Messenger RNA (mRNA) is a type of RNA molecule that carries genetic information from DNA to the ribosomes, where it is translated into proteins. mRNA synthesis is the process of generating artificial mRNA molecules for various applications, such as vaccines, therapeutics, and gene editing. mRNA synthesis has attracted considerable attention in recent years, especially after the development and approval of mRNA-based vaccines for COVID-19. However, using cellular mRNA for exogenous purposes poses several challenges and limitations, such as low yield, instability, degradation, and immunogenicity. Therefore, there is a need for developing efficient and reliable platforms for mRNA synthesis that can overcome these challenges and produce high-quality and functional mRNA molecules.
In vitro transcription (IVT) and chemical synthesis are two main approaches for mRNA synthesis. IVT is a method that uses DNA templates and RNA polymerases to synthesize mRNA molecules rapidly and in large quantities. The DNA templates can be either plasmids or linearized DNA fragments that contain an RNA polymerase promoter upstream of the sequence of interest. The RNA polymerases are usually derived from bacteriophages, such as T7, T3, or SP6, and they can recognize specific promoters and initiate transcription.
IVT-synthesized mRNA requires some post-transcriptional modifications, such as adding a 5' cap and a 3' poly(A) tail, to enhance its stability and translational efficiency. The 5' cap is a modified guanine nucleotide that protects mRNA from degradation by exonucleases and facilitates its binding to the ribosome. The 3' poly(A) tail is a stretch of adenine nucleotides that stabilizes mRNA and increases its half-life. These modifications can be either co-transcriptional or post-transcriptional, depending on the type of cap analogs and poly(A) polymerases used.
Chemical synthesis is a method that uses synthetic nucleotides and enzymes to extend mRNA chains step by step, allowing precise control over the sequence and structure of mRNA. The synthetic nucleotides can be either phosphoramidites or triphosphates, and they can be modified with various functional groups, such as methyl, fluoro, or thiophosphate, to improve the properties of mRNA. The enzymes are usually terminal deoxynucleotidyl transferase (TdT) or reverse transcriptase (RT), and they can catalyze the addition of nucleotides to the 3' end of mRNA.
Chemically synthesized mRNA does not need post-transcriptional modifications as the cap structure and the poly(A) tail can be incorporated during the synthesis process. However, chemical synthesis has lower efficiency and yield than IVT—the reaction is slower and more prone to errors and side reactions. Moreover, chemical synthesis requires more purification steps to remove excess nucleotides and byproducts.
| mRNA synthesis method | IVT | Chemical synthesis |
|---|---|---|
| Principle | RNA polymerase catalyzes the transcription of a DNA template into RNA | Chemical reactions synthesize RNA from monomers or oligomers |
| Advantages | High yield, low cost, easy to introduce modifications | High purity, high accuracy, no transcription errors |
| Disadvantages | Low purity, transcription errors, limited length | Low yield, high cost, difficult to introduce modifications |
| Applications | mRNA vaccines, gene therapy, protein expression | mRNA therapeutics, diagnostics, nanotechnology |
Table 1. Comparison of IVT and chemical synthesis of mRNA
mRNA synthesis requires various enzymes to catalyze the reactions of transcription, capping, polyadenylation, and splicing. These enzymes are essential for the production of functional and stable mRNA molecules that can be translated into proteins in the target cells. The main enzyme for mRNA synthesis is RNA polymerase, which can synthesize RNA from a DNA template. There are different types of RNA polymerases for different organisms and cellular compartments, such as RNA polymerase II for eukaryotic nuclear mRNA. RNA polymerase can initiate transcription at specific promoter sequences and elongate the RNA chain by adding nucleotides complementary to the template strand. Another important enzyme for mRNA synthesis is capping enzyme, which can add a 5' cap structure to the nascent RNA. The 5' cap consists of a 7-methylguanosine (m7G) linked to the first nucleotide of the RNA by a 5'-5' triphosphate bridge. The 5' cap protects the mRNA from degradation by exonucleases and facilitates its recognition by the translation machinery. Poly(A) polymerase is an enzyme that can add a poly(A) tail to the 3' end of the mRNA. The poly(A) tail is a sequence of adenine nucleotides, usually about 200–250 in length, that enhances the stability and export of the mRNA from the nucleus to the cytoplasm. The poly(A) tail also interacts with poly(A) binding proteins that regulate the translation and degradation of the mRNA. Splicing is a process that removes the introns (non-coding regions) from the pre-mRNA and joins the exons (coding regions) together. Splicing is catalyzed by a complex of proteins and small nuclear RNAs (snRNAs) called the spliceosome. Splicing increases the diversity of the mRNA population and the protein products by allowing alternative splicing patterns. To optimize the activity and stability of the enzymes involved in mRNA synthesis, various factors need to be considered, such as the temperature, pH, salt concentration, cofactors, inhibitors, and substrates of the reactions. Moreover, some novel enzymes or enzyme-like molecules have been developed to improve the efficiency and fidelity of mRNA synthesis, such as reversible terminator nucleotides and the enzymes that can incorporate them.
mRNA synthesis platforms have shown great potential and versatility in producing functional and stable mRNA molecules that can be translated into desired proteins in the target cells. However, there are still some limitations and obstacles that need to be overcome, such as the scalability, quality, safety, and regulation of mRNA production and delivery.
Future developments of mRNA synthesis platforms may focus on the following directions: (1) developing new methods and materials for mRNA synthesis, such as enzymatic synthesis, microfluidic synthesis, and reversible terminator nucleotides; (2) improving the efficiency and fidelity of mRNA modifications, such as capping, polyadenylation, splicing, and pseudouridylation; (3) optimizing the purification and quality control of mRNA molecules, such as using affinity chromatography, mass spectrometry, and sequencing; (4) designing and testing new encapsulation and delivery systems for mRNA, such as biodegradable polymers, peptides, and exosomes; and (5) exploring new applications and indications of mRNA-based therapies and vaccines, such as cancer, infectious diseases, and genetic disorders.
