We would be happy to use your DNA plasmids for the synthesis of your mRNA sequence as this may save time and money.

For us to utilize your plasmid in the synthesis of high-quality mRNA, your plasmid should meet the following requirements:

• T7 promoter upstream of the 5′ UTR
• T7 promoter should preferably be followed by three G nucleotides ….TATA(GGG) in case you desire enzymatic capping.
• For capping by means of trinucleotide cap analogues, we require T7 promoter immediately followed by AG, as in ….TATA(AG)…, or followed by AU, as in ….TATA(AU)… depending on the variant of the cap analogue.
• A 5′ and 3′ UTR sequence flanking the gene of interest, optionally including a translation initiation site, such as Kozak, in the 5’UTR
• Optionally an encoded poly(A) tail of no longer than 120nt (please note that we cannot guarantee exact poly(A) length)
• Linearization site after 3’ UTR. Preferably a type IIS restriction site creating a 5’ overhang (Please check if the enzyme cuts at the correct position of the template strand (often the bottom strand reading 5′ to 3′ from the T7 promoter)).

Other requirements:

• Name: Please inform us of the name of the restriction enzyme to be used.
• Format: Circular plasmid. Please do not perform linearization yourself as residual restriction enzyme may cause issues in our process.
• Quality: single band on agarose gel of correct size. In particular, avoid nicked pDNA, which shows up on gel as secondary (higher running) bands.
• Quantity: Approximately 7 µg of plasmid for 1 mg of RNA production.
• Vector backbone: Provide the name and map. Please inform us on GMO classification. (Does the coding DNA sequence encode a harmful gene product or contain the complete genome of a viral pathogen classified as risk group 4 ,3 or 2?*)
• Insert Gene: Is it encoding a toxic or viral protein?

Plasmid Handling Guidelines:

• If possible, supply the plasmid lyophilized.
• If lyophilization is not feasible, please provide in RNase-free water at a concentration of 100 ng/µL.
• Ensure the plasmid is high-purity and endotoxin-free
• Please label the tube with the plasmid and insert names.

If you are unsure whether your plasmid meets the requirements, feel free to contact us.

At -20°C the mRNA is stable for approximately one month, while at –80°C the mRNA is stable for at least a year. We advise to aliquot the mRNA sample the first time you thaw the mRNA, so that the efficacy and stability of the product does not decrease by repeated freeze-thaw cycles.

We can synthesize custom mRNA sequences starting from 100nt up to 15k nt.

After the formulation and dialysis we measure the size distribution of the LNP sample via Dynamic Light Scattering (DLS), we also have the option to measure the zèta potential (electrokinetic potential) and encapsulation efficiency assay upon request. An internal control LNP is always formulated during a production run and allows us to validate the activity and toxicity of the LNPs on HeLa cells.

We measure the purity of mRNA by spectrophotometry (A260/280>2, A260/230>2.2). We also validate the size of the mRNA product and the absence of any by-products via RNA gel electrophoresis. An internal control mRNA is always synthesized during a production run and allows us to validate the activity of the sample on HeLa cells.

As the dose is cell-type-dependent, the dose you use for one cell-type might not give you equal results in another. Therefore, we advise to test and try at least 3 doses of mRNA and determine linearity for the specific cell type you are using. A safe range to stay in for a well-designed mRNA is around 50-70 ng/96-well, although some cells will tolerate up to 200-250 ng per 96-well. At RIBOPRO, we often use 10, 50, and 100 ng to determine the optimal dose. Keep in mind that the dose scales (linearly) with a larger surface of cells.
For in vivo use, the species and the target organ make a huge difference in the recommended dosing. For mice, we generally recommend staying below 1-2ug/25gram animal.