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Both methods are useful methods to visualize short ssDNA and ssRNA as a safe alternative to radioactive labelling. Any DNA of interest can be ordered as oligo with a HRP-mimicking DNAzyme sequence connected to its 5’ or 3’ end. If longer DNA or any RNA is to be labeled with this DNAzyme splinted ligation of two DNA stands using an complementary short DNA sequence called splint can be performed as described by Kershaw.kershaw2012

For the click chemistry application we showed that alkyne modified nucleotides can be added to the 3’ end of a RNA with yeast Poly(A) Polymerase. Which afterwards allowed specific labeling of the RNA of interest. This terminal modification can be turned into an internal modifications by ligating two RNA parts together using a DNA splint. Incorporating the alkyne opens up a variety of possibilities because via CuCCA every azide activated molecule or even protein can be covalently connected to it. Lorenz showed that an azide activated protein horseradish peroxidase (HRP) can be attached to an alkyne modified RNA.lorenz2015 This protein-enzyme HRP amplifies the signal and thus increases the sensitivity of the assay.

Figure 7. Splinted Ligation of RNA and DNA

T4 Ligase is capable of ligating single strand nucleic acids using a DNA splint (red).

Furthermore specific detection of RNA or DNA is possible with both strategies. For example the detection of a cleavage products of RNA-cleaving DNAzymes proofed to be difficult because the cleavage products were running at the same height as the DNAzyme itself. Thus the possible appearing of this product was hidden by the DNAzyme band. We tried to address this problem by the addition of DNase I to our samples to digest the DNA but the DNase I was not able to digest all of the ssDNA from the sample as it is more efficient for dsDNA. To visualize the RNA of interest only we planned to connect the HRP DNAzyme to it via splinted ligation (see Fig. 7). This would enable us to see bands on two different heights on the blot only: the cleaved part that contains the HRP DNAzyme and also the uncleaved substrate.

We faced the problem of detecting RNA of similar size again with the in vitro assay of the twin ribozyme. Here we could not use the HRP as readout because the sequence is conserved from the 5’ end throughout the 3’ end. Thus the internal modification of the RNA with a CuAAC activation seemed suitable.

Connecting RNA to the HRP be is a way to multiplex assays similar to the one we described in our small molecule detection project. We tested for one target in one sample by adding a switchable hybrid of HRP-mimicking DNAzyme and F8 DNA-cleaving DNAzyme. By the self-cleavage of this construct in presence of the ligand that triggers the activity of the F8 DNAzyme the HRP DNAzyme activity was recovered leaving two different cleavage products. Those can be separated on a PAGE and blotted afterwards. The blotting step allows for multiplexing of this system. Many different versions of the same construct switchable with different ligands can be added to one sample if the cleavage products are designed to have characteristic sizes.