Difference between revisions of "Template:Heidelberg/project/rd/copperclick"

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            Fig.4: Modification of RNA with Alexa 488 azide
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Revision as of 13:04, 18 September 2015

Copper-catalyzed azide-alkyne cycloaddition (CuAAC)

Sharpless described the copper-catalyzed azide-alkyne cycloaddition (CuAAC)kolb2001 also called click chemistry. This is due to the simple concept. It works under many different conditions with high yields and no byproducts.mckay2014 A highly energetic azide reacts with an alkyne enabling a selective reaction that links these reactive groups to one another via a dipolar cycloaddition (Fig. 1). However this reaction requires a lot of activation energy.zhang2005

The advantages of a click reaction are that it is very simple and works under many different conditions, as well as that the reaction results in high yields with no byproducts. The highly energetic azides react with alkynes enabling a selective reaction that links reactive groups to one another. To obtain the oxidation state of the copper sodium ascorbate is added to the reaction. Furthermore a ligand like THPTA is necessary to keep the Cu(I) stabilized in aqueous solution.

Fig.4: Modification of RNA with Alexa 488 azide

In order to use the above explained advantages of click chemistry for the labelling of DNA and RNA azide or alkyne modified nucleotides have to be incorporated into the sequence (Fig. 2). Martin et al. have shown that yeast Poly(A) Polymerase is able to incorporate modified nucleotides with small moieties martin1998 to the 3’ terminus. To obtain an internal modification it is necessary to ligate two part of DNA or RNA to each other via splinted ligation.Kershaw2012winz2012