Written by Dr, Kurt Sales December 2024, updated by Ian Dawson, July 2025
Oligonucleotides are rapidly emerging as a powerful class of therapeutics, with their ability to silence or modulate gene expression offering novel pathways for disease intervention. Since the approval of formivirsen (Vitravene) in 1998, the field has grown significantly, now encompassing over 20 FDA-approved therapies and a vibrant pipeline in clinical development.
At the core of many diseases lies aberrant protein activity, often the target of conventional therapeutics which aim to modulate and restrict it’s activity. In contrast, oligonucleotides strike at the genetic blueprint itself, inhibiting the production of malfunctioning proteins. This allows for a more upstream and targeted therapeutic approach. Furthermore, oligonucleotides present exciting possibilities in infectious disease, with compounds like formivirsen demonstrating efficacy in preventing viral replication. With rising antibiotic resistance, this modality is gaining traction as a promising new solution.
Oligonucleotide Classes and Structural Design
Therapeutic oligonucleotides are generally classified by their structure and mechanism of action. Anti-sense oligonucleotides (ASOs), typically single-stranded and comprising 12 to 25 bases, operate by binding to complementary RNA sequences. This binding can modulate splicing, inhibit translation, or recruit enzymes that degrade the target mRNA. Small interfering RNAs (siRNAs), on the other hand, are double-stranded and typically 20-25 base pairs. They function through the RNA interference pathway, where the antisense strand guides the RNA-induced silencing complex (RISC) to the target mRNA for degradation.
However, efficacy in vivo demands more than just a well-chosen sequence. Oligonucleotides must also be structurally fortified to resist enzymatic degradation. The incorporation of phosphorothioate (PS) linkages, which replace a non-bridging oxygen with sulphur in the backbone, is one such widely adopted modification. This change enhances resistance to endonucleases, although it also introduces chirality, adding a layer of complexity to characterisation. Modifications to the ribose sugar are also common. Alterations such as 2′-O-methylation, 2′-fluoro substitutions, or locked nucleic acids (LNAs) enhance stability by preventing exonuclease attack. These are often concentrated at the termini of the molecule in what’s known as a ‘gapmer’ design.
The Three Main Challenges of Oligonucleotide Bioanalysis
Challenge 1 — Poor Chromatographic Retention of Oligonucleotides
Despite their promise, oligonucleotides present formidable analytical challenges. First among these is their poor chromatographic retention. Due to their highly polar nature, oligonucleotides do not interact well with traditional reverse-phase columns. This makes achieving adequate retention, and separation from endogenous interferences a persistent hurdle.
Challenge 2 — Mass Spectrometry Complexity and Charge State Distribution
The second challenge lies in mass spectrometry. Oligonucleotides are large molecules with multiple sites for ionisation and adduct formation. This leads to a proliferation of mass-to-charge (m/z) signals, complicating data interpretation. The charge state distributions must be carefully understood and controlled to obtain clean, quantifiable spectra and deliver the required sensitivity.
Challenge 3 — Sample Preparation and Non-Specific Binding Issues
The third challenge is sample preparation. Oligonucleotides exhibit a strong tendency to bind non-specifically to laboratory containers and endogenous proteins. They also display poor solubility in many organic solvents typically used in clean-up procedures. These characteristics make it difficult to recover sufficient quantities of intact analyte from complex biological matrices.
Agilex’s Regulatory – Ready Solutions for Oligo Bioanalysis
At Agilex, we have built a suite of strategies that directly and effectively address each of these core challenges, enabling us to deliver high-quality, regulatory-compliant data.
To counter the retention issue, we employ ion-pair reverse phase chromatography. By introducing basic ion-pairing reagents such as triethylamine or diisopropylethylamine, we mask the negative charges on the oligonucleotide backbone with hydrophobic moieties, enabling the molecule to interact with and be retained on a reverse-phase column. Column temperature is also carefully controlled, particularly for siRNA analysis, to disrupt secondary structures and produce consistent and effective chromatography.
Our approach to mass spectrometry embraces the complexity of oligonucleotide ionisation. We use tandem mass spectrometry (LC-MS/MS) in combination with soft ionisation techniques such as electrospray ionisation (ESI) to minimise in-source degradation. Through precise method development, we map charge state distributions and identify signature transitions that provide specificity and sensitivity, even in the presence of complex matrix components.
Sample preparation is tailored to the unique characteristics of each matrix. For plasma, cerebrospinal fluid, or tissue samples, we choose from a toolbox that includes phenol extraction, guanidine or urea denaturation, and Proteinase-K digestion. Each method is selected and refined to maximise oligonucleotide recovery while maintaining compatibility with downstream analysis. Clean-up steps may involve liquid-liquid extraction to remove interfering substances or solid-phase extraction (SPE) for highly purified eluates. In some cases, hybridisation capture assays offer superior selectivity by targeting specific nucleotide sequences with complementary probes.
To address non-specific binding, which can significantly reduce analytical recovery, we employ blocking buffers and surfactants to coat laboratory surfaces and equipment, preventing oligonucleotides from adhering and ensuring that more of the analyte reaches the detector.
Why Agilex?
Agilex scientists are leaders in the field of oligonucleotide bioanalysis, with a proven track record of navigating complex challenges. We combine cutting-edge technology with deep scientific insight to deliver data that is both precise and meaningful. From early discovery through to regulatory submission, our customised assays are built to withstand scrutiny and support confident decision-making.
Whether your challenge lies in recovery, sensitivity, or matrix interference, we offer the expertise and the tools to overcome it. Partner with Agilex — and together, we will unlock the full potential of oligonucleotide therapeutics.
