Page id: 93

Reactive Linkers



An aminolink adds a terminal amino group (NH2) bound to a linker. The amino group can be used to couple additional molecules, e.g. dyes or proteins like HRP. The aminolink is used as well with DNA arrays to bind oligonucleotides to adequately prepared surfaces. There are linkers of variable length for use in different applications. Aminolinks can be coupled to both the 5´- and the 3´-end of the oligonucleotide. Modifications at the
3´-terminus of oligonucleotides make them more resistant against exonuclease digestion. Furthermore, it can be useful to link an aminogroup inside the oligonucleotide. For this purpose, a thymidine nucleotide's C5 methyl group can be easily replaced by a C6 linker with an amino group at its end. Thus the interaction between the amino group and the DNA is reduced as far as possible so that the modified oligonucleotide behaves in hybridisation comparable to a respective unmodified one.

The figures below show the deprotected final product as it will be finally delivered with the oligonucleotide.

   Amino link at the 3´- or 5´-Terminus of an oligonucleotide

Thiol / Maleimide

Thiol and Maleimide

Sulfur compounds are often used to bind molecules to gold particles. Like aminolinks, thiol links at the 5´-end of an oligonucleotide are linked to the respective end of the oligonucleotide using a chain of 6 carbon atoms (6xCH2). At the 3´-end a SH group is linked using a C3 linker. Thiol links are frequently used to bind an oligonucleotide as a thioether covalent to maleimides. Thiol and maleimides are easily reacted forming a stable covalent linkage with each other. Dependent on the reactive agent available for the modifier, the oligo can either be activated with a thiol or a maleimide function, both options are available. This offers various means to couple an oligonucleotide that has been modified in such a way to e.g. dyes or proteins.
In addition to a simple thiol, a thioctic acid modification is also suitable for binding oligonucleotides to gold surfaces. Due to the two sulfur atoms the bond to the gold is even stronger.

OPSS, the orthopyridyl disulfide, is a terminal modification that can form a stable disulfide bridge with a SH group. Here, proteins, peptides or other biomolecules, as well as surfaces, can be coupled to an OPSS-modified oligonucleotide. 

   Thiol and Maleimide

Click Chemistry

Click Chemistry

The term „click chemistry“ describes a fast and thermodynamically favoured reaction which enables an efficient and selective linkage of two molecules. In a more specific sense the click reaction is a cycloaddition between an azide and an alkyne either under copper catalyzed or copper free reaction conditions. Due to the fact that such click reactions work efficiently in aqueous media, they are very much suitable for modifying biomolecules or linking different biomolecules together. Furthermore the azide and alkyne reaction partners do not interfere with other functional groups like e.g. amino or carboxy which opens an additional degree of freedom for orthogonal coupling strategies.

A selection of alkine and azide linker as well as the copper-free variants (DBCO, TCO, Tetrazine) can be found at Click Chemistry.







Aldehydes are highly reactive molecules that can be used to link oligonucleotides to other biomolecules. In general, carbonyl compounds (aldehydes) react with nucleophiles (amino groups, thiol compounds, etc.). The amino groups can be simple amine linkers (primary and secondary amines), but also hydrazine or aminooxy compounds.
Due to the partly complex reactions with aldehydes, reactions with aminooxy compounds (e.g. aminooxyacetic acid) are preferred. The aminooxyacetic acid (AOA) reacts with aldehydes to form stable oximes.

Picture 1: Reaction scheme of an aldehyde-labelled oligonucleotide and an AOA-coupled protein.



1. Organic Chemistry, 4th edition. Carey FA; Chapter 22, p. 858.

Cyanobenzothiazole / Cysteine

CBT and Cysteine – selective coupling of two biomolecules 


By means of cyanobenzothiazole-labelled oligonucleotides, suitable proteins, peptides or other molecules can be rapidly and selectively labelled and prepared for detection in the cell without restricting or inhibiting their function. The reactivity and specificity of the cyanobenzothiazole oligonucleotide tag are primarily dependent on the sequence of the protein. In order to ensure a stable adduct and a fast and effective reaction of the cyanobenzothiazole-labelled oligo with a molecule, a terminal cysteine on the molecule is essential. 
Dependent on the reactive agent available for the modifier, the oligo can either be activated with a CBT or a cysteine function. Both options are available.

Picture 1: Reaction scheme of a cyanobenzothiazole-labelled oligonucleotide with a suitable protein carrying an N-terminal cysteine.

The current coupling of proteins and oligos via click chemistry cannot be applied intracellularly due to toxic copper ions. Cyanobenzothiazole, however, allows the coupling of a protein with an oligonucleotide also in vivo.
According to this, a specific labelling of the protein for the detection or analysis of the protein structure and function is ensured.

Possible applications:
- Labelling and localisation of specific proteins
- Luminescence analyses in vivo
- Detection and quantification of intracellular, biochemical processes in real time

The terminal cysteine can react with thioesters (native ligation) and aldehydes. 3



We offer cyanobenzothiazole and cysteine as a 5´-modification on the oligonucleotide.

Please note that the cysteine (tbuSS) is supplied in protected form and need to be treated with TCEP before further conjugation steps to reduce the disulfide and release the thiol group. The TCEP can be added, for example, to the conjugation buffer.


1. A Biocompatible In Vivo Ligation Reaction and its Application for Non-Invasive Bioluminescent Imaging of Protease Activity in Living Mice. Godinat A, Park HM, Miller SC, Cheng K, Hanahan D, Sanman LE, Bogyo M, Yu A, Nikitin GF, Stahl A, Dubikovskaya EA; ACS Chem Biol. (2013), 8(5): doi:10.1021/cb3007314.

2. Sequence-Specific 2-Cyanobenzothiazole Ligation. Ramil CP, An P, Yu Z, Lin Q; J Am Chem Soc. (2016), 138(17):5499-502. doi: 10.1021/jacs.6b00982.

3. New phosphoramidite reagents for the synthesis of oligonucleotides containing a cysteine residue useful in peptide conjugation. Stetsenko DA, Gait MJ; Nucleosides Nucleotides Nucleic Acids (2000); 19(10-12):1751-64.