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.

Key Technologies

Our enzymatic synthesis platform is built on a novel, template-independent approach that utilizes specialized enzymes, such as engineered terminal deoxynucleotidyl transferase (TdT), to build oligonucleotide sequences without the need for a DNA template. This technology relies on a carefully controlled, iterative process that incorporates 3'-blocked reversible terminator nucleotides one by one into the growing chain. These specially designed nucleotides act as single-base units, which are incorporated by the enzyme, and their terminating group is subsequently removed to enable the next coupling step. This "controlled enzymatic synthesis" mimics the precise, step-by-step nature of solid-phase chemical synthesis but within a more environmentally friendly, water-based medium.

Where Sugars Meet Sequences: Enzymatic Precision.

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.1 Advantages of enzymatic synthesis. (CD BioGlyco)

Workflow

Our enzymatic synthesis workflow is a streamlined, multi-step cycle designed for precision and efficiency, ensuring the creation of high-quality oligonucleotides.

• Primer Immobilization

The synthesis begins with a short initiator oligonucleotide, often a primer, which is anchored to a solid support. This provides a stable foundation for the chain's growth.

• Nucleotide Incorporation

In the presence of our specialized enzyme and a specific reversible terminator nucleoside triphosphate, a single nucleotide is added to the 3' end of the primer. The reversible terminator prevents the addition of any further bases in this step, ensuring single-base resolution.

• Terminator Removal

The blocking group on the newly added nucleotide is then cleaved, exposing the 3'-hydroxyl group and preparing the chain for the next cycle.

• Repetition

The incorporation and de-blocking steps are repeated iteratively, with a new nucleoside triphosphate added for each cycle, until the desired sequence is complete.

• Final Cleavage and Purification

The final, full-length oligonucleotide is cleaved from the solid support, and the final protecting groups are removed. The product is then purified to the client's specifications.

Publication Data

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.

Frequently Asked Questions

Associated Services

To further enhance functionality while preserving these biocompatibility benefits, our associated service provides seamlessly integrated post-synthesis modifications:

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.