All about oligos desalting!

During the process of oligonucleotide synthesis, purification, or storage, oligos can become contaminated with salts, which can interfere with downstream applications. Desalting methods are therefore essential to remove these salts and ensure the purity and quality of oligonucleotide samples. There are several methods available for desalting oligonucleotides, each with its advantages and limitations. Here are some of the more commonly used methods for oligonucleotide desalting:

Dialysis: Dialysis is a traditional method for desalting oligonucleotides. In this method, the oligonucleotide sample is placed in a dialysis membrane that allows small molecules like salts to pass through while retaining the larger oligonucleotide molecules. The sample is then dialyzed against a buffer solution to remove the salts. Dialysis is a gentle and effective method for desalting oligonucleotides but can be time-consuming and requires careful handling of the dialysis membrane.

Spin Columns: Spin columns are another popular method for desalting oligonucleotides. In this method, the oligonucleotide sample is loaded onto a spin column containing a desalting resin that selectively binds salts. The column is then centrifuged, and the purified oligonucleotide is collected in the flow-through. Spin columns are quick, easy to use, and can desalt oligonucleotides efficiently. However, the desalting capacity of spin columns may be limited and their price is relatively high, therefore they may not be suitable for large-scale desalting.

Precipitation: Precipitation is a simple and cost-effective method for desalting oligonucleotides. In this method, salts are removed by adding a precipitation reagent such as ethanol or isopropanol to the oligonucleotide sample. The sample is then centrifuged to pellet the precipitated salts, and the purified oligonucleotide is recovered from the supernatant. Precipitation is a rapid method for desalting oligonucleotides but may result in lower yields compared to other methods. Moreover, precipitating from sodium acetate also exchanges oligonucleotide counterions for sodium, which is preferred for oligonucleotides destined for biological applications.

Size Exclusion Chromatography (SEC): Size exclusion chromatography is a chromatographic method that separates molecules based on their size. In this method, the oligonucleotide sample is loaded onto a size exclusion column, and salts are eluted first, followed by the purified oligonucleotide. Size exclusion chromatography is a powerful method for desalting oligonucleotides and can also separate oligonucleotides of different sizes. However, this method may require specialized equipment and expertise.

Solid-phase extraction (SPE): il this method, the oligonucleotide sample is loaded onto a C18 cartridge in a triethylammonium acetate buffer. Due to the high lipophilicity of the triethylammonium cation, oligos a retained on resin while salts are eluted. The desired deslated oligos are then eluted using organic solvents and lyophilized. However, the process may be more time-consuming due to the number of washing steps that need to be performed.

In conclusion, desalting oligonucleotides is a critical step in ensuring the purity and quality of oligonucleotide samples for various applications. Researchers can choose from a variety of desalting methods based on factors such as sample volume, purity requirements, and equipment availability. By selecting the appropriate desalting method, researchers can obtain high-quality oligonucleotide samples free from salts for their experiments. Please note, however, that desalting does not remove short-mers resulting from incomplete sequence elongation. It is therefore always best to purify oligos using a secondary method, such as ion-pairing HPLC, anion exchange chromatography, or gel electrophoresis.