Tyrosine- or Y-clicking of oligonucleotides and proteins useful for functional studies

Click reactions are high-yielding, specific, and practical chemical reactions now applied in various chemical fields, including materials science, bioconjugation, pharmaceuticals, and nanotechnology. Efficient click reactions are compatible with different functional groups, making them valuable tools in chemical synthesis.

A "tyrosine click reaction" refers to a click reaction involving the amino acid tyrosine, one of the 20 standard amino acids commonly found in proteins. Tyrosine possesses a phenolic hydroxyl group, a nucleophile in click-type reactions. Click-like tyrosine labeling reactions enable chemo-selective labeling of target molecules.

The tyrosine click reaction or Y‐Click allows the conjugation of peptides, proteins, or antibodies with glycans, oligonucleotides, fluorescent dyes, PEG‐tags, or antiviral agents.

Ban et al. (2013) utilized 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones to create stabile conjugation linkages through tyrosine with the help of a tyrosine-click reaction (Figure 1).

Figure 1: Tyrosine selective labeling of proteins with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) (Ban et al. 2013).

According to Ban et al., the molecular linkage created by the tyrosine click reaction is stable in a wide pH range, temperature, and exposure to human blood plasma. This linkage is significantly more robust than maleimide-type linkages commonly employed in bio-conjugations.

The tyrosine click reaction allows chemo-selective modification of small molecules, oligonucleotides, peptides, and proteins under mild aqueous conditions over a broad pH range using various biological buffers.

Meyer et al. (2022) converted N,O‐Diacetyl tyrosine and 3‐(4‐hydroxy‐phenyl)‐propanoic acid into phosphoramidite derivatives, which the researchers introduced to the 5′‐end of oligonucleotides (Figure 2). The resulting oligonucleotides now contain a 4‐hydroxyphenyl alkyl group, allowing conjugation with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione using the tyrosine click reaction. The reported reaction is fast (<1 h) and efficient (>90 %).

Figure 2: Tyrosine selective labeling of oligonucleotides with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-dione (PTAD) (Meyer et al. 2022).

Meyer et al. showed that the tyrosine‐click reaction allows selective conjugation of oligonucleotides containing a tyrosine moiety using phenyl‐1,2,4‐triazoline‐3,5‐dione or its derivative. The reaction can be applied in a solution or on solid support.

Potential applications of a tyrosine click reaction include:

3'- and 5'-tyrosine oligonucleotide conjugates

Tyrosine-labeled oligonucleotides enable tyrosine click reactions. Tyrosine-modified phosphoramidites allow the automated synthesis of oligonucleotides modified with tyrosine on their terminal ends. Alternatively, tyrosine can be conjugated to the terminal ends of oligonucleotides. Oligonucleotides containing tyrosine on their 3’- or 5’-end can be used as substrates for topoisomerase I and relaxases for functional studies of nicking and repair enzymes during DNA breakage and repair.

Protein engineering and modification

Introducing specific modifications into proteins allows the study of their structure and function. A tyrosine click reaction allows attaching functional groups or reporter molecules to tyrosine residues in proteins, allowing researchers to study protein behavior, interactions, and localization.

Drug delivery systems

A tyrosine click reaction enables the development of drug delivery systems by attaching drug molecules to carrier systems, enhancing their stability and targeting capabilities.


Tyrosine click reactions can facilitate the coupling of biomolecules, such as enzymes, antibodies, or nucleic acids, to other molecules or surfaces to create diagnostic tools, biosensors, or biocompatible materials.

Surface functionalization

A tyrosine click reaction allows modifying surfaces of materials or nanoparticles with specific functional groups, enabling tailoring of their properties and interactions with biological systems.

Polymer chemistry

Incorporating tyrosine-containing monomers in polymer synthesis enables the development of functional materials with diverse applications, such as tissue engineering scaffolds or stimuli-responsive polymers.

Bio-orthogonal chemistry

If a tyrosine click reaction is bio-orthogonal, for example, if a modified DNA, RNA, or peptide molecule is delivered into a living system such as a cell, the reaction enables selective and precise labeling of biomolecules in living cells and organisms.

