Realize your dream project with mRNA

Affordable high-service

for all your mRNA needs

the fastest way to get high-quality, ready-to-use mRNA

 
 

 

 

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We design, synthesize and formulate high-quality mRNA

Full-service mRNA synthesis by mRNA-specialists

RiboPro is a full-service provider of custom and off-the-shelf messenger RNA (mRNA) and long-non-coding RNA (lncRNA). Our service contains 4 key elements, that you may order in combination or as stand-alone service.

mRNA design

We design mRNA with the desired expression profile (high-low, long-short, conditional) by exchanging UTRs and/or optimizing the codon usage in the coding sequence.

Particular attention is given to de-immunization of the mRNA, which can be achieved equally well with sequence engineering, as with modified nucleotides.

Effects of mRNA design

mRNA design influences expression a lot; the 5'UTR, the 3'UTR, the codon usage, secondary structure, type of capping and A-tail length all influence performance (total amount of protein expression) as well expression kinetics (expression duration, peak- vs - steady-state expression, etc.).

Sequence design by RiboPro

By choosing for RiboPro, you get access to mRNA experts that can turn any wild-type coding sequence into a high-performance mRNA. They will either design the complete mRNA, or just the parts (e.g. the UTRs) you request.

Our method

When designing your mRNA, our scientists use proprietary software to test in silico every combination of tried and tested UTRs and coding sequences designed with our patent pending de-immunization method until they find an mRNA that fits your application.

mRNA synthesis

We use highly innovative, proprietary methods to synthesize high-quality, high-purity full-length mRNA.

Our enzyme-based methods produce mRNA virtually devoid of dsRNA, with high capping efficiency and well-defined poly(A)-tails, from either de novo or customer delivered DNA templates. If needed, we can arrange de novo DNA template synthesis.

High purity full-length RNA synthesis

RiboPro has developed a proprietary system using in-house developed enzymes with enhanced stability, activity and fidelity combined with progressively optimized protocols to ensure efficient synthesis of pure, full-length mRNA. These protocols allows for the production of short (around 100nt) to extremely long (15k nt) RNA transcripts with a minimum of side-products and contaminants. Any residual impurities are removed by our triple purification system, reducing such impurities at least 1000-fold.

Capping

To turn the basic RNA transcript into a fully functional mRNA, capping (addition of a 5' cap) and A-tailing is required. We offer 2 methods of capping; with our enzymatic Vaccinia capping system >99% capped mRNA can be achieved for uncomplicated 5'UTRs, whereas complicated 5'UTRs (with major or very stable secondary structure) can be capped co-transcriptionally with ARCA (Anti-reverse cap analog) or another cap analog. RiboPro has developed a proprietary method to ensure >95% capping with ARCA (100% correct orientation), where traditional protocols provide 50-80% capping via ARCA.

A-tailing

For the poly(A)-tail, we offer an enzymatic method producing a narrow distribution of fully-customizable A-tail lengths, and a template-based method producing a highly defined A-tail length. Frequently requested lengths (120/150nt) can be provided from stock templates.

Formulation services

Any mRNA purchased from RiboPro can be formulated into a selection of nano-particle technologies.

These technologies include cationic peptides, cationic or ionizable polymers and lipids. Our specialty is formulation in lipid nanoparticles (LNPs) of which we have several standard formulations available that display high mRNA delivery efficiency.

Our formulation service includes:

  1. Producing nanoparticles of defined size
  2. Buffer exchange (via dialysis)
  3. Dilution or concentration
  4. Quality control (size by DLS)
  5. Packaging and shipping

You may choose from various options:

  1. Buffer
  2. Sterile filtering
  3. QC-assays: Zeta-potential/TNS/RNA-inclusion
NB. We do not formulate mRNA obtained elsewhere.
CRO services

Not everyone has access to a lab or the time to do basic mRNA testing. Or, you may lack mRNA-specific knowledge or equipment. When ordering mRNA from RiboPro, you don't have to have any of that; you have access to RiboPro's CRO services.

All our CRO services are executed by experienced scientists.

