Phosphoramidite Considerations

Oligonucleotides (oligos) are short pieces of DNA and RNA crucial for the development of diagnostic kits as well as nucleic acid therapeutics. Oligos are commonly manufactured by chemical synthesis, which relies heavily on phosphoramidites as building blocks. Here, we outline the key considerations regarding phosphoramidites for manufacturing oligos for diagnostic and therapeutic applications.

Oligos can be synthesized in two ways: chemically and biosynthetically. Chemical synthesis, which is the industry-wide standard for oligo synthesis and the focus of this article, occurs through chemical reactions using phosphoramidites as the building blocks. Biosynthesis occurs via enzymatic reactions and is still in early phases of development.

The process of solid-phase chemical synthesis of oligonucleotides consists of the steps summarized in Figure 1:

1. Deblocking of the solid support
2. 3′ activation and coupling
3. Oxidation of the 5′–3′ linkage to the phosphotriester bond
4. Capping of the unreacted 5′-OH
5. Repeating steps 1–4 until the final base is added
6. Cleavage of the oligo from the solid support and removal of all protecting groups

These steps are the same whether you are performing small-scale or large-scale synthesis of oligos for use in both diagnostic applications and therapeutic drugs.

DNA and RNA oligos can be used in various diagnostic applications for genetic diseases, infectious diseases, pharmacogenomics, and more. Oligos, which are commonly used as primers and probes in these diagnostic techniques, bind specific sequences—primarily nucleic acids but sometimes proteins and small molecules (as in the case of aptamers)—for target detection. These diagnostic assays commonly use oligo primers and/or probes in techniques such as PCR, microarrays, sequencing, and in situ hybridization.

Another application of oligos is in therapeutics, where short DNA and/or RNA molecules modulate the expression of target RNA through antisense oligos (ASOs) or small-interfering RNAs (siRNAs) or guide the Cas9 enzyme to its target via guide RNA (gRNA) in CRISPR technology. These therapeutic oligos have been approved or are currently going through clinical trials to treat diseases such as muscular dystrophy, polyneuropathy, and hypercholesterolemia.

DNA or RNA oligo manufacturing begins with designing an oligo of a specific nucleic acid sequence (with modifications as needed), to chemically synthesize, purify, and analyze. This is followed by formulation and production for its specific application. Automated oligo synthesizers complete sequential chemical reactions using phosphoramidites to produce the nucleotide chains of synthetic oligos. Typically, oligo synthesizers add phosphoramidites as the building blocks from the 3′ to 5′ direction, the opposite of enzymatic synthesis. One advantage of synthesizing oligonucleotides chemically is that modifications or labeling of the building blocks necessary for final oligo function can be more easily introduced than through enzymatic methods.

Certain aspects of phosphoramidites should be considered when they are used in the manufacturing of oligos for diagnostic purposes. At minimum, your phosphoramidite supplier should be able to accommodate small, manageable starting synthesis scales, with the eventual ability to scale to the industrial scale required for many diagnostic kits. Options for base modifications, linkers, and dye labeling should also be available. Table 1 describes some modifications that are common for phosphoramidites as used in diagnostic applications.

Table 1. Phosphoramidite modifications of oligos for diagnostic applications.

Phosphoramidites used for therapeutic applications should be considered with some attributes different from diagnostic usage in mind. First and foremost for therapeutics, the raw materials must be produced under a refined quality system (e.g., ISO 9001:2015) with extensive testing for consistency, purity, traceability, and stability, to help meet requirements set by regulatory bodies. In addition, manufacturing must be scalable, especially when a therapeutic is ready to go into clinical trials and eventually manufacturing.

Because of their therapeutic uses, phosphoramidites are often modified to increase nuclease resistance and thermal stability while lowering their immunogenicity. Such modifications include 2′-O-methyl (2′-OMe), 2′-O-methoxy-ethoxy (2′-MOE), and 2′-fluoro. For delivery to target organs, conjugating moieties such as N-acetylgalactosamine (GalNAc) to the oligos for uptake (e.g., in the liver, by hepatocytes) is a common approach.

In addition to these modifications, controlling impurities that can be generated during phosphoramidites synthesis is critical to the development of your therapeutic. Impurities are defined by their reactivity in oligonucleotide synthesis or by the ability to be removed during the manufacturing process. They can be categorized as:

a. Critical impurities

• Potentially are incorporated into oligonucleotides
• Contain amidite functionality and acid-labile protecting group

b. Noncritical impurities

• Cannot be incorporated into oligonucleotides
• Could also be reactive but are removed in processing

A top concern for developers of oligo therapeutics would be impurities that are reactive and critical. Nonreactive and noncritical impurities as well as reactive but noncritical impurities are less of a concern. The presence of reactive and critical impurities can be limited through manufacturing processes implemented by phosphoramidite suppliers, such as rigorous control of raw material specifications, in-process manufacturing controls and testing, and final release testing.

Phosphoramidites are critical raw materials in the synthesis of oligos for diagnostic and therapeutic applications. Considerations for phosphoramidites depend on intended applications of the oligos, whether they are for diagnostics or therapeutics. Partnering with a phosphoramidite supplier that has years of experience and knowledge in customization as well as providing high-quality, consistent, and scalable products can help accelerate your development of oligo diagnostics and therapeutics. With over 40 years of partnership with our customers, customization of new nucleic acid technologies, and scale-up experience, Thermo Fisher Scientific is fully set up to help meet your needs in nucleic acid chemistry for both oligo diagnostics and therapeutics.

For a more visual explanation of this topic, please access our phosphoramidite infographic ›