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 complete synthesis process of oligos includes preparation (reagents, phosphoramidites, system, and method prep, etc.), synthesis operation, and post-synthesis operation (de-protection, buffer exchange, etc.). This blog will only focus on the preparation part. The information on the synthesis operation can be found in Chemical Synthesis of Oligonucleotides – Part II. And the post-synthesis operation is introduced in Chemical Synthesis of Oligonucleotides – Part III.
Overall, the pre-synthesis preparation includes a couple of steps: material preparation, column packing, and system preparation.
1. Material preparation
- Reagents
| Reagents | Comments |
| Acetonitrile (ACN) | used for system storage, system prep, and as wash buffer for each of the steps in a cycle |
| Detritylation reagent | 3% dichloroacetic acid (DCA) in Toluene |
| Cap A | 20% N-Methylimidazole in ACN |
| Cap B1 | 40% Acetic Anhydride in ACN |
| Cap B2 | 60% Lutidine in ACN Cap B1 and B2 are to be mixed before a run starts |
| Oxidation reagent | 0.05 M I2 in Pyridine |
| Thiolation reagent | 20% Diethylamine (DEA) in ACN |
| Activator (BTT) | 0.3 M BTT in ACN |
- Phosphoramidites
Phosphoramidites are the building blocks for oligonucleotides. For DNA chemical synthesis, dA, dC, dG, dT phosphoramidites are used; For RNA chemical synthesis, rA, rC, rG, rU phosphoramidites are used.
There are different types of phosphoramidites with different protection groups or even base structures, and depending on the application and the specific oligo sequence, you may choose different amidites.
*Molecular sieves are recommended to be used in amidites, activator, and ACN bottles. Bottle/vial filters are recommended to be used in the reagent and solvent bottles except for the Detrit bottles.
- Support
Two different matrix supports are widely used: polystyrene and controlled pore glass (CPG). Polystyrene has a higher density of binding sites, so it’s good to get a higher yield for short oligo sequences. CPG, on the other hand, has fewer binding sites and is good for the synthesis of longer oligo sequences. gRNA, for example, is normally about 100 nucleotides long, and therefore it is better to be synthesized on the CPG. Also, polystyrene will swell after being exposed to reagents, and CPG, on the other hand, is rigid and non-swelling. Both the polystyrene and CPG support are pre-loaded with the first nucleotide. There are options of dA-, dG-, dC-, dT- or rA-, rG-, rC-, rU- loaded support to choose from depending on the nucleotide of the 3′ end of the target sequence.
- Inert gas supply is needed to keep the system at a positive pressure. Argon, nitrogen, or carbon dioxide can be used.
2. Column Packing
The column packing is very different from the traditional way, where the dry resin is scooped into the bottom part of the column with the pre-calculated amount, and then the top part is capped onto the bottom part. It’s critical to calculate how much resin to put into a specific size of a column, and how much oligo can be produced with the resin packed into the column. Figure 1 shows the calculation of polystyrene support needed at different scales.
3. System Preparation
System preparation is needed before starting a run. This step normally includes turning on the inert gas supply and making sure the system is at the target pressure, purging the lines/tubings with ACN, checking for any leaks in the tubing, etc. All the reagents and amidites are then connected to their specific ports accordingly. The method is drafted with the Unicorn system; the interface is very similar to the Unicorn software AKTA chromatography systems. There are pre-built methods in the software that can be used as a template. Text instructions are used, and the target sequence needs to be put into the method.
After everything is prepared, the synthesis is ready to start. Start the run in the Unicorn system, and then just wait for the run to finish. The processing time is highly dependent on the length of the oligo sequence. The longer the sequence is, the longer the run is. It’s also dependent on the coupling rate of a cycle as well. It may take anywhere from 1-2 minutes to 5-6 minutes per cycle, and the overall process time will then be very different depending on the time it takes to finish each cycle.
Reference:
- “AKTATM oligopilotTM plus User Manual” from GE Healthcare.