In the aspect of preparing mRNA in vitro transcription templates, the selection of restriction endonucleases is mainly based on the following factors:
1. The cut site should be such that the 5' end protrudes or is flush with the end. The presence of the 3' protruding end will increase the generation of false by-products during in vitro transcription (IVT) from the template body.
2. The recognition sequence of the restriction enzyme cleavage site should be as long as possible; the longer the recognition sequence, the less likely it is to appear in the gene fragment.
3. IIS-type endonucleases are the preferred enzymes for linearizing plasmids in vaccine development, as their cleavage sites are outside the recognition sequence, ensuring the integrity of the designed poly(A) tail. After linearizing DNA templates using IIS-type endonucleases, no "scars" will remain, and no unnecessary nucleotides will be added during the IVT process.
4. The endonucleases meet the pharmaceutical production grade requirements (GMP, without animal-derived components, etc.)
At present, for most mRNA development projects, especially vaccine projects, the endonucleases used for linearization of plasmids are still BspQI, BsaI, and XbaI. Among them, BspQI is the most frequently used. It is estimated that there is a certain relationship with the previous designed plasmid sequences from abroad. Of course, it is not limited to these enzymes; we have also encountered cases where other enzymes were used for linearization.