Difference between revisions of "Team:Heidelberg/project/rd"
Line 8: | Line 8: | ||
{{Heidelberg/project/rd/methods/clickreaction}} | {{Heidelberg/project/rd/methods/clickreaction}} | ||
{{Heidelberg/project/rd/methods/blotting}} | {{Heidelberg/project/rd/methods/blotting}} | ||
+ | {{Heidelberg/project/rd/results}} |
Revision as of 08:54, 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. 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
Copper-catalyzed azide-alkyne cycloaddition (CuAAC)
Sharpless described the copper-catalyzed azide-alkyne cycloaddition (CuAAC)
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.
HRP-mimicking DNAzyme
The versatile HRP-mimicking DNAzyme forms a G-quadruplex structure in which hemin can be bind.
Methods
Alkyne modification and CuAAC
Entre HERE
Blotting
All blotting was performed with ssDNA or ssRNA that was separated on a denaturing PAGE. The DNA and RNA after staining with ethidium bromide (EtBr)was transferred onto a nylon membrane in a semidry blotting apparatus from Biorad in 0.5x TBE by applying 10-20 V for 35 min. Samples were immobilized with NaOH. Refolding of HRP-mimicking DNAzyme was performed in HRP-mimicking DNAzyme buffer that was supplemented with 100 mM hemin for 10 min. Excessive hemin was washed away by washing with the same buffer without hemin for 5 min. Blots were developed 5 min in in SuperSignal™ West Pico Chemiluminescent Substrate and imaged on a ChemoCam from Intas.