A Complete Guide to Phosphoramidite Chemistry

Since its development, phosphoramidite chemistry has remained the best method for DNA oligonucleotide manufacturing. It is facilitating the chemical synthesis of DNA and RNA universally due to its efficiency. The technology has revolutionized biochemical research and enabled numerous other technologies such as DNA forensics. Therefore, phosphoramidite chemistry is central to developing synthetic methods to create biological molecules.

Here is a look at how phosphoramidite chemistry makes the process of DNA synthesis efficient.

What is DNA synthesis? 

DNA synthesis, also known as RNA oligonucleotides, is the chemical synthesis of structured nucleic acid fragments in the DNA. Within the oligonucleotide, you will find a linear arrangement of nucleotides that are subunits. Natural DNA comprises these nucleotides that form a chemical chain linked by the action of enzymes.

In recent times, the study of nucleic acids has grown into a dynamic scientific enterprise. The nucleic acids are crucial in all biological systems due to the importance of genes in hereditary. In physical terms, nucleic acids are found in higher ordered structures of the DNA. Therefore, an understanding of phosphoramidite chemistry helps trace a person’s genetic sequence during DNA synthesis.

Development of oligonucleotide synthesis

The development of oligonucleotide synthesis started with the phosphoramidite method. It is the primary process used in synthetically creating DNA. The technique is used in synthetic biology and metabolic engineering to synthesize people’s DNA with high efficiency. Moreover, recent technological advancements in solid phase-based synthesis have facilitated large-scale DNA synthesis through phosphoramidite.

The further development of this method for the chemical synthesis of deoxyribose-oligonucleotide created a genetic code that led to the first DNA synthesis since genes are made of DNA. Today, it is easier and cost-effective to synthesize oligonucleotides thanks to the availability of commercial DNA synthesizers.

Scientists developed the technique of solid-phase synthesis and applied it to peptide synthesis. They optimized the process to become efficient when used in automated oligonucleotide synthesizers. Based on this process, successful synthesis lies in anchoring the first monomer to an insoluble polymeric support.

The phosphoramidite method

This is the standard DNA synthesis method used in automated synthesizers. It is popular due to its ability to achieve the high coupling efficiency needed to synthesize longer oligonucleotides. The building blocks used for this synthesis method are phosphoramidite, which is an activated DNA nucleoside. They are the chemical used in modern DNA synthesis since they allow the sequential addition of new bases to the DNA chain. During the synthesis, there are protecting groups that help prevent any undesirable side reactions from occurring.

Before this method, DNA synthesis focused on the quality, length, and yield of DNA produced. However, the synthesis had undesirable side reactions such as branching and mutated oligonucleotide sequence. This was caused by the inefficient use of protecting groups to mitigate the instability of the growing strand. When there is no protection, the process will synthesize undesirable molecules that won’t conform to the recognized DNA structure.

In the phosphoramidite method, we synthesize oligonucleotides on a solid support. The synthesis cycle includes condensation, capping oxidation, and deprotection, which produces a single-stranded oligonucleotide product per column. Therefore, phosphoramidite differs from other methods because of optimal protecting groups.

Phosphoramidite chemistry has been successful due to its simplicity and high efficiency. It produces large volumes of oligonucleotide sequences, which is why it stands as the only commercially viable chemistry. It is essential to know that synthesis provides large volumes of DNA for the synthetic biology market. The current methods of DNA synthesis perform this process using phosphoramidite chemistry as a reliable method of DNA synthesis.

Automation and miniaturization

Automation technology has improved with the phosphoramidite chemistry in DNA synthesis. However, automated RNA synthesis is a little more complex than DNA synthesis due to the hydrolytic instability of RNA. As a result, modern RNA synthesis methods are using different phosphoramidite monomers with protective groups.

In DNA synthesis using phosphoramidite chemistry, you still need automation and miniaturization to manufacture large-scale DNA. Good chemistry is not enough to make enough synthetic DNA. It requires combining phosphoramidite chemistry with proprietary silicon support through robotics, fluid mechanics, and miniaturization.

This is where solid-phase chemistry comes in. The technology in DNA synthesis removes the need for extensive purification of DNA strands at every step. The silicon provides solid support that allows synthesizing multiple oligonucleotides simultaneously. It can be aligned and patterned with ease to create a unique sequence, meaning we can synthesize each differently. Synthesizing modified oligonucleotides involves selecting the right strategy and planning the correct synthetic approach. It starts with retro-synthesis analysis followed by on-support modifications and post-synthetic modifications.

Oligonucleotide synthesis refers to the chemical synthesis of short fragrance. In this field, you cannot separate silicon DNA synthesis technology from phosphoramidite chemistry. The properties of silicon technology allow the adoption of robust commercial structure and incorporate into any technology to improve speed.

Final Thoughts

Researchers are enjoying the benefits of using phosphoramidite chemistry to synthesize DNA. This phosphoramidite chemistry is a highly efficient synthetic method that allows easy access to large-scale DNA synthesis. The discovery of nucleoside phosphoramidite offered a powerful tool in DNA synthesis by providing more stability. This method involved phosphite with four protecting groups that provide stable protection. It allows DNA chemists to produce only DNA-like molecules when desired and have control over the synthesis chemistry.