Comprehensive Guide to Oligonucleotide Synthesis: Techniques, Optimization, and Purification Strategies

Oligonucleotide synthesis is a sophisticated chemical technique used to construct short-stranded DNA or RNA molecules for a wide range of applications in biomedical research, molecular diagnostics and therapeutic drug development. The process mainly relies on solid-phase phosphoramidite chemistry, in which nucleotide monomers are added step by step through a solid-phase carrier to generate the target sequence. In the following, we will discuss its development and industrialization progress in detail from the synthesis principle, carrier selection, optimization method and purification technology.

Synthesis Principle and Core Steps

Oligonucleotide synthesis relies on solid-phase phosphoramidite chemistry on controlled pore glass (CPG) or other solid-phase carriers.

Figure 1. Solid phase phosphoramidite oligonucleotide synthesis reaction cycle^[1].

The core steps of oligonucleotide synthesis are as follows:

A. Solid phase carrier preparation

Solid-phase carriers immobilize the 3' end of the oligonucleotide chain, and a common material is CPG, whose high porosity and surface area provide ample active sites for the reaction. Novel carriers such as highly loaded polymers (NittoPhase HL) and magnetic nanoparticles (MNP) are also gradually being applied for different needs.

B. Deprotection

Removal of the 5'-hydroxyl protecting group (e.g. DMT) of nucleotides using trichloroacetic acid (TCA) or other acidic reagents provides the reaction site for coupling. Optimization of conditions is key to improving synthetic efficiency.

C. Coupling

Reaction of activated nucleotide monomers with exposed 5'-hydroxyl groups to form phosphate bonds. Tetrazoles are commonly used as activators in conjunction with organic solvents (e.g., THF or DMSO) for efficient coupling under controlled moisture conditions.

D. Closure

The unreacted 5'-hydroxyl group is blocked by acetic anhydride and 1-methylimidazole to prevent false extensions and reduce n-1 impurity generation.

E. Oxidation

Phosphite bonds are oxidized to phosphate bonds by iodine solution to enhance chain stability. This step directly affects the physicochemical properties of the target product.

F. Ammonolysis and protecting group removal

Upon completion of the synthesis, the solid phase carrier bonds are severed and residual protecting groups are removed using ammonia treatment.

G. Purification

Oligonucleotide synthesis may produce impurities such as single base deletions (N-1 impurity) and incorrect conversion of phosphodiester bonds to phosphorothioate bonds (P=O impurity). These impurities may affect the purity and function of oligonucleotides. Purification by methods such as HPLC or PAGE removes single base deletions or incorrectly modified strand impurities generated during synthesis to ensure high purity.

Selection and Comparison of Solid Phase Carriers

Solid-phase carrier is a key component of oligonucleotide synthesis, and its performance directly affects the synthesis efficiency, yield and purity. The following is a comparison of different carriers:

Optimization of Key Steps

During oligonucleotide synthesis, optimization of the synthesis steps to reduce impurities and increase yields is at the heart of technology development. Optimization of deprotection and coupling steps to improve synthetic efficiency and reduce impurities can be done in the following ways.

Selection of suitable deprotection conditions:

• Use a weak base solution such as 28% ammonia for deprotection at appropriate temperature for a short period of time to reduce the risk of degradation.
• For oligonucleotides with 3'-amino joints and basic variable nucleotides, potassium carbonate methanol solution can be used at room temperature can efficiently remove the protecting groups at room temperature.

Optimize coupling reaction conditions:

• Use of high purity tetrazole or iodine-TMP activators ensures reaction efficiency and rate of phosphate bond formation.
• Maximize coupling completion by drying the reaction mixture (<100 ppm residual water) to reduce n-1 impurity levels.
• Increasing the amount of thiourea to 1.5 and 3 molar equivalents in the coupling reaction while maintaining 3 molar equivalents of base improves coupling efficiency.

Optimization of capping and oxidation steps

• The use of efficient oxidizing agents such as iodine rapidly converts the phosphite to phosphate and enhances the chemical stability of the oligonucleotide chain.

Improvement of purification steps:

The Role and Advantages of Automated Equipment

Modern oligonucleotide synthesis relies on automated equipment such as the Applied Biosystems 394 or the Versatile Integrated Platform. The working principle is mainly based on the solid-phase phosphoramidite method, which is an automated synthesis of oligonucleotides (DNA or RNA) via a computer-controlled reagent delivery system. Specifically, the device adds different bases such as A, T, C, and G sequentially to the solid support according to a preset program, and after the steps of decapsulation, activation, coupling, capping, and oxidation, the target nucleic acid is finally obtained by cutting off and removing the protective groups from the solid-phase carrier.

In order to improve the accuracy and efficiency of the synthesis process, automated oligonucleotide synthesis equipment employs a variety of technological tools:

• Microreactor chip technology.
• Solid phase synthesis method.
• Automated control and monitoring.
• Purification and recovery technology.
• Versatile integrated platform.

Conclusion

The continuous development of oligonucleotide synthesis technologies has significantly increased the efficiency of their application in research and industry. From carrier selection to optimization of key steps to the introduction of modern purification and automation equipment, each improvement has had a profound impact on enhancing the quality and economy of synthesis. In the future, through the further integration of new chemical reagents, efficient carriers and intelligent equipment, oligonucleotide synthesis will provide more powerful technical support for precision medicine and nucleic acid drug development.

Related Products & Services

Our products and services are for research use only and cannot be used for any clinical purposes.

Online Inquiry

* Name

Phone

* Email

* Services & Products of Interest Comprehensive Guide to Oligonucleotide Synthesis: Techniques, Optimization, and Purification Strategies

Project Description

* Verification Code

Submit