Click Chemistry in Oligonucleotide Synthesis

Click chemistry is a versatile reaction that can be used to synthesize a wide range of compound conjugates. The click reaction allows for the convenient introduction of designed small molecules into oligonucleotides and imparts a variety of additional properties to the oligonucleotide, such as optical, physical, and chemical properties.

What is Click Chemistry in Oligonucleotide Synthesis?

Currently, the application of click chemistry in oligonucleotide synthesis involves the following two main reactions.

  • Copper-catalyzed Azide-alkyne Cycloaddition (CuAAC) Reactions
    In the presence of a reducing agent and a stabilizing ligand, a copper(I)-catalyzed cycloaddition reaction between an azide and an alkyne forms a stable triazole portion [1,4-disubstituted (trans)-1,2,3-triazole] similar to an amide bond in a reaction known as the CuAAC reaction. With high efficiency and relatively fast kinetics, this reaction has been widely used in bioconjugation and organic synthesis, including labeling of oligonucleotides and generation of libraries of analogs of biologically active molecules.

Solid-phase CuAAC modification of oligonucleotides.Fig 1. Solid-phase CuAAC modification of oligonucleotides. (Farzan et al., 2017)

  • Strain-promoted Azide-alkyne Cycloaddition (SPAAC) Reaction
    The reaction does not require the use of metal catalysts, reducing agents or stabilizing ligands. Instead, the reaction utilizes the enthalpy released from the cyclic strain to become cyclooctyne (e.g., OCT, BCN, DBCO, DIBO, and DIFO) to form a stable triazole. Although SPAAC has slower reaction kinetics than CuAAC, its biocompatibility in living cells is unquestionable.

SPAAC click DNA ligation between labeled oligonucleotidesFig 2. SPAAC click DNA ligation between labeled oligonucleotides. (Shelbourne et al., 2011)

Types of Oligonucleotides Available to Click Chemistry

Advantages of Click Chemistry for Oligonucleotides

  • High specificity and selectivity
    Click chemistry is a highly specific and selective chemical reaction that occurs only between molecules containing alkyne and azide groups.
  • Mild reaction conditions
    Click chemistry is usually carried out under mild conditions, such as at or near room temperature, and does not require special environments or reagents.
  • Fast reaction
    Click chemistry is a fast reaction, usually completed within minutes to hours.
  • Wide compatibility
    The click chemistry reaction has broad compatibility with different types of oligonucleotides. It can be applied to different types of oligonucleotides such as DNA and RNA and is suitable for oligonucleotides of different lengths and sequences.

Application of Click Chemistry in Oligonucleotide Synthesis

  • Click Oligonucleotide Labelling
    The CuAAC reaction provides a method for labeling oligonucleotides with a range of functional groups, including chimeric molecules, epigenetic modifications, and fractions that may be unstable to enzymatic ligations. The CuAAC reaction is orthogonal to standard amino-bond labeling, and typically produces high yields under mild reaction conditions. Click chemistry allows for the introduction of various markers such as fluorescent dyes, biotin, and gold nanoparticles. These markers can be used to detect, image and quantify the presence and activity of oligonucleotides in biological samples.
  • Metallization of Oligonucleotides
    Metallization of DNA increases the electrical conductivity of its nanostructures, allowing them to be used as molecular wires. The process involves the doping of DNA with acetylene-modified nucleotide triphosphates using DNA polymerase, followed by a click reaction directly on a polyacrylamide gel to obtain aldehyde-modified DNA, and finally the metallization of the DNA by a silver microscopy reaction.
  • Cross-linking of Oligonucleotides
    Click chemistry can also be used for oligonucleotide cross-linking. Two complementary DNA strands modified with alkyne and azide groups, respectively, can undergo a CuAAC reaction followed by fast and efficient cross-linking. Cross-linked double strands obtained in this way are much more thermally stable than non-cross-linked double strands. This is because the interstrand interactions of cross-linked double strands are intramolecular and the shorter distance between the cross-linked complementary strands is more favorable for hydrogen bond formation.
  • Modification of Oligonucleotides
    Click chemistry can be used to introduce various modifying groups on oligonucleotides, such as polymers, polyethylene glycol (PEG) chains, and lipid chains. Modifications to oligonucleotides include artificial modifications including backbones, bases, sugars and inter-nucleotide bonded oligonucleotides.


  1. Farzan V M, et al. Automated solid-phase click synthesis of oligonucleotide conjugates: From small molecules to diverse N-acetylgalactosamine clusters[J]. Bioconjugate Chemistry, 2017, 28(10): 2599-2607.
  2. Shelbourne M, et al. Fast copper-free click DNA ligation by the ring-strain promoted alkyne-azide cycloaddition reaction[J]. Chemical Communications, 2011, 47(22): 6257-6259.
* Only for research. Not suitable for any diagnostic or therapeutic use.
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