RNaseAlert Readout
To characterize our Cas13a, we first turned to the standard of the field, namely the RNase Alert detection kit. This was used by Gootenberg and Doudna to characterize the Cas13a and detect pathogen RNA sequences1. In the absence of Cas13a activation, the physical proximity of the quencher dampens fluorescence from the Fluor and there is no fluorescence activity. When Cas13a is activated, the RNA substrate is cleaved, and the Fluor and quencher are spatially separated in solution, emitting a bright green signal when excited by light of the appropriate wavelength. We did most of our experiments using the RNaseAlert system and the corresponding results are in the Cas13a and target subsections.
The fluorescence signal quality of the RNaseAlert assay was very good, however one cannot use this readout system without the fluorescence detector. Also, since the RNaseAlert is a modified RNA, it is somehow expensive in comparison to other readouts.
Figure 1: The fluorescence activity of the Spinach aptamer decreased with the increase in the target concentration.
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RNaseAlert Readout
After the successful experimentation of the Cas13a with the RNaseAlert, we also tried out RNA aptamers for our readouts. For this, we used the Spinach aptamer which binds to the DFHBI changing its 3D structure2. We activated the Cas13a by the specific target, which then cleaved the Spinach aptamer bound to DFHBI and were able to show that the fluorescence activity slowly decreases (Figure 1).
Although we could see a clear decrease in the fluorescence activity as soon as the Cas13a is activated, the original level of fluorescence is lower than in case of RNaseAlert. This could be due to the fact that as soon as the spinach aptamer binds to the DFHBI, the fluorescence is already released. And regarding the time factor needed to mix all the reaction components, we lose some fluorescence before the Cas13a cleavage activity starts.
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