mRNA Preparation

If you want to synthesis your own mRNA in lab, NEB HiScribe® T7 High Yield RNA Synthesis Kit is the most stable and cost-effective kit to use.

Materials:

1. DNA template encoding your mRNA of interest
2. Enzymes (T7 polymerase, DNase)
3. Nucleotide triphosphates (ATP, CTP, GTP, UTP)
4. Reaction buffers

Procedure:

1. Linearize your plasmid DNA: Use a restriction enzyme to linearize your plasmid. Purify the linearized plasmid (this blog specifically talks about the mRNA production plasmid).
2. In Vitro Transcription (IVT): Set up IVT reaction with DNA template, T7 polymerase, and nucleotides. Incubate the reaction to synthesize mRNA.
3. DNase Treatment: Remove template DNA by treating the reaction with DNase. Purify mRNA using methods such as column purification or precipitation.
4. Quality Control: Assess mRNA integrity using gel electrophoresis. Measure concentration using a spectrophotometer.

Lipid Mix Preparation

We have an amazing summary about the development of lipid formulation. Feel free to check out!

Materials:

1. Lipids:

– ionizable lipid:

– helper lipid

– PEG-lipid

2. Organic solvents (ethanol, chloroform)
3. Buffer solution

Procedure:

1. Lipid Dissolution: Dissolve lipids in an organic solvent mixture to create a stock solution.
2. Mixing Lipids: Combine ionizable lipid, helper lipid, and PEG-lipid in the desired molar ratio. Evaporate the organic solvent to form a thin lipid film.
3. Hydration: Hydrate the lipid film with an appropriate buffer to form a multilamellar vesicle (MLV) suspension.
4. Liposome Formation: Extrude the MLV suspension through a membrane to form small unilamellar vesicles (SUVs).

mRNA-Lipid Mixing

Materials:

1. Prepared mRNA
2. Liposome suspension

Procedure:

1. Lipid-mRNA Complex Formation: Mix the mRNA with the liposome suspension at the desired charge ratio. Incubate the mixture to allow the formation of mRNA-LNP.
2. Optimization: Test different charge ratios to optimize mRNA encapsulation efficiency.

mRNA-LNP Purification

Materials:

1. Purification columns or methods (size exclusion chromatography, ultrafiltration)
2. Buffer solutions

Procedure:

1. Purification: Purify the mRNA-LNP mixture to remove excess lipids and impurities. Use size exclusion chromatography or other suitable methods.
2. Concentration: Concentrate the purified mRNA-LNP solution using ultrafiltration if necessary.

mRNA-LNP Size Measurement and Storage

Materials:

1. Dynamic light scattering (DLS) instrument
2. Cryoprotectant (e.g., sucrose)
3. Storage containers

Procedure:

1. Size Measurement: Use DLS to measure the hydrodynamic size of mRNA-LNPs. Confirm a narrow size distribution.
2. Cryoprotection and Storage: Add a cryoprotectant to the mRNA-LNP solution. Store aliquots at -80°C or in liquid nitrogen for long-term storage.
3. Quality Control: Periodically assess mRNA-LNP stability, size, and encapsulation efficiency during storage.