Oligonucleotides have been researched in the field of life sciences and biopharma for their therapeutic properties for decades and have surfaced as promising therapeutic agents for the treatment of various diseases, including neurodegenerative disorders, respiratory disorders, cancer, and diabetic retinopathy.
Although gene silencing has been the focus of most oligonucleotide therapies, alternative approaches, such as splicing modification and gene activation, are being studied, broadening the universe of potential targets beyond those typically accessible to conventional pharmacological methods.
There are several classes of nucleic acids that are being investigated for oligonucleotide-based therapies, such as antisense, small interfering ribonucleic acid (siRNAs), and aptamers.
What are oligonucleotides?
Oligonucleotides are short deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules that have several uses in forensic science, genetic research, and molecular diagnostics. These tiny pieces of nucleic acids, which are frequently produced in the lab by solid-phase chemical synthesis and can be produced as single-stranded molecules with any user-specified sequence, are essential for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, molecular cloning, and the use of molecular probes.
In nature, oligonucleotides are typically found as small RNA molecules such as microRNAs that control the expression of certain genes or as degradation intermediates produced by the breakdown of larger nucleic acid molecules.
Importance of Phosphoramidites Oligonucleotide Synthesis
For the development of oligonucleotide therapeutics, DNA and RNA oligonucleotides are chemically synthesized using nucleic acid monomers known as phosphoramidites.
Phosphoramidites are highly reactive derivatives of nucleosides and the building blocks of oligonucleotide sequences. They are the best option when it comes to raw materials because they have the best protective groups.
Usually, the chemical yields oligonucleotide sequences that are 200–300 bases long. However, stability and precision decline as the length of the produced oligonucleotide sequence increases.
The best method for producing short sequences is phosphoramidite chemistry. It has been holding this position as the industry benchmark for DNA synthesis for the past 30 years.
For the creation of synthetic oligonucleotides that can then be used in a variety of different sectors, such as primers for a polymerase chain reaction, oligonucleotide-based therapeutics, and genetic engineering, phosphoramidite chemistry is fundamentally important.
Hospitals, pharmaceutical companies, and research labs use millions of synthetic oligonucleotides.
Since the demand for primers and short-length DNA sequences is increasing within the pharmaceutical, synthetic biology, and molecular diagnostic industries, it is doubtful that phosphoramidite chemistry will be displaced by future technologies.
DNA synthesis was developed in the last century and is essentially used for the discovery and engineering of biological processes. A sequence of oligonucleotides made up of individual phosphoramidite monomers is typically 60–100 nucleotides long.
The global phosphoramidite market is anticipated to expand significantly due to the rising need for oligonucleotides, the expanding synthetic biology market, and the rising prevalence of many diseases creating an urgent need for novel therapeutic solutions.
As per the BIS Research analysis, the global phosphoramidite market is projected to reach $2.9 billion by 2032 from $900.3 million in 2021, growing at a CAGR of 7.78% during the forecast period 2022-2032.
Types of Phosphoramidites Used for Oligonucleotide Synthesis
A typical phosphoramidite consists of four different protecting groups that provide the structure stability, along with a phosphite rather than a phosphate.
When necessary, the protective groups are eliminated, allowing DNA chemists to exert tight control over the synthesis process and expose just reactive centers to form an oligonucleotide.
Currently, several modified phosphoramidites are being offered, each with distinctive characteristics optimized for custom oligonucleotide synthesis. A few of them are discussed further in the article
1. DNA Phosphoramidites: DNA forms the basis of living organisms. The most often employed phosphoramidites in the sector are those derived from DNA. Solid-phase DNA synthesis is made easier and quicker due to the stable nucleic acid monomer structure that allows oligonucleotides to be activated into highly reactive components.
They possess a variety of chemical configurations and are capable of chemically reconstructing the resulting structure into the four synchronized phosphate groups present in DNA molecules.
The speed at which the internucleosidic network and selectivity form has significantly increased because of DNA phosphoramidites. Hence, it is appealing to use synthetic DNA for oligonucleotide synthesis.
2. RNA Phosphoramidites: Automated chemical ribonucleic acid (RNA) synthesis was introduced in the early 1990s by Cruachem Ltd. Demand for RNA phosphoramidites was initially constrained by development difficulties and unfavorable outcomes. The level of difficulty increased in comparison to conventional DNA synthesis as one more 2'-hydroxy group was added.
When bases are deprotected and synthesized, it is essential to preserve the protective group of 2' hydroxyl in ribose since, under normal circumstances, RNA reveals hydrolytic instability. Due to this, the 2' hydroxyl group location has protective groups in all RNA synthesis methods.
RNA has emerged as a major force in the pharmaceuticals sector in recent years. Following the development of a successful messenger ribonucleic acid (mRNA) COVID-19 vaccine, interest in RNA as a medicinal modality has surged.
3. Labeled Phosphoramidites: Typically, labeled phosphoramidites are used to create labeled oligonucleotides with emission spectra and absorption for a wide variety of applications, including microscopy and polymerase chain reaction (PCR).
They are substantially more expensive than DNA and RNA phosphoramidites and are not easily accessible commercially. Incorporating residues at certain positions in RNA and DNA sequences makes labeled phosphoramidite synthesis more beneficial than enzymatic synthesis.
They can be produced chemically by combining ribose and nucleobases either enzymatically or chemically or by sourcing labeled nucleosides from bacteria on a labeled medium. Additionally, rather than labeling the nucleoside evenly, the method can be used to incorporate isotopes in specific places.
Conclusion
In the fields of research, therapy, and diagnostics, there is a growing need for oligonucleotides. The demand for synthetic DNA is rising as the fields of gene sequencing, synthetic biology, next-generation data storage, and pharmaceuticals advance.
As phosphoramidites serve as raw materials for DNA synthesis, the global phosphoramidite market is subjected to grow significantly in the DNA and RNA oligonucleotide industry.
