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Reactive Linker
 

Aminolink

Aminolink


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. 


   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.

 

Carboxylinker

Carboxylinker


     Carboxylinker
 

Surface immobilisation 

Modifications for the immobilisation on surfaces 


Carboxymethylaniline (4-CMA) allows to immobilise coupled biomolecules to appropriate prepared surfaces. In this way, the binding of oligonucleotides to conductive surfaces (e.g. graphite) enables the detection of currents. By coupling, either to the 5 'or 3' end of oligonucleotides, the molecules are covalently bonded in the desired orientation to the surface. 

 


4-CMA at the 3´- or 5´-end of an oligonucleotide
Bindung von Oligonucleotiden an Oberflächen über 4-CMA
 

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Literature:
- Diazonium-Protein Adducts for Graphite Electrode Microarrays Modification: Direct and Addressed Electrochemical Immobilization. Corgier BP, Marquette CA, Blum LJ; Journal of the American Chemical Society, (2005), 127, 18328-18332.

- A versatile method for direct and covalent immobilisation of DNA and proteins on biochips. Corgier BP, Laurent A, Perriat P, Blum LJ, Marquette CA; Angewandte Chemie International (2007), 46, 4108-4110.

- On-Chip Chemiluminescent Signal Enhancement using Nanostructured Gold-Modified Carbon Microarrays. Corgier BP, Li F, Blum LJ, Marquette CA; Langmuir (2007), 23(16), 8619-8623.