Oligonucleotide Therapeutics—Complementing Modern Medicine

Oligonucleotide therapeutics are short, synthetic oligonucleotides that modulate the expression or function of target RNA for the treatment of disease. These oligos are:

• Usually about 20 nucleotides long
• Single- or double-stranded
• Manufactured almost exclusively using phosphoramidite chemistry

View infographic: Oligonucleotide therapeutics

Common types of oligonucleotide therapeutics, a drug family, include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs) involved in RNA interference (RNAi), small activating RNAs (saRNAs), aptamers, and guide RNAs (gRNAs) involved in CRISPR gene editing technology. The number of approved oligonucleotide therapeutics continues to grow globally, with numerous additional candidates advancing through clinical development. Oligonucleotide therapeutics treat diseases including muscular dystrophy, polyneuropathy, and hypercholesterolemia, with many more therapeutics currently in development and other diseases being targeted.

• Antisense oligonucleotides (ASOs): Short single-stranded DNA or RNA molecules that bind complementary messenger RNA (mRNA) sequences to block translation, alter splicing, or promote RNA degradation. ASOs are widely used to reduce expression of disease-causing genes.
• Small interfering RNAs (siRNAs): Double-stranded RNA molecules that trigger RNA interference (RNAi) pathways. siRNAs guide the RNA-induced silencing complex (RISC) to complementary mRNA targets, resulting in sequence-specific mRNA cleavage and gene silencing.
• Small activating RNAs (saRNAs): Short double-stranded RNAs that increase gene expression through RNA activation (RNAa) mechanisms. Unlike siRNAs, saRNAs upregulate transcription of specific target genes and are being explored for regenerative medicine and cancer therapy applications.
• Aptamers: Structured single-stranded nucleic acids that fold into three-dimensional shapes capable of binding proteins, small molecules, or cells with high affinity and specificity. Aptamers function similarly to antibodies and can be used for targeted therapeutics or drug delivery.
• Guide RNAs (gRNAs): RNA molecules used in CRISPR gene editing technology to direct CRISPR-associated nucleases (such as Cas9) to specific genomic DNA sequences. gRNAs determine the targeting specificity of CRISPR systems for gene knockout, insertion, correction, or regulation.

ASO and RNAi technologies act on target RNA, resulting in RNA cleavage and ultimately helping prevent protein translation. However, their structure and mechanisms are different. ASOs are single-stranded oligos that recruit RNase H1 to cleave their RNA targets in the nucleus or the cytoplasm. On the other hand, RNAi involves double-stranded siRNAs that recruit the RNA-induced silencing complex (RISC) to cleave their RNA targets in the cytoplasm.

The success of mRNA-based SARS-CoV-2 vaccines has brought renewed interest to other nucleic acid drugs, including ASO and RNAi therapeutics. In the next 5 years, the oligo therapeutics market is expected to grow by 25–30%; more than one thousand oligonucleotide therapeutics have entered clinical or preclinical development.

As both ASOs and siRNAs are short oligonucleotides, their stability in the body, immunogenicity to the host, specificity to their target sequences, and delivery to target organs have always been challenges.

As the building blocks for oligonucleotide therapeutics, modifications to phosphoramidite structures have been developed to improve performance and mitigate these issues. For example:

• 2′-O-methyl (2′-OMe) and 2′-fluoro (2′-Fl) modifications are commonly incorporated into siRNA oligonucleotides to improve resistance to nucleases and thermal stability and reduce off-target effects.
• For ASOs, 2′-O-methoxyethyl (2′-MOE)–modified amidites, among others, can be incorporated to produce similar effects.
• For delivery, encapsulating oligonucleotide therapeutics in lipid nanoparticles (LNPs) or conjugating them to N-acetylgalactosamine (GalNAc) moieties that have been developed in recent years facilitate improved durability and specificity of organ targeting for nucleic acid drugs.

