Team:Heidelberg/project/rd

iGEM Team Heidelberg 2015

• Achievements
• Results
• Project
• Software
• Notebook
• Parts
• Team
• Modeling
• Outreach

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. Both methods bear the potential of sensitive and specific readout and could be used in many applications.

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.

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 Without catalyst the reaction is slow and results in a 1,4, 1,5 triazole regioisomer. To increase the reaction rate and to avoid this byproduct it is necessary to add copper as a catalyst. Copper (Cu(I)) proofed to be a suitable catalyst that rapidly yields a 1,2,3 triazole heterocycle.

Figure 1. Copper-catalyzed azide-alkyne cycloaddition (CuAAC)

Reaction scheme of the CuAAC. Adapted from: Source

Figure 2. Alkyne modification and click reaction of RNA

RNA was 3' modified with alkyne modified nucleotides using yeast Poly(A) Polymerase at 37 °C. Afterwards RNA was precipitated and used for copper click reaction with Alexa 488 azide.

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.

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 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. Winz2012

HRP-mimicking DNAzyme

The versatile HRP-mimicking DNAzyme forms a G-quadruplex structure in which hemin can be bind.Travascio1998 This enables it to catalyze the fission of hydrogen peroxide to water and a reactive oxygen species (ROS). Thus it can be used to catalyze chemiluminescence and a series of colorimetric reactions, known from the horseraddish peroxidase from Amoracia rusticana (Fig.3). As we show in our AptaBody project the HRP-mimicking DNAzyme can be used to generate a signal on a western blot. Here we directly blot the DNA onto the membrane and recover its activity after denaturing polyacrylamide gel electrophoresis (PAGE). For this after the transfer the catalytically important hemin is added to the DNAzyme which results into catalytic activity on the membrane.

Figure 3. HRP-mimicking DNAzyme

The HRP-mimicking DNAzyme forms a G-quadruplex that binds hemin in its active center. In this conformation it is able to activate classical HRP substartes.