Oligonucleotide synthesis basics: Introduction to phosphoramidite chemistry
Oligonucleotide synthesis is the foundation of many molecular biology applications, including polymerase chain reaction (PCR), gene editing, DNA sequencing and therapeutics/drug delivery. The scientific progress we’ve seen in diagnostic testing, biotechnology and pharmacology over the past 35 years was supported, in part, by advances in oligonucleotide synthesis. Let’s take a deeper dive into oligonucleotide synthesis powered by phosphoramidite chemistry and examine the benefits and limitations of in-house and outsourced production.?
How are oligonucleotides synthesised?
Today, commercial manufacturers create oligonucleotides using the phosphoramidite chemistry method introduced more than 35 years ago.1?Its straightforward 4-step cycle is simple and efficient, and
?is the basis of modern automated oligonucleotide synthesis instruments.?
The method relies on coupling DNA phosphoramidite nucleosides on a solid support to build a growing?oligonucleotide chain. A four-step cycle that consists of deblocking, activation, capping and oxidation commences to attach each additional nucleoside to the oligonucleotide. After the entire oligonucleotide is built, it is cleaved from its solid support and post-synthesis processing, including deprotection, initiates.?
Figure 1. The oligonucleotide synthesis cycle using phosphoramidite chemistry.
Synthetic production of oligonucleotides is possible due to the protecting groups on the nucleoside phosphoramidites, which prevent unwanted side reactions during synthesis. The four classic protected DNA nucleoside phosphoramidites are benzoyl-dA, benzoyl-dC, iso-butyryl-dG and dT (which requires no base protection). Acetyl-dC is also widely used. In addition, UltraMILD monomers, phenoxyacetyl (Pac)-dA, and iso-propylphenoxyacetyl (iPr-Pac)-dG are nucleoside phosphoramidites that use alternative protection groups when sensitive labels and tags are incorporated into an oligonucleotide.
Production considerations: in-house or outsourced?
Deciding whether to produce oligonucleotides in-house or outsource the work to a commercial manufacturer requires a thorough analysis of project objectives, available resources and economics. There are benefits and limitations of both in-house and outsourced oligo production, and the most appropriate choice may not always be obvious.?
In-house oligo production can be an economical option in situations where a large-scale product is required. With the right equipment, expertise and protocols in place, the ability to scale production in an on-site manufacturing facility is relatively simple. In addition, there is likely more flexibility and control when operations remain in-house.?
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As both a component supplier and an oligo manufacturer, Biosearch Technologies maintains a strict separation between systems and services supporting external oligo producers, and the systems and teams supporting our oligo design and production offerings. Reach out to our technical services team with specific questions
Alternatively, outsourcing oligo production might be the best option if rigorous quality standards are required or if internal resources limit production capabilities.?Most researchers and many therapy/diagnostic developers may prefer to outsource oligonucleotide manufacturing operations instead of building in-house capabilities for those reasons. Some large pharma companies, despite having proprietary manufacturing capabilities, outsource more than half of their clinical stage oligonucleotide manufacturing operations.2?
We support our customers with a comprehensive portfolio of products and customisation options whether they choose in-house production or rely on us for manufacturing. Our technical experts can provide guidance to help determine whether in-house oligo production is a viable, economical option for your project needs.???
Boost in-house oligonucleotide production success?
As you know, there are many decision points during the oligonucleotide synthesis cycle that can affect the quality and yield of the end product. Here are some recommendations that can help establish a solid foundation for in-house oligonucleotide production.
We control and check our entire manufacturing process, repeatedly, at every stage of production. In addition, our Quality Management System is ISO 9001 certified to meet rigorous production standards.
Check out our?blog post?that deep dives into the best practices for oligonucleotide synthesis. While there are benefits to producing oligonucleotides in-house, thorough analysis and thoughtful planning are required for successful operations.?Contact us?to connect with a specialist who can help match your needs with the appropriate synthesis scale and throughput setup.
Learn more?about our comprehensive product portfolio that offers end-to-end support for nearly any oligonucleotide synthesis application
If you are thinking about bringing oligo synthesis in-house, there are many considerations to account for beyond instrument selection. But the benefits of synthesising your own oligos are clear.?
Download this guide?that summarises all the practical and technical information in a single place, to ensure that your synthesizers run at full capacity and you maximise your return on investment.?
CEO-Azemidite Biopharm
1 年HI dear Sir nice to meet you ! Our company provides phosphoramidites to the world, see if there is any cooperation opportunity in this regard?
Specialist in Oligonucleotide and Peptide Synthesis | Automated Solid Phase Chemical Synthesis of Biomolecules
1 年Oligo synthesis is indeed an elegant chemistry!
I made Oligos for years at #Illumina
Milena Trevisan Pelegrino