For short strands of either RNA or DNA that are around or fewer than 100 nucleotides, chemical synthesis is the most commonly used method. The complete synthesis process of an oligo includes preparation, synthesis operation, and post-synthesis operation. This blog will only focus on the post-synthesis operations. Information on the preparation and synthesis operation can be found in “Chemical Synthesis of Oligonucleotides – Part I” and “Chemical Synthesis of Oligonucleotides – Part II“.
Assuming everything ran successfully during the synthesis process, after that, the column is taken down from the system and dried through vacuum. The beads, together with the synthesized oligos attached to them, are collected into either a bottle or tube. Following that, the two most important steps are cleavage and deprotection. Cleavage is to detach the oligos from the solid support (beads), and deprotection is to remove the protection group on the 2′-hydroxyl site.
For DNA, cleavage and deprotection can be completed in a single step with ammonium hydroxide incubation overnight at 55°C. Then filter the content through a glass filter, and collect filtrate containing the detached oligos. Through buffer exchange to ethanol/water, and drying on a rotary evaporator, the oligonucleotide is ready to be processed through downstream purification steps.
For RNA, cleavage is completed in the same manner, with overnight ammonium hydroxide incubation. However, to deprotect the 2′-hydroxyl protecting group of RNA, hydrofluoric (HF) or HF-related acid is often needed, which poses a very dangerous chemical hazard. For example, the widely used 2′-OTBDMS protection needs HF or TBAF (fluoride) for the 2′ deprotection. There are other options with different protection groups that require much milder reagents. PivOM, for example, only requires a base to deprotect, TC needs ethylenediamine, and ALE uses hydrazine hydrate. The table below shows a list of different protection groups and 2′ deprotection methods. However, they may have different coupling efficiencies and therefore need to be selected based on specific applications.
| 2′-PG | 2′ deprotection |
| privaloxyloxymethyl (PivOM) | Base (NH4OH) |
| 2-(4-Tolysulfonyl)ethoxymethyl (TEM) | TBAF (fluoride) |
| 2-cyanoethoxymethyl (CEM) | TBAF (fluoride) |
| Cyanoethylated (CE) | Base (NH4OH) |
| tert-butyldimethylsilyl (TBDMS) | TBAF (fluoride) |
| tris-iso-propylsilyloxymethyl (TOM) | TBAF (fluoride) |
| tert-butyldithiomethyl (DTM) | 1,4-dithiothreitol or tris(2carboxyethyl)phosphine (following NH4OH treatment) |
| bis(2-acetoxyethyloxy)methyl (ACE) | pH 3, 10 min., 55 °C |
| thionocarbamate (TC) | ethylenediamine |
| (o-nitrobenzyloxymethyl) (2-Nbom) | Photolabile |
| p-nitrobenzyloxymethyl (4-Nbom) | TBAF |
| acetal levulinyl ester (ALE) | Hydrazine hydrate |
| 2-cyano-2,2-dimethylethanimine-Noxymethyl (CDEO) | TBAF |
| iminooxymethyl ethyl proponate (IEP) | TBAF |
Reference:
- Table adopted from: Flamme, Marie, et al. “Chemical methods for the modification of RNA.” Methods 161 (2019): 64-82.