DNA. Polymerase moves forward along the DNA template chain, reads codons on the DNA strand, and adds corresponding nucleotides to the mRNA strand being synthesized.

Termination: The termination process refers to the cessation of mRNA synthesis by RNA polymerase and its dissociation from DNA.

Transcription termination signal: There are specific sequence signals on the DNA template strand that indicate that RNA polymerase stops synthesizing mRNA.

RNA polymerase dissociation: Once RNA polymerase recognizes the termination signal, it stops transcription and dissociates the newly synthesized mRNA strand from the DNA template.

It should be noted that there may be some differences in the transcription process for different types of RNA, such as mRNA, tRNA, and rRNA. In addition, the transcribed mRNA needs to undergo subsequent processes such as splicing, modification, and transportation in order to become functional and mature mRNA.

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Transcriptional modification of mRNA

The transcriptional modifications of mRNA mainly include splicing, capping, and tail modification. These modification processes occur in the early or middle stages of transcription, aiming to generate mature and stable mRNA molecules.

Splicing: Splicing is the process of removing introns (non coding regions) from precursor mRNA and connecting encoded exons (coding regions). Splicing is the most important step in mRNA processing. The splicing process is catalyzed by spliceosome, a complex composed of snRNPs (small ribonucleoprotein particles) and other auxiliary proteins.

Splicing site recognition: snRNPs in spliceosomes can recognize the splicing site sequences of introns and exons. The splicing sites include the donor site and the acceptor site.

Splicing reaction: The splicing process involves two steps to complete splicing. Firstly, the adenylate at the donor site of splicing is linked to the adenylate at the recipient site of splicing through a ribosomal ester bond, forming a structure of splicing site adenylate transposition. Then, the phosphodiester bond between the intron and the splicing donor site is cleaved, causing the intron to be cleaved.

Selective splicing: The splicing process can also selectively select different exons for connection, thereby generating different mRNA subtypes. This allows a gene to encode multiple proteins with different functions.

Capping: Capping involves adding a compound called 7-methylguanosine (m7G) to the 5 ‘end of mRNA. This hat structure has the following functions:

Improve the stability of mRNA to prevent degradation by nucleases.

As a recognition marker for mRNA, it assists in transport to the cytoplasm.

Promote the beginning of translation.

The addition of the hat structure is completed through three steps:

The addition of m7G: Methylguanosine triphosphate (m7GTP) is linked to the 5 ‘end of mRNA through enzymatic reactions, forming a 5’ -5 ‘triphosphate chain.

Methylation: After adding the cap structure, m7G is methylated by methyltransferase to form 7-methylguanosine (m7G).

Addition of methylated adenosine monophosphate: On the cap structure, a series of methylated adenosine monophosphates are also added to form a 2 ‘- O-methylated adenosine monophosphate cap. This step is completed by another methyltransferase.

Tail modification: Tail modification is the addition of a poly-A tail at the 3 ‘end of mRNA. This tail has the following functions:

Improve the stability of mRNA.

Promote mRNA transport and ribosome recognition.

Related to transcription termination and mRNA degradation processes.

The tail modification process includes the following steps:

Oval ribosomal signaling sequence (CPS) recognition: There is a CPS located on a specific sequence at the 3 ‘end of mRNA, which is recognized by auxiliary protein complexes.

Methylation: CPS adenosine is first methylated by methylase during tail modification.

Addition of multiple adenosine monophosphates: Multiple adenosine monophosphates are sequentially added to the 3 ‘end of mRNA via phosphodiester bonds, forming a poly-A tail.

RNA binding proteins: RNA binding proteins on the tail can enhance the stability and transport of mRNA.

These transcriptional modifications play an important role in the synthesis of mRNA, enabling stable and accurate expression of protein coding information.

Editing and modifying mRNA

Editing and modifying mRNA refers to the process of altering and modifying synthesized mRNA molecules, typically including the following:

RNA Editing: RNA editing refers to the chemical modification of mRNA molecules, resulting in changes in bases. This modification can alter the coding sequence of mRNA, resulting in mRNA molecules that are not completely consistent with the DNA template after transcription. RNA editing typically involves the conversion of adenosine (A) to inosine (I), especially in the translation region.

ADAR enzyme catalyzed editing: ADAR (adenosine deaminase acting on RNA) enzyme is the most common RNA editing enzyme. It can catalyze the conversion of adenosine to inosine, forming U-I pairing in mRNA.

C to U editing: In some cases, other RNA editing enzymes in cells can also catalyze the editing of cytosine (C) to uracil (U) in mRNA, forming C-U pairing.

RNA Modifications: RNA modifications refer to chemical modifications that occur on mRNA, usually by adding specific chemical groups to the nucleotides of RNA. These modifications can affect the stability, translation efficiency, and subcellular localization of mRNA.

Methylation modification: On mRNA, some nucleotides are added with methyl groups by methyltransferase to form methylated nucleotides. These modifications can affect the stability and transport of mRNA.

Glycation modification: On mRNA, some nucleotide glycosides can also be modified, such as adding N-acetylglucosamine groups, which can affect the degradation rate and translation efficiency of mRNA.

Pseudouridine modification: Some nucleotides of uridine can be converted into pseudouridine, which can affect mRNA translation and transport.

RNA Splicing Factors Regulation: In addition to the splicing bodies during the splicing process, other RNA splicing bodies can also play a role in transcribed mRNA by binding to specific sequences to regulate mRNA stability, subcellular localization, and translation efficiency.

5 ‘Spliceosome: It can bind to the 5’ terminal region of mRNA and affect its stability and translation initiation.

U small ribonucleoprotein (snRNP): can bind near the splicing site of mRNA, affecting the accuracy and efficiency of splicing and modification.

Editing and modifying mRNA is a complex and precise process that can regulate protein expression and function by altering the sequence and structure of mRNA. These changes and modifications are of great significance for the biological processes of cells and the development of diseases.

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