The success and application of any click reaction, including one involving tyrosine, depends on the development of suitable and efficient reagents, catalysts, and conditions that ensure high selectivity, yield, and biocompatibility. Therefore, further research and validation are required to determine the practical applications of a tyrosine click reaction in oligonucleotide and peptide chemistry

The reversible nicking reaction is catalyzed by DNA topoisomerse I

Champoux (1981) showed that DNA strand breakage by the rat liver DNA nicking-closing enzyme resulted in the covalent attachment of the 3’-end of the broken strand to the enzyme. Labeling experiments using 32P-labeled complexes revealed that a covalent linkage was associated with O4-phosphotyrosine, providing direct evidence that tyrosine was the amino acid at the end of the DNA chain it was attached to.

Shuman and Prescott (1990) showed that the Vaccinia topoisomerase formed a cleavable complex with duplex DNA in which the enzyme is covalently attached to a 3’-phosphoryl group at the site of an enzyme induced single strand nick. The study mapped the cleavage site to a pentameric consensus sequence, 5’-(T/C)CCTT.

Pluta et al. (2017) reported that DNA-nicking reactions involve a nucleophilic tyrosine residue in the active site of the enzyme. The reaction proceeds via nucleophilic attack at the scissile phosphate to result in a transient covalent intermediate. 
A relaxase-mediated nucleophilic attack at the nicking site generates a covalent linkage between a tyrosine residue and the 5′-phosphate DNA of the cleaved dinucleotide, leaving a free 3′-hydroxyl end that serves as a primer for DNA replication by conjugative rolling-circle replication.

The current model for this type of conjugation hypothesizes that the covalent phosphotyrosine DNA-relaxase complex is pumped to the recipient cell by the coupling protein and the type IV secretion system (T4SS). In the recipient cell, the transferred ssDNA is converted into double-stranded (ds) DNA molecules by replication from a lagging strand origin, followed by a second relaxase-mediated reaction to close the newly synthesized strand and supercoiling of the dsDNA by the recipient gyrase.


Ban H, Nagano M, Gavrilyuk J, Hakamata W, Inokuma T, Barbas CF 3rd. Facile and stabile linkages through tyrosine: bioconjugation strategies with the tyrosine-click reaction. Bioconjug Chem. 2013 Apr 17;24(4):520-32. [PMC]

Champoux JJ.; DNA is linked to the rat liver DNA nicking-closing enzyme by a phosphodiester bond to tyrosine. J Biol Chem. 1981 May 25;256(10):4805-9. [

Champoux JJ.;
  (2001). DNA topoisomerases: Structure, function, and mechanism. Annual Review of Biochemistry, vol. 70, no. 1, p. 369-413.

Claeboe CD, Gao R, Hecht SM. 3'-modified oligonucleotides by reverse DNA synthesis. Nucleic Acids Res. 2003 Oct 1;31(19):5685-91. [

DNA Repair [

Li M, Liu Y. Topoisomerase I in Human Disease Pathogenesis and Treatments. Genomics Proteomics Bioinformatics. 2016 Jun;14(3):166-171. [

Meyer, A., Baraguey, C., Vasseur, J-J., and Morvan, F., Oligonucleotide Conjugation by Tyrosine‐Click Reaction. European Journal of Organic Chemistry. Volume 2022, Issue 21, 55-64. ISSN 1434-193X, https://doi.org/10.1002/ejoc.202101361. [

Pluta R, Boer DR, Lorenzo-Díaz F, Russi S, Gómez H, Fernández-López C, Pérez-Luque R, Orozco M, Espinosa M, Coll M. Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance. Proc Natl Acad Sci U S A. 2017 Aug 8;114(32): E6526-E6535. [PMC]

Reversible Nick Reaction [

Shuman S, Prescott J. Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I. J Biol Chem. 1990 Oct 15;265(29):17826-36. [

Bio-Synthesis provides a full spectrum of bio-conjugation services including high quality custom oligonucleotide modification services, back-bone modifications, conjugation to fatty acids and lipids, cell-penetrating peptides, cholesterol, tocopherol, other peptides as well as biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs, miRNA, or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

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