Enzymatic Synthesis Method for Oligonucleotide

Oligonucleotide in Medicine Research

With the completion of human genome sequencing and the in-depth study of functional genomics and proteomics, molecular targets related to diseases have been continuously discovered and understood, which provides a premise for gene therapy. Oligonucleotides, relatively short nucleic acid fragments with a defined chemical structure (sequence), are fundamental tools for modulating gene expression in biomedical and life science research. In the past few decades, artificially synthesized oligonucleotides such as antisense oligonucleotides (ASO), small interference ribonucleic acid (siRNA), transcription factor decoys, etc. have been used in the medical research of viruses, tumors, and genetic diseases. Among them, ASO and siRNA are commonly used gene regulation tools and have been developed as gene therapy drugs.

Fig.1 ASO-mediated gene regulatory mechanisms. (Roberts, et al., 2020)

Enzymatic Synthesis for Oligonucleotide at CD BioGlyco

At CD BioGlyco, we have developed two methods of Oligonucleotide Synthesis, including Chemical Synthesis and enzymatic synthesis. The chemical synthesis is carried out by solid-phase phosphoramidite triester method, and separated and purified by High-Performance Liquid Chromatography (HPLC). We also use novel thermocycling enzymatic techniques to amplify and mass-produce oligonucleotides. This method uses double-stranded deoxyribonucleic acid (DNA) containing repeated target sequences and enzyme cleavage sites as "seeds". Through a unique "sliding-cutting mechanism", it is extended with a heat-resistant DNA polymerase, cut with a heat-resistant restriction endonuclease, and then processed in a thermal cycle. The oligonucleotides are made to self-replicate like a virus under control, amplifying specific target oligonucleotides. The number of molecules of its amplification products continues to increase exponentially and is not limited by the number of molecules of the initial "seed" oligonucleotide input.

DNA polymerases and endonucleases of the reaction have a wide range of choices and are used to prepare a variety of natural and modified oligonucleotides, such as thio- and fluoro-modified oligonucleotides. Then, the target oligonucleotides in the product are purified and detected by HPLC and mass spectrometry (MS). For double-stranded oligonucleotides, there is no need for HPLC separation, and it is purified by ordinary column chromatography. Therefore, the technology is particularly suitable for the preparation of double-stranded oligonucleotides, such as transcription factor decoys.

Fig.2 Advantages of enzymatic synthesis. (CD BioGlyco)

Applications

• Nucleic acid drug development, such as siRNA and ASO
• Synthesis of oligonucleotides deoxythymidine (dT), bonded to substrates for isolation of mRNA
• Pathogenic microorganism detection, tumor detection, etc.
• Nucleic acid structural studies

Advantages of Us

• CD BioGlyco provides clients with chemical and enzymatic oligonucleotide synthesis services, and oligonucleotide fragments are purified by techniques such as HPLC.
• Our enzymatic synthesis technology has the characteristics of simple operation, high efficiency, and stability.
• The method is suitable for producing large quantities of oligonucleotides of purer composition and less cost.
• We also provide clients with Oligonucleotide Modification services to improve their biological effects.

Relying on advanced equipment and technology, CD BioGlyco provides you with high-quality, customized, and large-scale oligonucleotide synthesis services. We not only provide standard, unmodified oligonucleotides but also produce complex oligonucleotides requiring multiple modifications. If you are interested in our services, please feel free to contact us.