Oligonucleotides: From design to characterisation

Solid-phase chemical synthesis, pioneered by Bruce Merrifield in the 1960s, is an established and efficient method for the synthesis of oligonucleotides. In this post, Quentin Vicentini from ATDbio will explain the synthesis lifecycle of oligonucleotides.
Oligonucleotides have diverse applications in diagnostics and therapeutics, and new applications require ever more complex designs, often including chemically modified DNA bases. Incorporating multiple chemically modified bases into an oligonucleotide can greatly increase the complexity of the synthesis, and specialized chemical knowledge is required to assess the compatibility of these modifications, and the effect of possible side reactions on the likely quality of synthesis.

A series of snapshots from a typical oligonucleotide synthesis lifecycle are described below.

Design and synthesis

Every oligo synthesis starts with a design. Depending of the functionality sought by scientists, the strand can be designed to hybridize with genomic DNA (as the basis for a diagnostic test) or bind to mRNA and switch off the production of a harmful protein.

Once the strand is designed, the sequence is sent to an oligo synthesizer. The synthesis is then performed in an automated fashion.


Though solid-phase oligo synthesis is highly efficient, although errors do occur, leading to truncated products (missing one base or more). Purification by chromatography, in which the crude oligonucleotide is passed through a column on which different components of the mixtures are retained to different degrees, giving separate UV peaks, separates the pure product from undesired impurities. Preparative HPLC (high performance liquid chromatography) is an efficient and automated method of purifying oligonucleotides.

Purified oligonucleotides look, after lyophilization, like cotton balls. If one is curious to see actual DNA, DIY extraction can be safely performed at home e.g. by extracting it from a banana (see video on YouTube here) or even strawberries (see video here).

Analysis and characterisation

After purification, two important questions must be answered: how pure is the product (i.e. what is the ratio of the desired product to all other impurities that were not removed during purification), and what is the nature of the product (i.e. is it really the desired product or something else entirely).

Gel electrophoresis is a common low-cost method of assessing purity, but its lack of resolution makes it unsuitable for anything other than the most cursory analysis. Analytical HPLC (on a smaller scale than preparative HPLC) gives the most definitive analysis of an oligonucleotide’s purity.

Electrospray ionization mass spectrometry (ESI-MS), in which the sample is converted into a stream of gas-phase ions, and the mass-to-charge ratio of each ion measured, provides detailed information on the composition of a sample. Comparison of the processed data with predicted information on the desired product and likely impurities allows the individual peaks to be assigned.

The coupling of analytical HPLC with ESI-MS means that ESI-MS data can be collected for each component in a mixture, helping to build up a full picture of the sample composition.

In our example, the sample looks pure on the UV chromatogram (red spectrum) but several different peaks can be observed after in the mass spectrum (right). These additional peaks are sometimes the result of chemical reactions that occur during the ESI-MS ionization process, so chemical knowledge is needed to fully understand what is observed.

Importantly, this information can be used to make changes to the synthesis process (e.g. by changing the chemistry or redesigning the oligonucleotide itself). This iterative process allows very pure complex oligonucleotides to be produced and allows scientists to develop new diagnostic and therapeutic methods using synthetic oligonucleotides.

If interested, more information about oligonucleotides can be found in the Nucleic Acids Book published by ATDBio (see here).


Writte by Quentin Vicentini, employed at ATDbio

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