Helping you to understand and design oligonucleotides

Oligonucleotide synthesis uses solid-phase phosphoramidite chemistry, a process refined over decades into a highly reliable, automated cycle that builds the sequence one nucleotide at a time from the 3′ end to the 5′ end. Here is what happens at each stage.

Step 1: Attachment to solid support

Synthesis begins with the 3′-terminal nucleoside already attached to a solid support, typically controlled-pore glass (CPG) or polystyrene. This support holds the growing chain in place throughout the entire synthesis cycle and allows reagents to be washed through efficiently.

Step 2: Detritylation

The 5′ hydroxyl of the support-bound nucleoside is protected by a dimethoxytrityl (DMT) group, which prevents unwanted reactions during synthesis. Acid removes the DMT before each coupling step, exposing the hydroxyl for chain extension. Each incoming monomer carries its own DMT group, so the cycle can repeat.

Step 3: Coupling

The incoming phosphoramidite monomer is activated and coupled to the free 5′ hydroxyl of the support-bound chain. Coupling efficiencies in modern synthesis routinely exceed 99% per step, but because the shortfall is compounded over many cycles, longer sequences naturally yield a greater proportion of truncated products. This is why purification becomes increasingly important as sequence length increases.

Step 4: Capping

Any unreacted 5′ hydroxyl groups that failed to couple are chemically blocked (capped) in this step. This prevents them from participating in subsequent couplings and generating deletion sequences that would be difficult to remove during purification and ultimately detrimental to target specificity.

Step 5: Oxidation

The phosphite triester bond formed during coupling is unstable. Oxidation with iodine converts it to the more stable phosphate triester (P(V)), which forms the backbone of the final oligonucleotide. For phosphorothioate linkages, a sulfurising reagent is used at this step in place of iodine. Steps 2 to 5 are then repeated for every base in the sequence.

Step 6: Cleavage and deprotection

Once the full sequence has been assembled, the oligonucleotide is cleaved from the solid support and the remaining protecting groups are removed using aqueous ammonia, methylamine/ammonia or similar reagents. This yields the crude oligonucleotide ready for purification.

A note on modified oligonucleotides: modifications on the backbone, the sugar, the base, or as terminal labels, are incorporated in the synthesis cycle using specialised phosphoramidite reagents or post-synthesis conjugation chemistries (for example, a dye NHS ester reacted with an amino-modified oligonucleotide). The choice of modification strategy affects both yield and performance, and is something our team can help you with.

After synthesis, every oligonucleotide contains a mixture of the desired full-length product and shorter truncated sequences from incomplete couplings, as well as residual reagents and protecting group fragments. Purification removes these impurities, but different methods offer different levels of resolution. The right choice depends on your application. Tell us what the oligo has to do and we will advise which method suits it.

Desalting

The simplest form of clean-up. Removes salts, solvents and small molecule contaminants, but does not separate full-length product from truncated sequences. Suitable for straightforward applications where absolute sequence purity is not critical.

Suitable for: standard PCR primers, screening libraries and high-throughput work, and simple unmodified oligonucleotides up to approximately 30 bases. It is also the quickest of the three methods to turn around.

HPLC purification

High-performance liquid chromatography is the industry standard method for all applications requiring the highest purity, and the method of choice at Assemblon. Our HPLC methods are optimised for different types of modified oligonucleotides, including dual and heavily modified sequences.

Importantly, HPLC achieves high-purity separations without exposing oligonucleotides to conditions that can cause chemical damage.

Suitable for: dual-labelled probes, therapeutic oligos, long sequences, siRNA, CRISPR components, any application requiring the highest purity and integrity.

Anion-exchange HPLC

Anion-exchange HPLC separates oligonucleotides of different lengths based on charge rather than hydrophobicity. It is particularly effective for phosphorothioate oligonucleotides and modified therapeutic sequences where reversed-phase methods may be less discriminating.

Suitable for: phosphorothioate ASOs, some therapeutic sequences.

Not sure which to choose? If your application involves a fluorescent probe, a therapeutic sequence, or an oligo longer than 40 bases, HPLC purification is our strong recommendation.

At Assemblon we carry out oligonucleotide synthesis on various scales, from 200 nmol to 16 µmol. The smallest scale is sufficient for most biological experiments, but for therapeutic and some diagnostic projects scales of 1 µmol to 16 µmol are required. As a rough guide, a 1 µmol synthesis will yield 25–80 nmol of purified oligonucleotide after HPLC purification.

Synthesis scale is the amount of starting nucleoside on the solid support, in nanomoles (nmol): a 1 µmol synthesis starts with 1 µmol of the 3′-nucleoside, and everything that follows is a fraction of it. A higher scale generally means more final product, but the yield you receive depends on sequence length, modification content and purification method.

The table below gives typical yield ranges as a guide. These are estimates. Actual yields can vary, and complex or heavily modified sequences will typically yield less than a simple unmodified oligo of the same length.

OD = optical density units at 260 nm. One OD corresponds to approximately 33 µg for a typical 20-mer.

Typical figures for unmodified sequences. Modified or difficult sequences may yield less, and we quote per project.

Ordering by minimum required yield

Rather than choosing a synthesis scale, you can specify the minimum yield you require, for example "I need at least 500 nmol of final purified product." Tell us your minimum yield requirement in your order form or enquiry, and the synthesis scale is selected to meet it and quoted accordingly.

Every oligonucleotide we supply is analysed by UPLC-MS before dispatch, and only material that meets our release criteria is released.

Free, no-registration calculators we maintain for the common chemistry questions that come up during experiment planning. Each is also available as a deep-linkable page so you can bookmark or share.