• Solid-phase phosphoramidite synthesis: The most widely used method for synthesizing therapeutic oligonucleotides; builds oligonucleotides stepwise on a solid support using protected nucleotide phosphoramidites in automated synthesis cycles.
• Phosphotriester synthesis: An earlier chemical synthesis approach using protected phosphate triesters; largely replaced by phosphoramidite chemistry due to lower efficiency and scalability.
• H-phosphonate chemistry: A chemical oligonucleotide synthesis method that forms internucleotide H-phosphonate linkages prior to oxidation; useful for certain backbone modifications.
• Enzymatic oligonucleotide synthesis: Uses DNA or RNA polymerases and enzymatic reactions to generate oligonucleotides; emerging as an alternative to purely chemical synthesis.
• Liquid-phase oligonucleotide synthesis: Synthesizes oligonucleotides in solution rather than on a solid support; explored for large-scale manufacturing and process simplification.
• Microarray-based oligonucleotide synthesis: Produces large numbers of oligonucleotide sequences in parallel on chip surfaces; commonly used for high-throughput screening and library generation.
• Flow-based or continuous synthesis systems: Automated continuous-flow chemistry platforms that improve scalability, reproducibility, and throughput of oligonucleotide manufacturing.
• Click chemistry–assisted oligonucleotide assembly: Uses bioorthogonal chemical ligation methods to assemble modified oligonucleotides or conjugates with high specificity.
• Hybrid chemical-enzymatic synthesis: Combines phosphoramidite chemistry with enzymatic extension or ligation approaches to improve efficiency and enable complex modifications.
• Modified nucleotide incorporation methods: Specialized synthesis workflows designed to incorporate therapeutic modifications such as phosphorothioates, 2′-MOE, LNA, GalNAc conjugates, or fluorescent labels.

Therapeutic oligonucleotides are typically synthesized using solid-phase phosphoramidite chemistry, an efficient and automated method for building short nucleic acid sequences one nucleoside phosphoramidite at a time. In this process, the first nucleoside is attached to a solid support, and protected nucleoside phosphoramidites are sequentially added through repeated synthesis cycles consisting of deprotection, coupling, capping, and oxidation steps. During coupling, an activated phosphoramidite reacts with the growing oligonucleotide chain to form a phosphite linkage, which is then chemically oxidized to create a stable phosphate backbone. After synthesis is complete, protecting groups are removed and the oligonucleotide is cleaved from the solid support, followed by purification and quality analysis. Phosphoramidite chemistry also enables incorporation of common modifications—such as phosphorothioate linkages, 2′-O-methyl, or locked nucleic acid (LNA) residues—which improve nuclease resistance, binding affinity, and therapeutic stability. This scalable synthesis approach forms the foundation of modern oligonucleotide manufacturing for research and therapeutic applications.

Oligotherapeutic developers look for quality assurance, documentation support, capacity, scalability, and batch-to-batch consistency in their products.

• Quality: Offering quality products starts with solid manufacturing processes housed within a well-established quality system for quality assurance, traceability, and documentation support.
• Capacity: Drug developers want to make sure their phosphoramidite supplier is capable of meeting their capacity needs, to help minimize interruption in their drug development pipelines.
• Scalability: Scale-up and future capacity are also important when drug developers are starting with a custom modification but also looking toward their large-scale manufacturing down the road.
• Raw material consistency: Batch-to-batch consistency is very important for process developers and manufacturers. Minimizing batch-to-batch variation by the supplier starts from raw material qualification and incoming raw material testing to help ensure quality is maintained in manufacturing and final product release specifications via in-process testing.

We offer Thermo Scientific TheraPure phosphoramidites, which are suitable for oligonucleotide research supporting the development of therapeutic applications. Our TheraPure phosphoramidites undergo additional quality control release testing compared to our standard phosphoramidites, helping ensure that impurities and residual solvents are controlled to the stringent levels required by our customers for their oligo therapeutics.

In addition to our on-shelf products, we offer services for custom oligonucleotide chemistry such as customization of existing phosphoramidites and the development of new compounds or oligonucleotide delivery chemistries.

Outside of our phosphoramidite team, Thermo Fisher Scientific offers reagents for oligonucleotide synthesis and analysis tools for synthesized oligonucleotides, as well as the ability to synthesize large-scale oligonucleotides.