Origin of Solid-phase Chemical Synthesis Method

In the field of biology, the synthesis of DNA and RNA primarily relies on DNA/RNA polymerase enzymes. While the replication of DNA (as observed in techniques like the polymerase chain reaction) or RNA can be accomplished in substantial quantities through in vitro simulations, certain challenges arise. These include issues with sample purity, the requirement for specific templates during synthesis, and the complexity of introducing chemically modified nucleotides. In response to these challenges, researchers have embarked on investigating the creation of short-chain DNA/RNA molecules (referred to as oligonucleotides) exclusively through organic chemical processes, excluding the need for DNA/RNA polymerases and templates.

Fig.1 Synthesis of oligonucleotides on the solid phase. (Qiu, et al., 2013)

Key Technologies

Our synthesis platform is built on the robust and highly efficient phosphoramidite method, a solid-phase technique that is the industry standard for oligonucleotide production. This method involves anchoring the first nucleotide of the sequence to a solid support, which acts as a stable foundation for the growing chain. This approach allows for the use of excess reagents to drive each reaction to near-completion, while unreacted reagents and impurities can be simply washed away.

Solid-Phase Precision, Nucleotide by Nucleotide.

CD BioGlyco has a professional and constantly updated Glyco™ Synthesis Platform to provide Custom Carbohydrate Synthesis services for clients around the world, including Custom Sugar-nucleotides Synthesis, Custom Oligosaccharides Synthesis, Custom Polysaccharides Synthesis, and other high-quality services. At CD BioGlyco, we provide high-quality Oligonucleotide Synthesis services based on chemistry methods for clients. Chemistry method-based oligonucleotide synthesis techniques often require the use of controlled pore glass (CPG). CPG is covalently bonded to the 3'-OH of the initial nucleotide ribose through a linker, while the 2'-OH of ribose is protected with a protecting reagent. The 5'-OH is protected with dimethoxytrityl (DMT). In addition, adenine, guanine, and cytosine also require protection with acyl reagents (such as benzoyl) due to the presence of primary amino groups. The solid-phase synthesis service we provide mainly includes the following four steps.

• Deprotection
  Use trichloroacetic acid (TCA) or dichloroacetic acid (DCA) dissolved in dichloromethane/toluene to remove the DMT group at the 5' ribose, exposing the 5'-OH for the next coupling step. Deprotection time depends on flow rate and column dimensions.
• Coupling
  The phosphoramidite monomer of the next nucleic acid reacts with the tetrazole to form a reactive intermediate. The 5'-OH formed during deprotection attacks the active intermediate nucleophile, then forms a new phosphorus-oxygen bond, and removes the tetrazole to extend the nucleotide chain.
• Oxidation
  After the coupling reaction, the newly added nucleotides are connected to the oligonucleotide chains on the CPG through trivalent phosphorus. The 2-cyanoethyl protecting group in the phosphodiester linkage makes the phosphite bond more stable in the subsequent synthesis. The commonly used oxidizing agent is tetrahydrofuran//pyridine/water solution of iodine. It is also converted to P (pentavalent) by transferring a sulfur atom to P (trivalent), thereby forming a phosphorothioate bond.
• Capping
  Since it is impossible to achieve 100% coupling efficiency, there are still 5'-OH active groups that have not reacted after deprotection. If not treated, these groups are still coupled in the next cycle to generate impurities. Two reagents are usually used (capping reagent A, usually acetic anhydride; capping reagent B, usually N-methylimidazole, the catalyst for the acylation reaction) to acylate the 5'-OH. Reagents A and B, stored separately, are mixed during the capping step and delivered to the system.

Workflow

Fig.2 Process of oligonucleotide synthesis by phosphoramidite method. (CD BioGlyco)

Publication Data

Applications of Solid-phase Chemical Synthesis Method

• The technology is helpful for the research on the synthesis of nucleic acid primers.
• The technology is applied in the field of nucleic acid probes, such as the rapid detection of viruses.
• The technology is applied in the development of nucleic acid medicines such as gene therapy.

Advantages of Us

• The technology allows the efficient synthesis of large numbers of oligonucleotide sequences.
• Oligonucleotides synthesized by the solid-phase synthesis technology are highly pure and specific. This is very important in genetic engineering and drug development.
• The technology allows the incorporation of artificially modified nucleic acids that do not occur in nature.

Frequently Asked Questions

Associated Services

To extend functionality for advanced therapeutic, diagnostic, and research applications, we offer specialized associated services enabling targeted modifications:

CD BioGlyco has first-class technology and well-trained technicians. Our dedicated team of experts works tirelessly to provide the Chemical-based Oligonucleotide Synthesis Service that meets clients’ needs and requirements. CD BioGlyco provides high-quality oligonucleotide synthesis services based on solid-phase chemical synthesis method, especially solid-phase phosphoramidite chemistry method. If you are interested in our services, please feel free to contact us for more details.