For short strands of either RNA or DNA that are around or less than 100 nucleotides, chemical synthesis is the most commonly used method. The entire process involves adding one nucleotide at each cycle onto a solid support, and the number of cycles depends on the length of the RNA or DNA strand. For each cycle, there is a coupling efficiency that indicates the success rate of adding that nucleotide to the existing “pool” of RNA or DNA sequences. The coupling rate is usually higher for DNA synthesis, which can be as high as 99 – 99.5%. For RNA synthesis, the coupling rate decreases to about 98 – 98.5%. Although this difference may seem small, with an increase in cycles, the yield can vary significantly. Figure 1 shows the yield vs. cycles for 98% and 99.5% coupling efficiencies. With a 20mer, the difference is only between 90% and 67%. However, with a 100mer, we are talking about 60% vs. 13% purity. Then after 200 cycles, it’s 37% vs. 1.8%.
The complete process of oligo synthesis includes preparation (reagents, phosphoramidites, system and method prep, etc.), synthesis operation, and post-synthesis operation (de-protection, buffer exchange, etc.). This blog will focus only on the synthesis operation. The other two steps will be covered in later posts.
During oligo synthesis, there are multiple similar cycles, with the only difference being the type of phosphoramidite for that specific cycle, which depends on the sequence of the oligo. The commonly used solid support, controlled pore glass (CPG) or polystyrene, already has the first nucleotide loaded onto the support. Therefore, if the system is provided with a 20mer sequence, it will start the first cycle from the second nucleotide on the 3′ end. And the total number of cycles will be 19 rather than 20. There is also an alternative support that comes “naked”, and one of them is the Unylinker. With these alternative supports, the number of cycles equals the length of the sequence.
In each cycle, there are four different steps: detritylation, activation and coupling, oxidation, and capping. There are phosphoramidites of different protection groups, but the process of each cycle is very similar (see Figure 1 for a typical RNA chemical synthesis).
dimethoxytrityl; Bp – natural or modified, protected, nucleobase; PG- protecting group1
- Detritylation – The 5′ DMTr group on the fifth carbon in the sugar ring is removed from the terminal residue on the solid support, making the 5′ hydroxyl group available for coupling with the incoming amidite.
- Activation/coupling – The incoming amidite is activated with an activator (5-benzylmercaptotetrazole (BMT) is often used due to high coupling efficiency) to allow for subsequent coupling. In the activation process, the pronated diisopropylamino was replaced by the nucleophilic tetrazole, which then in the coupling process is replaced by the nucleophilic 5′ hydroxyl group on the terminal residue. This forms a 3′-5′ phosphite triester link to the growing oligonucleotide.
- Oxidation – The phosphite triester is oxidized to a phosphotriester using iodine/water in pyridine. This step changes the unstable P(III) phosphite triester link into a more stable P(V) structure.
- Capping – Because the coupling efficiency is not 100%, there are still some terminal sequences that don’t have the nucleotide added at each cycle. If these unreacted species aren’t capped, they will continue to participate in the following cycles, and there will be heterogeneous n-1, n-2 … oligo products at the end that are hard to be removed in the purification process. Therefore, after each cycle, unreacted 5′-hydroxyl groups are converted to acylates, which is a permanent protection group, and the capped sequences wouldn’t continue adding new nucleotides in the following steps.
After one cycle, the terminal sequences on the solid support will enter the next cycle, where detritylation, activation/coupling, oxidation, and capping steps happen again to add the next nucleotide onto the current sequence. After all the cycles are completed, the operation on the system is finished, and the product is ready to be taken off from the system and for the subsequent de-protection procedure.
Reference:
- Flamme, Marie, et al. “Chemical methods for the modification of RNA.” Methods 161 (2019): 64-82.