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iGEM Team Heidelberg 2015

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Click Chemistry

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 hein2010

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.

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-1zhang2005, 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.

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 lauria2014.

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 presolski2011.

Martin et al. have shown that yeast PolyA Polymerase is able to incorporate certain modified NTPs martin1998 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.

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 Kershaw2012.

Introduction

As our project revolved around functional nucleic acids we had to face the challenge of visualizing short ssRNAs and ssDNAs. When testing the activity of our designed ribozymes or DNAzymes it was especially important for us to distinguish between reacted and unreacted equivalents even if their sizes were very similar. Most scientists choose a radioactive labeling technique to visualize little amounts of RNA or DNA. We wanted to develop new approaches to give scientists the possibility to detect little amounts of nucleic acids without the necessity to employ radioactive isotopes. On the one hand we decided to use copper-catalyzed azide-alkyne cycloaddition to specifically label RNA or DNA with fluorophores as described in Winz2012. On the other hand we came up with the idea of using the HRP-mimicking DNAzymeTravascio1998 connecting it to our DNA or RNA of interest to make it visible on a Southern/Northern blot.