RiboPro offers the following mRNA-focused (standard) CRO services.

  1. mRNA activity testing
  2. Protein expression profiling
  3. Immunological activation testing
  4. Toxicity testing (in vitro only)
  5. Release from bio-material testing

 

Other tests/assays may be available upon discussion. Contact our RiboProfessionals to learn what we can do for you!

Custom mRNA synthesis to your specifications

What is included in our custom mRNA synthesis service?

Hover on item to learn more

  1. Free mRNA designAny modification made by RiboPro’s mRNA specialists to your RNA sequence is free of charge, regardless whether it is a single nucleotide mutation, amino acid mutation, replacement of the UTRs and/or sequence optimization of the coding sequence. With a single exception; for commercial use of an RNA sequence optimized according to our patent-pending sequence de-immunization method, we offer a customer-friendly license against an affordable royalty.
  2. De novo DNA synthesis if needed

    If you choose to use our coding sequence de-immunization, and/or a high number of other modifications, and/or if you do not have a suitable DNA template available, de novo DNA synthesis may be the fastest and most affordable way to go. In each of these cases, we would be happy to arrange de novo DNA synthesis as part of our service. Once we have the DNA template on file, next orders of the same sequence can be delivered in 1 week and at reduced cost.

    If you do have a suitable template available, you may send us 1ug of high purity DNA for each 200ug of RNA you order. Once the DNA arrives, we perform several quality checks, linearize the DNA and start RNA synthesis.

  3. RNA synthesisThis is the core of our service; synthesizing long (>100nt) RNA molecules using RNA polymerases from a DNA template. Crucial benefits from our service include a very low amount of dsRNA, improving safety and protein expression, and a very high percentage of full-length RNA molecules. The product of this step can already contain an A-tail (template based) and/or a cap (co-transcriptional capping with ARCA). When these are not added yet, they can be added in the next steps.
  4. A-tailing to desired lengthThe poly(A)tail is a crucial element of any messenger RNA, because it stabilizes the binding of the Ribosome during the very first steps of translation. During translation, the A-tail is typically shortened, placing an expiration date on the mRNA functionality. By choosing for longer A-tails, the expression duration can be enhanced. However, longer A-tail make the entire RNA longer and therefore more fragile to intra-molecular degradation and/or damage. A good balance is often between 120 and 150nt of poly(A)tail.
  5. Capping (ARCA or enzymatic)Next to a proper A-tail, the cap is another essential structure for mRNA translation to protein. Thus a high capping efficiency ensure good protein expression. Even more so, RNA molecules missing a 5’cap often display a 5’triphosphate which can be highly immunogenic, resulting in lower protein expression and pro-inflammatory cytokine release. Therefore, RiboPro uses 2 different methods to ensure high capping efficiency; 1) our enzymatic capping system capable of capping >99% of RNA molecules with a native cap in the proper orientation, and 2) our co-transcriptional capping method using anti-reverse cap analog (ARCA) that is particularly suited for 5’UTRs with complicated or very stable secondary structure. Using our proprietary protocol, called ARCA-Active, we can achieve >95% capping efficiency, whereas the typical ARCA capping efficiency lies in the 50-80% range.
  6. Triple purificationFor most applications it is important to remove as many reaction components, such as salts, buffers, stabilizing proteins and enzymes, from the final (m)RNA product to ensure proper folding, transfection and/or protein expression. In our process, the (m)RNA is purified 3 times with 3 different methods, each designed to remove a particular type of potential contamination, to deliver the most pure RNA possible. Our triple purification system sets itself apart by the high quality of the final product, as well as its flexibility in scalability and desired concentration.
  7. Quality control (length, A-tail length, purity)Since mRNA synthesis is a complex multi-step process, quality control is extremely important to ensure consistent quality. We perform quality control on the length (agarose or PAGE gel against known controls/ladders), A-tail length and purity (spectrophotometrically against known controls with similar nucleotide composition). De novo DNA is Sanger sequence verified or NGS verified by our supplier. Additional QC is available upon request.
  8. Packaging and shipping

    The finished product is aliquoted in 1 or more screw cap tubes, depending on preference. We typically recommend aliquoting in multiple tubes as a countermeasure against RNAse-contamination. The tubes are individually labelled and allow visual inspection of the content.

