On July 17, 2023, Novo Nordisk announced that it would enter into a collaboration with a startup company founded in 2020, Eleven Therapeutics, to “identify new medicines that can deliver nucleic acids”. Today, let’s take a close look at the technology offered by Eleven Therapeutics, including both its advantages and challenges.
The idea of the “DE-Liveri” technology is to use a high throughput concept to quickly generate a lot of RNA sequences with different combinations of modifications of nucleotides on the “TERÅ beads”, while having the DNA sequence on the same bead signifying the specific sequence of RNA. There are two novel aspects of this technology:
- High Throughput Production Technology
Their website only uses uridine as an illustrative example of the whole process, so we’ll just focus on that. There are two types of modified uridine, 2’F-uridine and 2’OMe-uridine. By splitting the beads into two pools at each cycle, it is possible to generate different RNA sequences. The theoretical total number of different RNA sequences is dependent on the number of cycles. With one cycle, there are 2^1=2 distinct RNA sequences, either 2’F-uridine or 2’OMe-uridine. With n cycles, there will be 2^n distinct RNA sequences. The exponential effect of splitting is what makes the technology high throughput.
- Signaling RNA Sequence with DNA
The signaling of the RNA sequence is achieved through grouping modified uridines with DNA molecules in each cycle, i.e., 2’F-uridine RNA is always added together with pyrimidine DNA, and 2′-OMe-uridine with purine RNA. Therefore, the DNA sequence will be directly correlated with RNA in a certain manner on the same bead.
Novel though the thought is, there are many challenges or many questions to bring up for more discussions:
- Anyone familiar with chemical synthesis of oligos knows that the two have different coupling efficiencies. With DNA chemical synthesis, we could expect >99.5% efficiency in each cycle, but in RNA, the number is only around 98%. Though it seems to be close, if we are synthesizing a long chain, it can make a whole lot of difference. Let’s do a simple math here. If we are to produce a chain of 100 nt for both DNA and RNA, and we assume 99% efficiency for DNA, and 98% for RNA, the final yield after 100 cycles would be 36.6% for DNA and 13.3% for RNA! This also means that we cannot guarantee that we get the same length of DNA and RNA on a single bead. For example, it is very possible that the addition of a new RNA nucleotide is not successful at the 10th cycle, and the sequence is capped to avoid further addition. While the DNA synthesis doesn’t stop until it has reached the 20th cycle. Also, another thing to note is that, on the same bead, it’s also possible to have two or multiple different lengths of RNA molecules as well. With all these discrepancies, it is crude to say that we can rely on the “barcode”.
- Not much information was found about the “TERÅ beads”. However, in the traditional method with controlled pore glass (CPG) or polystyrene beads, once DNA or RNA is synthesized, there will be extra steps for cleavage and deprotection, which often involve very harsh treatment with ammonium hydroxide, HF acid, etc. Eleven Therapeutics mentioned on their website about incubation of beads with cells and then release the RNA oligos and DNA barcodes from the beads. It would be hard to imagine cells unaffected by any of those treatments.
- By adding DNA and RNA nucleotides together to a chemical synthesis cycle, how can we avoid creating a chain of mixed DNA and RNA nucleotides? With the similar structural and chemical properties of DNA and RNA nucleotides, it is very likely to produce a chain of both. It’s not clear what measures Eleven Therapeutics is taking on this potential issue.
Overall, the technology Eleven Therapeutics is offering is very interesting and has the potential for high throughput RNA screening to identify promising candidates. However, there are still many challenges that the new startup company needs to address. Looking forward to more updates on this “DE-Liveri” technology.