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Click-Chemistry based oligonucleotide labeling technology

Currently, labeled and dual labeled oligonucleotide probes are mainly manufactured using either dye-labeled amidites, or activated esters. Both of these methods have their limitations.

For companies and departments involved in custom oligonucleotide synthesis, we offer a Click-Chemistry based solution for oligo labeling.

process scheme

Just as in the case of NHS esters, Click-Chemistry labeling is a two-step protocol. First, an alkyne moiety is introduced into the oligonucleotide using an alkyne amidite. Second, the deblocked oligo is labeled with a dye azide under appropriate conditions. The labeled oligo is then precipitated with acetone and purified (HPLC is the most appropriate method).

Compared to amidite-based labeling, Click Chemistry yields more pure probes, with higher yield, and the probes require less effort to purify. The sensitive and expensive dye is not subjected to reactive deblocking reagents, and not wasted. There is no need to use ultra-mild protective groups.

Compared to the activated ester method, Click Chemistry is more scalable, and requires less dye. The probes are purified more easily. The excess of the dye (about 0.2 to 0.5 eq above required amount) is nearly completely removed during acetone precipitation. This is often not the case for activated ester labeling, where multiple precipitations sometimes required to remove the excessive dye. Click Chemistry allows efficient labeling of hundreds of OD260 units of oligonucleotide in a single centrifuge tube.

The cost-efficiency of the labeling method is comparable to amidite and NHS ester labeling.

 Dye amiditeNHS esterClick-Chemistry
Sensitive dye exposed to
deblock conditions
yes no no
Scalability good limited excellent
Raw labeling mixture purity 30-90% 60-95% 95%+
Excess of dye used for the labeling 10 eq. 10 eq. 1.2-1.5 eq
Completeness of the labeling 98%+ 60-100% 99-100%
Features simple protocol
low oligo yield
difficult to purify
low stability of amidites
high purity
large excess of dye
incomplete labeling
high purity
high efficiency
low dye excess
competitive price

We provide all necessary reagents for oligo labeling: alkyne amidites (5'-terminal, and Dmt-protected for 3'-labeling on universal CPG), alkyne CPG, dye azides, and Click Chemistry catalyst. Use our recommended protocol for oligonucleotide labeling for optimal results!

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