    Because of the sensitive nature of (m)RNA molecules, we ship on dry ice (-80oC), which is the golden standard for (m)RNA storage. We pack our products so that they survive at least 5 days at ambient temperature. Therefore, we only ship on Mondays to avoid materials being thawed over the weekend. Our ultra-pure RNA is stable for multiple days at 4oC and for weeks at -20oC, so alternative shipping solutions are available upon request.

You may choose from various options:

  1. Cap modification (Cap1)The 5’cap is an important single (inverted) nucleotide structure at the 5’end of the mRNA, which serves as an achoringpoint for the Ribosome during translation. The majority of mammalian mRNAs, in particular those of higher animals like humans, are modified on the first 1 or 2 nucleotides after this cap. The reason for this is not completely known, but it at least facilitates the recognition of ‘self mRNA’ versus viral RNA. Therefore, for specific more-sophisticated cell-types (usually immune cells), modification of the first nucleotide after the standard cap results in a higher protein expression and less intracellular innate immune activation. Without modification the cap is known as Cap0, with the first nucleotide modified it is known as Cap1.
  2. DephosphorylationThe fraction of RNA that is not capped; either by-design or as a consequence of the production process, typically carries a triphosphate. A 5’triphosphate is a strong activator of RIG-I, resulting in reduced protein expression and pro-inflammatory cytokine release. Therefore, we recommend the optional dephosphorylation for all custom RNA products with a significant portion of uncapped RNA.
  3. Desired concentration and bufferThe RNA is standard delivered in RNAse-free water at a concentration of either 0.5 or 1mg/ml. If you require another buffer and/or concentration, this is possible.
  4. Aliquoting into multiple tubesAs a countermeasure against RNAses, we generally provide the RNA product into multiple aliquots. You may request the number and size of aliquots during the ordering process. We recommend not to make any aliquot smaller than 20ul.
  5. Custom tube labelling for identificationIf you require a special annotation on your tubes, you may request that during ordering. Up to 30 characters will be printed on the label, alongside the regular information, which includes manufacturing date, gene name, concentration and solvent.
What is our turn-around time for custom mRNA?

As scientists ourselves, we understand the pressures you are under, and time is of the essence. Therefore, we optimized our process to finish mRNA synthesis in 2-3 days.

If you send us a ready-to-use template arriving Wednesday latest, you may expect your product to be send the next Monday.
If your order requires de novo DNA synthesis, it typically takes 2-3 weeks (incidentally up to 4 weeks due to COVID-19 related busyness at our DNA synthesis partner) to complete your order. Please be aware that certain sequences present difficulties, due to secondary structure formation, repeats and other sequence features, and may require revised synthesis conditions, resulting in some delay. Of course, we always update you if and when this happens.

Because mRNA is a sensitive molecule, we ship on dry ice and are therefore bound to shipping on Mondays.

How does it work?

At RiboPro, we are committed to making your life easier. Thus, we are working hard on also making the ordering of mRNA quick and easy. At the moment, you may order in the following ways:

  1. Assemble your mRNA with the our mRNAssembler™
  2. Send us an email via the contact form detailing your needs

Next, we get to work: we check your sequences, assess the work to be done and provide you with a quotation within 24h (mostly within 2 hours, so monitor your mailbox).
If you agree with the quotation, we get started immediately with at-risk production while you arrange a P.O. number, or we wait for the P.O. number to be arranged (based on your preference).

During production, we do the following things:

  • Optimize the sequence if desired
  • Communicate optimized sequences back to you (optional)
  • Order DNA if needed
  • Perform RNA synthesis
  • Perform Quality Control
  • Arrange shipping
  • Check if material has arrived complete and correct
  • Send invoice
  • Follow-up to help you with your experiments if needed

For planning purposes, you will receive updates at steps 2, 4 and 6.

Our 3 service levels explained