Purity Analysis of Oligonucleotides

Oligonucleotides are typically composed of short-chain nucleotides (deoxyribonucleotides or ribonucleotides) of less than 20, serving as short RNA and DNA oligomers. They can be used as probes to determine the structure of DNA or RNA, and are used in processes such as gene chips, electrophoresis, and fluorescence in situ hybridization.

 

Oligonucleotides are widely used in biology, agriculture, medicine, and other fields mainly through chemical synthesis. Currently, automatic oligonucleotide synthesizers have been developed to quickly synthesize various oligonucleotide fragments and fluorescently labeled oligonucleotides. In this process, each step can introduce impurities, including unreacted raw materials, reaction by-products, degradation products, and oligonucleotides of different sequences. These impurities not only cause inaccuracies in the quantification of oligonucleotides but may also affect subsequent biological experiments. Therefore, the synthesized oligonucleotides need to be purified to ensure the accuracy of subsequent experiments.

 

The current methods of oligonucleotide purification are roughly divided into two types. One is the conventional control of oligonucleotide quality and length for separation, and the other is the specific adsorption of the 5′ hydroxyl incompletely removed dimethoxytrityl (DMT) protecting group.

 

Control of Oligonucleotide Quality and Length

The existing methods for controlling oligonucleotide quality and length include polyacrylamide gel electrophoresis (PAGE) technology and high-performance liquid chromatography (HPLC) technology. Both of these methods are based on the speed difference of oligonucleotides of different lengths moving in the medium to separate the target oligonucleotides, and the throughput is usually not high.

 

1. PAGE

Due to the difference in charge and size of each molecule, it overall affects their migration speed in the gel. Larger fragments move slower, and after a certain electrophoresis time, fragments of different sizes can be separated.

 

Characteristics: The operation is complex, the analysis time is long, and it is difficult to realize automated separation and analysis; the resolution is high, and there is no limit to the molecule length.

 

2. HPLC

HPLC is currently the main method for separating and purifying oligonucleotides. The separation and purification index is the separation degree between the target and the impurities. The current HPLC method uses TEAB as an ion pair reagent to separate and purify oligonucleotides. This method is also called reverse phase ion pair (IP-RP) chromatography.

 

Characteristics: It has a high degree of automation and saves manpower, and the product purity is high. However, the purification volume is small, the speed is slow, the cost is relatively high, and the efficiency of mass production is not high. Also, due to the memory effect of the chromatographic column, cross-contamination needs to be considered.

 

Specific Adsorption Based on the DMT Group

The target oligonucleotide can selectively carry the DMT group after the synthesis of the last base, and the prematurely terminated oligonucleotide does not carry this group. This difference can be recognized by the medium that specifically binds the DMT group, thus only harvesting the target oligonucleotides. This method has a high throughput but is only suitable for shorter oligonucleotides, and an additional step to remove the DMT group is needed after purification.

 

1. OPC Column Purification

It uses the affinity of the column material and the DMT at the 5′ end of the synthesized primer to specifically adsorb the oligonucleotide molecules containing DMT. Therefore, during the subsequent processing after synthesis, care should be taken not to remove the DMT and remove it during the purification process.

 

Characteristics: Salts and short fragments can be efficiently removed, and the purified product can achieve high purity, which can be used for most molecular biology experiments. This method is fast, efficient, and can achieve automation. However, it is limited by length, column capacity, and limited purification.

 

Desalination Purification

For oligonucleotides without DMT protecting groups, the collected oligonucleotide solution after electrophoresis separation and anion exchange liquid chromatography purification needs further desalination. If the synthesized oligonucleotide has a very high quality, it can be directly desalinated and purified, that is, only non-DNA impurities are removed, which will not have much impact on downstream operations. The desalination method generally uses solid-phase extraction (SPE) technology.

 

1. SPE

The principle is that oligonucleotides can specifically adsorb on reverse phase adsorbents, can be eluted by organic solvents, but cannot be eluted by water. Therefore, they can effectively remove salts.

 

Characteristics: Suitable for oligonucleotides with higher purity, effectively removes salts, cannot remove fragments, and is a fast and simple desalination method.

 

Oligonucleotide purity analysis is an important link in the field of biomedicine and plays a key role in ensuring the quality and safety of oligonucleotides. With the continuous development and innovation of biotechnology, methods for analyzing oligonucleotide purity are also constantly improving and optimizing. MtoZ Biolabs has developed and validated methods for analyzing oligonucleotide purity based on the high-resolution liquid chromatography platform and the principle of purification analysis. Depending on the different oligonucleotide samples, appropriate column lengths, mobile phase flow rates, separation temperatures, and ion pair reagent buffer components can be selected to identify impurities such as n-1 short bodies, n+1 long polymers, incomplete sulfation, and side reaction produced diesters caused by reaction failure. For more oligonucleotide services, Free project evaluation!