Part:BBa_K1614025
Ketamin-dependent self cleaving F8 - readout DNAzyme
Construct consisting of a ketamin-switched F8 DNAzyme (Wang et al. 2014) linked to a stemmed HRP mimicking DNAzyme.
It contains the HRP mimicking DNAzyme, which can bind hemin as a cofactor to catalyse the degradation of hydrogen peroxide to water and reactive oxygen species. For use as a readout system, luminol, TMB, or other substances suitable to detect radicals can be used.
The F8 DNAzyme is a copper ion dependent DNAzyme, which can catalyse the excision of a thymine from its substrate strand, and does so with enhanced efficiency and faster kinetics in the presence of hydrogen peroxide, which should be present in all reactions involving the HRP mimicking DNAzyne, as well as this construct.
A software designed, aptamer-containing stem-loop is inserted into the DNAzyme's active site, which contains parts of the active site necessary for catalysis, resulting in an inactive DNAzyme. Upon binding of ketamine to the software-designed aptamer, the stem displaces and releases the active site, resulting in a catalytically active conformation.
The resulting degradation of hydrogen peroxide to water and reactive oxygen species can be detected using luminol, TMB, ABTS or any other analogous method.
Stems and Aptamer generated by MAWS and JAWS software.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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