The performance of a messenger RNA, to be added to cells via standard transfection methods, greatly depends on the design of the sequence, the quality of the synthesis, chemical modifications of specific bases and the purity of the end-product. Introducing a sub-optimal mRNA into cells is known to trigger the innate immune response; resulting in lower protein expression, cellular toxicity and the release of pro-inflammatory cytokines.
High performance mRNA
For the best performance, we recommend the following combination of features to be selected for your custom mRNA:
• 5’UTR: no upstream ATG, kozak or similar present directly upstream of the start codon. For example RiboPro catalogue 5’UTRs.
• 3’UTR: selected from mRNAs known to be extra-ordinary stable.
• Cap1 – 95%.
• 120-150nt A-tail.
• dsRNA reduction.
These parameters will provide you with a decent expressing mRNA. However, for any of the following situations:
• therapeutic applications,
• a demand for high protein-expression,
• experiments incompatible with pro-inflammatory cytokine release,
• or any other application requiring the best protein expression and the lowest toxicity,
we recommend additional mRNA-specific sequence-optimization. Using RiboPro’s patent-pending technology allows for 5x more protein (as high as 40x more in specific applications) and significant reduction in pro-inflammatory cytokine production.
If you like to understand why these settings lead to the best performance, read more below.
The trouble avoided by RiboPro’s high-quality RNA synthesis
Synthesizing a high-performance messenger RNA requires optimization all through-out the multi-step synthesis, including specialized purification.
First of all, during the enzymatic production process, dsRNA can be formed. dsRNA is one of the main triggers of innate immunity, primarily via TLR3 (endosomal recognition during transfection), OAS1, PKR and MDA5 (cytoplasmic sensors). RiboPro’s mRNA synthesis process results in the lowest amount of dsRNA possible. The method used to reduce the amount of dsRNA, also negatively affects the yield of ssRNA, so limited additional costs do apply compared to the standard method, because we increase the scale in order to obtain the guaranteed yield. Without dsRNA reduction, protein expression is typically 10-fold lower than with dsRNA reduction.
Uncapped, triphosphorylated RNA
Secondly, currently no supplier can promise that 100% of all mRNA molecules will be capped. When uncapped, the 5’UTR of the mRNA contains a 5′ triphosphate, which can be a ligand for the cytoplasmic innate immune sensor RIG-I. RiboPro offers 2 capping grades (~80% and 95%+). If selecting the lower grade (cheaper), additional phosphatase treatment may be required to reduce RIG-I activation.
Cap methylation status
Third, if a cap is present, but no additional methylation of the first nucleotide (Cap0), then IFIT1 may be activated in certain cells, leading to around 30-50% reduction in activity in some experiments. This seems to be particularly relevant in cells of the immune system. RiboPro offers optional methylation of the first nucleotide (Cap1).
Finally, template DNA, salts and small RNA species (formed during abortive cycling of the RNA polymerase) must be carefully removed to avoid interfering with uptake and interpretation of results. RiboPro uses extensive DNAse I-based DNA removal, reducing the residual DNA to far less than 1 millionth of the RNA concentration.
Next to an optimized production process, RiboPro also has also developed a series of test for quality control, offering you peace of mind about the identity, purity and modification status of your order.
Do you have additional questions? Please contact us directly below or later via the contact page. We are happy to help!