| Synthesis Method | DNA | RNA |
| Short Sequence | Chemical synthesis with oligo synthesizer | Chemical synthesis with oligo synthesizer |
| Long Sequence | Chemical synthesis with oligo synthesizer + DNA assembly and cloning | In Vitro Transcription (IVT) |
For RNA, there are basically two different options: chemical synthesis and IVT. The former one is only good for synthesizing short RNA sequences, normally around or shorter than 100 nt due to the relatively low coupling efficiency. For longer sequences of RNA, IVT is used, which is an enzymatic reaction to synthesize RNA molecules from a DNA template (plasmid DNA) outside of a living cell. mRNA is generally a very large molecule with more than 1000 nucleotides. Therefore, IVT is widely adopted for mRNA synthesis.
A typical IVT reaction includes the following steps:
- Preparation/processing of DNA template: Plasmid DNA (pDNA) is the template used for the transcription. It contains the RNA sequence to be synthesized as well as a promoter sequence where the RNA polymerase can recognize and bind to in order to start the transcription process. Sometimes pDNA comes in circular format and will need to be linearized to be used. Linearization will increase the transcriptional efficiency as well as control over the size and quality of the synthesized mRNA.
- Mixing of components: The DNA template is mixed with RNA nucleotides (ATP, GTP, CTP, UTP, or modified UTP), a DNA-dependent RNA polymerase enzyme that recognizes the promoter, and reaction buffer solution.
- Incubation: The reaction mixture is then incubated at 37°C for a certain amount of time. During the incubation process, the RNA polymerase recognizes and binds to the promoter sequence on the pDNA and starts to synthesize the mRNA molecule. RNA NTPs that are complementary to the pDNA sequence are added one by one to the growing mRNA strand.
- Termination and purification: After mRNA has been synthesized, EDTA is added to the mixture to quench the reaction. Then DNase is added to digest large pDNA molecules into multiple small fragments. The solution is then ready to be further purified to remove all the impurity species, like DNA fragments, enzymes, free NTPs, and buffer components.
There are two steps in mRNA synthesis that can be completed during the transcriptional process or post-transcription: polyadenylation and capping. Capping and tailing of mRNA molecules are crucial for both increasing translational efficiency and molecule stability. The poly(A) tail of mRNA can be encoded directly onto the pDNA sequence, saving an extra step of adding the tail after the transcription process. Co-transcriptional capping of mRNA can be done using modified cap analogs such as CleanCap and Anti-reverse cap analog (ARCA), etc. Post-transcriptional capping can be achieved with enzymatic reactions using enzymes like Vaccinia virus capping enzyme and 2’ O-methyltransferase enzymes.
IVT is widely used for mRNA synthesis for both vaccine and therapeutic applications. One of the key advantages of IVT is that it can generate large amounts of RNA in a short amount of time.