Oligonucleotide synthesis is a process central to molecular biology, particularly polymerase chain reactions (PCR), gene editing, DNA sequencing and therapeutic deliveries. Over the past 35 years, we’ve seen significant scientific progress in diagnostic testing, biotechnology and pharmacology, due to advances in oligonucleotide synthesis. 

Below, we take a further look into this process and explore its importance in modern medicine.

What is oligonucleotide synthesis?

Oligonucleotide synthesis is the chemical process used to create short strands of nucleic acids, such as DNA or RNA, with a specific sequence. When synthetically produced, these strands are an essential part of molecular biology, enabling applications in gene editing, diagnostics, therapeutics, and research.

For enzyme-free synthesis, the process involves sequentially adding nucleotide units to a growing chain, typically using solid- or liquid-phase synthesis. It’s a precise and controlled approach that ensures high purity and accuracy, making it a cornerstone technique in biotechnology and pharmaceutical development.

What is an oligonucleotide? 

Oligonucleotides are segments of nucleic acids, either single or double-stranded, intricately connected to form single-chain biological polymers. Research has shown that the individual nucleotide bases can be likened to the monomers constituting traditional chemical polymers. Moreover, a nucleotide comprises three components: a nitrogen-containing base, a five-carbon sugar molecule (together forming the nucleoside) and one or more phosphate groups. 

Alternatively, nucleotides may incorporate non-natural or non-canonical bases, such as LNA (locked nucleic acids), Morpholino, and bases or backbones with structural modifications.

Furthermore, the intricate linkage of nucleotides results in the formation of biologically crucial RNA and DNA biomolecules. In RNA, a single-stranded biopolymer, the sugar is ribose, while in DNA, a double-stranded molecule, the sugar is deoxyribose. 

As a result, it is the concise sequences of deoxyribonucleic acids and ribonucleic acids that serve as the fundamental components of significant oligonucleotides. In fact, this is what gives rise to the target biomolecules essential in various biological, medical, forensic and clinical applications.

Oligonucleotide synthesis process and steps

Oligonucleotide synthesis typically involves a series of chemical steps to build the nucleotide chain. The process begins with attaching the first nucleotide to a solid support. Each subsequent nucleotide is added in a stepwise fashion through processes like deprotection and coupling, where the growing chain is extended. After the full sequence is assembled, the oligonucleotide is cleaved from the solid support, purified, and verified for accuracy. These steps ensure the oligonucleotide has the desired sequence and purity for its intended application.

What is solid-phase oligonucleotide synthesis and how does it work? 

In the field of modern medicine, commercial manufacturers are using the phosphoramidite chemistry method, created over 35 years ago, to perform oligonucleotide synthesis. This straightforward 4-step cycle, known for its simplicity and efficiency, forms the fundamental process for modern automated oligonucleotide synthesis instruments.

The process involves anchoring the first nucleotide to a solid support, typically a resin, and then sequentially adding nucleotides one at a time. Each nucleotide is added in a controlled manner, ensuring the precise sequence of the oligonucleotide. The solid support enables the purification of intermediates and facilitates automation. 

The synthesis follows a repetitive cycle of steps, including deblocking, coupling, capping, and oxidation, allowing for the stepwise extension of the oligonucleotide chain. 

Once the desired sequence is achieved, the oligonucleotide is cleaved from the solid support and subjected to post-synthesis processing, including deprotection. 

What are the alternative methods of oligonucleotide synthesis?

Solid-phase oligonucleotide synthesis presents many challenges for molecular biologists, including the decrease in yield as chain length increases. As a result, other forms of oligonucleotide synthesis can be used. 

One such method is solution-phase synthesis, where nucleotides are sequentially combined in solution without the need for solid support. This method allows for larger-scale reactions but can be challenging in terms of purification and yield.

Another process is enzymatic synthesis, which involves using enzymes, such as DNA polymerases, to synthesise oligonucleotides. This technique is highly selective and can produce longer sequences, but it is limited by the need for specific enzymes and has a greater potential for error.

Array-based synthesis involves the simultaneous synthesis of multiple oligonucleotides on a solid surface, such as a microarray. This approach is advantageous for high-throughput applications but may have limitations in sequence length and yield.

Finally, the phosphite-triester method uses solution-phase chemistry. It involves the reaction of nucleotide phosphoramidites with phosphites, leading to the formation of oligonucleotides. Although less commonly used, it offers an alternative to solid-phase synthesis.

Each alternative method has its advantages and limitations, and the choice depends on factors such as scalability, sequence length, and the specific requirements of the intended application. 

What is phosphoramidite oligonucleotide synthesis?

Phosphoramidite oligonucleotide synthesis is a widely used method for creating oligonucleotides. This technique involves attaching a phosphoramidite derivative of a nucleotide to a solid support. Each nucleotide is added one at a time to the growing chain, with protective groups removed during each step to enable the next nucleotide addition. The process is highly efficient and allows for precise control over the sequence of the oligonucleotide. Once synthesis is complete, the oligonucleotide is cleaved from the support, purified, and validated for its intended use.

Why choose to work with Bachem?

Trust in Bachem for our expertise in oligonucleotide process development, GMP production and meeting regulations. Our commitment to innovation and decades-long experience permeates every phase of the process, enhancing the potential of your product. We employ the latest engineering solutions to ensure precise process control and high throughput. Rest assured, we also possess the capability to get your product to market!

With extensive expertise, our team specialises in the development and validation of test methods tailored for large and structurally intricate molecules. Bachem’s proficiency extends to solid-phase synthesis, protecting group chemistry, chromatography-based purification, as well as ultra/diafiltration techniques, precipitation and lyophilisation.​

Leverage the complete advantages of our cutting-edge chemistry, manufacturing and control (CMC) development services tailored for oligonucleotide-based APIs. We are here to help you with everything from the creation of scalable manufacturing processes to supporting IND and NDA applications.

Elevate your projects with Bachem’s unparalleled expertise in oligonucleotide manufacturing. Simply contact us to find out more about our innovative solutions, precision in synthesis and commitment to quality. 

Looking for a convenient tool to determine oligonucleotide molecular weight? Try our helpful oligonucleotide calculator today.