Spotlight on: 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. 

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.