Spotlight on: Oligonucleotides

What are oligonucleotides?

Oligonucleotides (also referred to as oligos) have become one of the most important tools in modern-day biology. They are single or double-stranded fragments of DNA or RNA molecules, with varying lengths of nucleotides per strand. Using phosphoramidite chemistry, they are synthesized by adding one of the five nucleic acids (A, T, C, G and U) until the desired sequence is achieved.

The first method of synthesis was introduced in the late 1950s by Dr. Har Gobind Khorana and colleagues. Solid phase synthesis of oligos began in 1965, with additional efforts to create longer strands in the 1970s and 1980s. Today, many companies synthesize oligos for academic, research, or commercial use, including another company under the Danaher umbrella, IDT.

The synthesis of oligonucleotides provides a versatile and powerful tool for modern science. From advancing research capabilities to improving diagnostic accuracy and expanding therapeutic options, synthetic oligonucleotides have become indispensable in numerous scientific and medical endeavors.

Why are oligonucleotides both challenging and interesting?

Due to their inherent negative charge and structural complexity, a high level of expertise is needed to understand their physiochemical behavior. Specialized optimization of sample preparation is vital, especially for PK/PD and bioanalysis studies in different matrices.

Frequently, the backbone and bases are modified so they are more stable, but this adds to their polarity and makes them difficult to retain on conventional reversed-phase (RP) columns – the most typical columns used in LC-MS analysis. Oligonucleotides display multiple charges and love to take up adducts, limiting sensitivity and creating complex MS spectra. They also like to stick to components of the LC system, leading to sensitivity losses and ultimately affecting the accuracy of analyses negatively. Because of this, it’s common practice to use ion-pairing agents to combat their hydrophilic nature during LC separation, which often leads to dedicated instrumentation.

How are oligonucleotides impacting the world?

Oligos are incredibly versatile biological tools thanks to their ability to hybridize specifically with complementary nucleic acid sequences–leading to utility in gene manipulation, detection, and analysis.

While the first approvals of oligonucleotide therapeutics started in the late 1990s, it took another twenty years before the first small interfering RNA (siRNA) drug was approved (used in the treatment of genetic disorders, cancers, and other diseases). Oligonucleotide therapeutics mark a revolution of mechanism of action (MoA) by targeting the messenger RNA (mRNA). Today, more than a dozen antisense oligonucleotides (ASOs) and siRNA drugs are on the market, most designated orphan drugs for treating rare genetic diseases with many more in different stages of drug development. [1]

1 Collotta, D. et al. Antisense oligonucleotides: a novel frontier in pharmacological strategy. Front Pharmacol. 2023 Nov 17;14:1304342. doi: 10.3389/fphar.2023.1304342. PMID: 38044945; PMCID: PMC10690781.

How does SCIEX help overcome key challenges?

SCIEX offers several solutions that uniquely assist scientists in analyzing oligos. Launched just last year, the high sensitivity and resilience of the new SCIEX 7500+ system permits pharmacokinetic or impurity studies with limited sample availability that require quantitation at trace, negative levels. Furthermore, matrices for bioanalytical studies can be extremely challenging. The SCIEX 7500+ system excels in negative ionization mode with enhanced robustness, exceling in the quantitation of oligonucleotides, its impurities, or metabolites.

As we mentioned above, oligos can be quite “sticky” so to limit undesired binding, it is key to consider the surfaces with which oligonucleotide samples come in contact, especially metal surfaces. Thus, inert LC systems can be tremendously helpful in oligonucleotide analytics. We have partnered with Shimadzu Corp to offer a new inert LC option: the Shimadzu Nexera XS Inert LC.

Key benefits of oligonucleotides

Oligos have became a great interest in drug development due to binding specificity of complementary sequences
Oligo-based therapies can be used to inhibit gene expression, or slow protein function or are the basis for novel cancer-fighting drugs
Previously undruggable conditions can be addressed with oligo therapeutics