Template:Heidelberg/project/rd/clickchemistry

Abstract

Detection of short nucleic acids is mainly done using a radioactive labeling strategy because of the high sensitivity it offers. We wished to bypass the risks that are connected to working with radioactivity, therefore we aimed to establish two alternative readouts with suitable sensitivity. The first method we worked on is based on click chemistry and has been previously proposed. The second method, based on the HRP-mimicking DNAzyme, is novel and was established by us. Firstly, we show that ssRNA can be elongated by an alkyne modified nucleotide with yeast Poly(A) Polymerase at the 3’ end. This alkyne modification can specifically be connected with a fluorophore under copper-catalyzed azide-alkyne cycloaddition conditions. As novel visualization method for the labeling of DNA and RNA we propose the HRP-mimicking DNAzyme. We show that the activity of the HRP-mimicking DNAzyme can be recovered after denaturing polyacryl amid gel electrophoresis and transfer to a membrane. Both methods bear the potential of sensitive and specific readout and could be used in many applications.