Commercial RNA is typically synthesized by chemical synthesis (phosphoramidite chemistry) or biosynthetic approaches (in vitro transcription; IVT). Chemical RNA synthesis is the only widely available approach for introducing site-specific modifications and facilitates the incorporation of a vast number of modifications. However, due to the numerous chemical steps required for the sequential addition of nucleotides, synthesis of RNA greater than 100 bases quickly becomes impractical and cost prohibitive. In vitro transcription utilizes natural biological processes for greater than 10,000 base long RNA. This is the primary RNA synthesis process used by the RFC.
AcceleRNA: Accelerated RNA Manufacturing
There is a growing need for research and discovery grade RNA for screening and early validation studies. We have developed a two cell-free manufacturing platforms to dramatically reduce production timelines and facilitate high-throughput, small-scale production. Typical scales range from 100 µg to 1 mg.
High-throughput AcceleRNA (HT-AcceleRNA) can provide custom RNA synthesis in about 12 business days. We also offer a higher-fidelity, more flexible HF-AcceleRNA platform that can provide custom RNA synthesis in about 16 business days.
Traditional RNA Production
While AcceleRNA provides rapid production, high scale and high purity RNA products benefits from traditional cell-based DNA production processes. There are also multiple options for DNA production, given the budget and needs of your research.
(1) New Gene Synthesis
This is the most common approach, where you simply provide a protein or gene sequence. The initial time and cost involved in DNA synthesis process is only required once.
(2) Provide Gene Template Only
If you already have the gene template, we may be able to clone it into our standard backbone in-house. If it does not fit with our rapid in-house protocols, we have our templates archived with commercial vendors for outsourcing. While this is more time consuming, it affords cost savings. Please provide us the sequence for us to evaluate.
(3) Provide DNA Template
Most teams do not have a gene template that works with our workflows; however, we offer a kit for groups to insert and assemble compatible templates in-house. Users can then provide the DNA template for RNA synthesis. While there are some limitations, this is preferred for many academic groups with molecular biology capabilities.
Lab-Scale RNA Production, Big-Pharma Approach
Many research labs lack strict regulatory controls over in-house RNA production, leading to untracked or unknown variables that can lead to inconsistent RNA production and uncertainty in results. Using industry-level production standards, the RFC offers high batch-to-batch consistency and quality in RNA products. Strict quality control measures and proprietary workflows ensure that the desired product is delivered with high quality and reproducibility .
Flexibility in RNA Synthesis
Capping
The Cap structure in mRNA refers to the 5′ cap, a crucial modification at the beginning of the molecule. Cap1 involves methylation of the initial nucleotide, providing intracellular stability to the mRNA and is essential for efficient translation initiation, contributing to the overall regulation of gene expression. In the RFC, co-transcriptional capping methods are typically used for mRNA synthesis. Compared to enzymatic capping methods, this minimizes RNA handling to ensure high integrity products. However, intellectual property surrounding these methods may be undesirable during commercialization. Therefore, intellectual property (IP)-free enzymatic capping is also available upon request.
Untranslated Regions
The untranslated regions (UTRs) of mRNA play crucial roles in gene expression. The 5′ UTR primarily contains regulatory elements influencing translation initiation. While the 3′ UTR also plays a role in translational control, it is also critical for stability, localization, and is subject to antisense RNA regulatory processes. Importantly, UTRs can be cell-specific and several UTR designs are restricted by IP. Therefore, we offer RNA production using IP-protected, IP-free, or your own designs.
Poly-A Tailing
The poly-A tail consists of a string of adenine nucleotides at the 3′ end. It protects against canonical nuclease degradation pathways and facilitates efficient translation. It can also pose a challenge for template production, as homopolymer tracts such as the poly-A tail are hotspots for recombination events. Typically, the tail is template-encoded, as it simplifies synthesis and ensures RNA is produced with a discrete-length tail, but post-transcriptional tailing is also possible.
Modified Nucleotides
Modified nucleotides play diverse and complex roles in RNA, such as translocation, translation, and immune tolerance. While numerous naturally occurring modifications exist, their roles are largely yet to be understood. Immune tolerance modification such pseudouridine and N1-methyl pseudouridine have become popular in mRNA production to increase the therapeutic window of mRNA. In fact, the discovery of such immune-modulating modifications by Karikó and Weissman unlocked the potential for the COVID-19 mRNA vaccines, leading to the 2023 Nobel prize in Physiology or Medicine for the pair. Generally, the RFC incorporates N1-methyl pseudouridine into mRNA products, but modalities such as saRNA and circRNA rely on secondary structures that can be disrupted by such modifications. A variety of modifications are available at the RFC and can be introduced at any ratio compared to their natural NTP counterparts.