mRNA synthesis from pDNA template

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mRNA synthesis from pDNA template by Mind Map: mRNA synthesis from pDNA template

1. Components

1.1. 5' cap

1.1.1. different styles, most commonly used one (m7GpppN)

1.2. mRNA itself

2. Essential considerations for mRNA vaccine development

2.1. optimizing the translation and stability of IVT mRNA

2.1.1. 5' and 3' UTR (derived from viral/eukaryotic genes)

2.1.2. 5' caps

2.1.3. 3' poly (A tails)

2.1.4. codon optimization enriching the G:C content replacing rare codon with synonyms codon that have abundant cognate tRNA in the cytosol.

2.2. modulating the immunogenicity of mRNA

2.2.1. purification of IVT mRNA

2.2.2. introduction of modified nucleotides

2.2.3. complexing the mRNA with carrier molecules (adjuvants)

2.3. in vivo mRNA vaccine delivery

2.3.1. ex vivo loading of Dendritic cells (DCs)

2.3.2. parenteral injection of mRNA w/ or w/o carriers (polymer particles, liposomes, lipid nanoparticles)

3. Procedures - mRNA synthesis

3.1. In vitro transcription with bacteriophage enzymes (commercial kits available)

3.1.1. within the kit, should have cap sequence available, directly add the cap into the reaction

3.2. Chemical modification

3.2.1. e.g. pseudouridine or other modified nucleotides to reduce the immunogenicity of mRNA itself (the pattern recognition receptors (PRRs) for RNA in cells)

3.3. Purification

3.3.1. lab-scale Commercial purification kits, with precipitation steps followed by the use of ethanol/isopropanol denaturing gel to remove remaining dsRNA or short transcript residue

3.3.2. large scale high performance liquid chromatography (HPLC)