Of course as a life-science researcher you have the knowledge and tools to produce any bio-molecule yourself. However, several issues make this difficult for (m)RNA.

The disadvantages of self-synthesis of (m)RNA

So also for the synthesis of mRNA; the multi-step enzyme-based reaction, with 1 or more purifications, in addition to acquiring a suitable DNA template, can be time-consuming; taking 2-3 days per synthesis.

In addition, especially when a limited amount or number of mRNAs is required, the up-front cost for the reaction ingredients, the quality control, the certified RNAse-free buffers and consumables is high.

But perhaps more than the reasons listed above, very few labs are well-equipped to work with such a delicate molecule, which is subject to degradation to omni-present and hard-to-destroy RNAses. Working with mRNA as a final product in an experimental setting is certainly doable, but multi-step manipulations during synthesis in a sub-optimal environment present a significant risk to the integrity of the final product.

And finally, designing a high-performance mRNA requires very specific knowledge on 5’UTR and 3’UTR functioning, secondary structure formation of RNA, the induction of innate immunity by RNA, codon optimization, ideal A-tail length, etc.

Why choose RiboPro custom mRNA synthesis instead?

By ordering your desired mRNA from RiboPro’s custom mRNA synthesis service you can get started with your experiment as quickly as possible, without hassle! No need to worry about RNAse contamination during production, no need to setup complex quality control, no need to invest in the equipment and materials; we take care of it all. Our RNA experts make sure you get the best results.

Order now with the RNAssembler™

RiboPro’s custom mRNA service is very flexible; we include as much or as little as you need.

Generally speaking, we have 2 flavors:
A. You provide us with a (semi)suitable template, which we use as input for the RNA synthesis. Minor changes to the template to make it suitable are no problem for us.
B. You provide us with the desired sequence, and we perform de novo DNA synthesis to obtain a suitable template for the RNA synthesis.

What is included?

So, in the full-service variant (B) the following is included:
1. mRNA design and optional sequence optimization
2. de novo DNA synthesis
3. DNA prep
4. RNA synthesis
5. RNA capping (up to 99% capped, and Cap1 modification possible)
6. A-tailing to desired length (up to 500nt)
7. 2 purification round
8. (optional) dsRNA removal procedure
9. Quality control (length, sequence verification (DNA), A-tail length, purity)
10. (optional) Aliquotation
11. Packaging & shipping
12. After-care to ensure success in your experiments

This very complete package is available from €660 + shipment cost (typ. €50-€150) + DNA synthesis (typ. €100-€200)

Order now with the RNAssembler™

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.

dsRNA

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).

Contaminants

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.

QC

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!