Oligonucleotides are short, synthetic strands of nucleic acids (DNA or RNA) used in a wide range of biopharmaceutical applications, including:
Their versatility, customizability, and targeted mechanism of action have made them increasingly important in personalized medicine and genetic therapies.
At BioPharmaSpec, we support companies developing oligonucleotide-based drugs and tools by offering robust, regulatory-aligned analytical characterization at every development stage.
Oligonucleotide synthesis is the chemical process used to construct short DNA or RNA sequences, most commonly through solid-phase synthesis. This stepwise technique involves the sequential addition of individual nucleosides to a solid support enabling high-fidelity and custom sequence design for research and therapeutic applications.
In this method, nucleoside phosphoramidites act as the primary building blocks. Each synthesis cycle includes a carefully controlled series of chemical reactions (activation, coupling, capping, oxidation, and deprotection) that add and secure one nucleoside at a time. The oligonucleotide chain is covalently attached to a solid support, typically composed of controlled pore glass (CPG) beads or macroporous polystyrene, which provides stability and simplifies purification.
The phosphoramidite and hydroxyl groups play essential roles in forming the nucleoside linkages in the growing chain. This solid-phase setup ensures that unreacted materials/impurities can easily be washed away between steps - a major advantage which significantly helps with purification.
While several oligonucleotide synthesis methods exist, including solution-phase, enzymatic, and array-based techniques, solid-phase synthesis remains the gold standard due to its efficiency, scalability, and suitability for producing high-purity sequences.
Because this process works at both research and therapeutic scales, it has become indispensable in the development of tailored DNA or RNA strands for targeted diagnostics, genetic tools, and novel treatments. From early-stage discovery to clinical manufacturing, the ability to precisely design and build oligonucleotides has revolutionized modern molecular science.
Synthetic oligonucleotides are found in:
Many therapeutic applications now rely on chemically modified oligonucleotides, such as phosphorothioate backbones or 2’-O-methyl substitutions, to improve stability, binding affinity, and resistance to nucleases.
BioPharmaSpec provides analytical support for oligonucleotide synthesis in fields ranging from oncology and rare diseases to infectious disease diagnostics.
Most conventional oligonucleotide synthesis methods rely on solid-phase phosphoramidite chemistry to construct a desired oligonucleotide, with nucleosides sequentially added to a growing strand attached to a solid support.
Here’s how the process works:
We help clients interpret results, set appropriate specifications, and design a testing plan that aligns with their development timelines and filing goals.
Design effective structural characterization studies for oligonucleotide therapeutics.
As the oligonucleotide chain grows longer, maintaining efficient synthesis becomes increasingly difficult. Each step in the synthesis cycle involves a chemical reaction that must proceed nearly to completion before the next nucleoside can be added. Even small inefficiencies in the coupling reaction yield can compound over multiple cycles, drastically reducing the proportion of full-length product.
While modern synthesis platforms often achieve coupling efficiencies greater than 99%, the cumulative effect of even slight losses becomes significant as chain length increases. For example:
These reduced yields present downstream challenges for purification, quantification, and regulatory compliance. At BioPharmaSpec, we help clients identify and assess truncated sequences using high-resolution mass spectrometry, ensuring accurate product characterization even in complex oligo mixtures.
Chemical synthesis can result in a complex mixture of full-length products, truncated sequences, and side products (e.g. depurination, dephosphorylation, or incomplete deprotection). Impurities may interfere with biological function or produce off-target effects.
This is where high-resolution analytical techniques like LC-MS and ion-pair reverse-phase chromatography become vital. BioPharmaSpec supports clients by identifying and quantifying minor impurities, helping ensure that batches meet both internal and regulatory standards.
Even with automated oligonucleotide synthesis, errors such as base misincorporations, deletions, or unwanted chemical modifications can occur, especially when scaling up or working with modified sequences. Unlike biologically synthesized sequences, chemical synthesis offers no built-in proofreading.
BioPharmaSpec’s sequence confirmation workflows, including mass spectrometry-based oligonucleotide mapping, give clients confidence in the fidelity of their synthetic product.
Oligonucleotides intended for therapeutic use must meet strict regulatory expectations from agencies like the FDA and EMA. These include:
Regardless of how well a synthesis platform is designed, not all are equally suitable for oligonucleotide synthesis aimed at regulated therapeutic applications. Careful analysis ensures the final product meets necessary quality standards.
At BioPharmaSpec, we offer:
With over 15 years of experience in oligonucleotide characterization, we use high-performance mass spectrometry instrumentation, like Orbitrap, Triple Quad, and Q-TOF mass spectrometers, to generate the insights you need, backed by direct support from our PhD-level scientists to help you understand your data and move your project forward.
Speak directly with one of our scientists to discuss your project and receive a tailored analytical proposal.
