Chemical synthesis of siRNA: synthesis route, purification method, and quality control

RNA interference (RNAi) is a process in which small RNAs inhibit the expression of specific genes. Among them, siRNA is an RNA molecule that can highly specifically and efficiently silence gene expression. Therefore, it is widely used in biomedical research and drug development. Chemical synthesis is an important method for obtaining siRNA molecules, which can provide a large number of uniform and pure siRNA products.

synthetic route

The chemical synthesis of siRNA usually begins with solid-state synthesis, which involves the use of a series of chemical reactions on solid support to synthesize siRNA. The two main methods for solid-phase synthesis of siRNA are phosphate amide ester method and H-phosphonate ester method. Antiviral mechanism

Phosphate amide ester method

The phosphate amide ester method is one of the most commonly used methods in the chemical synthesis of siRNA. Its basic principle is to use phosphate diester or phosphate triester reagents to undergo esterification reaction with nucleotides, forming phosphate amide ester bonds. This reaction requires the use of nucleophilic base catalysts (such as tetraethylamino catalysts) and organic solvents (such as dimethyl sulfoxide or acetonitrile). The advantage of the phosphate amide ester method is that the reaction conditions are mild, the product is easy to purify, and it is suitable for the synthesis of long chain siRNA. However, due to the slow esterification reaction speed and relatively unstable phosphate amide ester bonds, the product may have problems with decomposition and degradation.

H-phosphonate method

The H-phosphonate method is another commonly used chemical synthesis method in siRNA synthesis. Its basic principle is to use H-phosphonate reagents to react with nucleotides to form H-phosphonate bonds. This reaction requires the use of nucleophilic base catalysts (such as triethylamine or isopropylamine) and organic solvents (such as DMF or ACN). The advantages of the H-phosphonate method are fast reaction speed, relatively stable products, and suitability for the synthesis of short chain siRNAs. However, due to the ease of hydrolysis of H-phosphonate bonds, they are easily affected by moisture during the preparation and purification process, which may affect the purity and activity of the product.

Main steps

Solid phase support: Before the synthesis begins, nucleotides and other reagents need to be fixed on the solid phase support material. Solid phase support materials are typically polymer microspheres, such as triethylvinylstyrene (TEVB) or carboxymethyl acrylic acid (CM).

Deprotection: Nucleotides are protected by protective groups during the synthesis process to prevent them from reacting in inappropriate positions. After completing the synthesis of siRNA, these protective groups need to be removed. The method of deprotection usually uses nucleophilic reagents (such as ammonia or methanol) or acidic conditions (such as trichloroacetic acid).

Synthesis: After removing the protective group, nucleotides undergo chemical reactions with other reagents to gradually synthesize siRNA sequences. Common reactions include phosphorylation, phosphorylation, nucleophilic substitution and complementary hybridization.

Washing: After each step is completed, a detergent (such as acetone or methanol) needs to be used to remove the remaining reagents and intermediate products. The washing process helps to ensure the purity and purity of the final product.

Purification: After the synthesis process is completed, purification methods need to be used to remove byproducts and impurities to obtain pure siRNA. Common purification methods include HPLC, ion exchange chromatography and gel electrophoresis.

Purification method

Reversed phase high-performance liquid chromatography (RP-HPLC) is one of the most commonly used purification methods in the chemical synthesis of siRNA. It is a chromatographic separation technique based on polarity differences, which can separate siRNA products from impurities, thereby improving the purity and quality of the products.

principle

RP-HPLC is a chromatographic technique that uses reverse phase as the separation mechanism, and its separation principle is based on the polarity difference of solvents. The stationary phase in the reverse phase column is a highly hydrophilic polar matrix that requires the use of non-polar organic solvents for elution. In RP-HPLC, after the sample is dissolved in an organic solvent, as the polarity of the solvent in the mobile phase decreases, the sample molecules gradually enter the stationary phase and are eventually separated.

application

RP-HPLC is a commonly used purification method in chemical synthesis of siRNA. It can separate siRNA products from the reaction system while removing impurities and unreacted substances. RP-HPLC can be applied in various stages of siRNA synthesis, such as deprotection, synthesis, deprotection, and other steps. In some cases, RP-HPLC can also be used to monitor reaction progress and product purity. The use of RP-HPLC can achieve a high level of purity of siRNA products, thereby ensuring their accuracy and reliability in cell and in vivo experiments.

Its advantages include good separation effect, fast separation speed, good repeatability, high separation degree, and the ability to perform batch operations. At the same time, RP-HPLC also has some drawbacks, such as high equipment cost, requiring a large amount of solvents and columns, and different siRNA products may need to be purified under different conditions. In addition, improper operation of RP-HPLC may lead to issues such as product degradation or loss.

Quality Control

1. Synthesis reaction control: The process of chemical synthesis of siRNA requires multiple chemical reactions, including esterification, phosphorylation, deprotection, and other steps. In each reaction step, it is necessary to strictly control parameters such as reaction time, reaction temperature, and reactant molar ratio to ensure the integrity of the reaction and the purity of the product.

2. Deprotection control: In the process of siRNA synthesis, various protective groups need to be used to protect nucleophilic sites to prevent unnecessary reactions from occurring. During the deprotection process, it is necessary to strictly control the reaction conditions to ensure that the removal of protective groups does not damage the chemical structure of the product.

3. Solvent control: In the process of chemical synthesis of siRNA, different solvents are required for different reactions. In the process of selecting solvents, it is necessary to consider the purity, stability, and impact on reactants and products of the solvents, and strictly control the amount and concentration of solvents used in each step of the reaction.

4. Purification and detection control: For chemically synthesized siRNAs, strict purification and detection are required. During the purification process, it is necessary to choose appropriate methods, such as RP-HPLC, ion exchange chromatography, etc., to remove impurities and impure products. During the detection process, reliable techniques such as mass spectrometry, electrophoresis, etc. are required to verify the purity and sequence of the product.

5. Structural identification control: For chemically synthesized siRNAs, strict structural identification is required, including base sequence, phosphorylation position, etc. During the identification process, reliable techniques need to be used, and the results need to be compared and verified.