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

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                         <h3 class="basicheader"> Click Chemistry </h3>
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                                     Hein et al. describe the copper-catalysed azide-alkyne cycloaddition (CuAAC) and the positive aspects of it as well as the importance of using copper as a catalyst <x-ref>hein2010</x-ref>
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                                     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.
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                                    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.
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An azide anion is a strong nucleophile that easily substitutes potential leaving groups. Thus, azides react with alkynes via a dipolar cycloaddition. The reaction itself is a highly exothermic one with a ∆H° between -50 and -60 kcal mol-1. However, on the contrary, the activation energy is high too (24-26kcal mol-1<x-ref>zhang2005</x-ref>, which is why the reaction rate is low. Without catalyst the reaction would result in a 1,4, 1,5 triazole regioisomer. Therefore, it is necessary to add copper as a catalyst. Additionally, Zhang et al. have shown that a ruthenium catalysed reaction (RuACC) between organic azides and alkynes is possible, too, leading to a 1,5 disubstituted triazole.
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With regard to the choice of catalyst used, copper (Cu(I)) proves to be the most thermodynamically stable one. Hence, the thermal reaction proceeds rapidly into a 1,2,3 triazole heterocycle which provides many positive features. Its aromatic character, strong dipole moment, high chemical stability and capability of accepting hydrogen bonds make it highly applicable for many chemical reactions in a broad field of science <x-ref>lauria2014</x-ref>.
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Additionally, ascorbate is added to the reaction as a reductant maintaining the oxidation state of copper. Furthermore, a ligand is important to prevent the unwanted influence of ROS (reactive oxygen species) by protecting the biomolecules from oxidation <x-ref>presolski2011</x-ref>.
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Martin et al. have shown that yeast PolyA Polymerase is able to incorporate certain modified NTPs <x-ref>martin1998</x-ref> to the 3’ terminus. As terminal alkynes and azides are relatively small, Poly A Polymerase can easily incorporate them, while bigger molecules cause steric hindrance leading to a deficiency in enzymatic activity.
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The transformation of a terminal label into an internal label, one can be achieved by splinted ligation using a DNA template that is complementary to the two RNA templates that are to be connected to each other <x-ref>Kershaw2012</x-ref>.
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Revision as of 08:07, 18 September 2015

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.