Difference between revisions of "Part:BBa K1614025"

Latest revision as of 19:11, 20 September 2015

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 __NOTOC__
 <partinfo>BBa_K1614025 short</partinfo>
-Construct consisting of a ketamin-switched F8 DNAzyme linked to a stemmed HRP mimicking DNAzyme.
+Construct consisting of a ketamin-switched F8 DNAzyme (Wang <i>et al.</i> 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.
-If there no ketamine binds to aptamer the F8 is inactive and HRP mimicking DNAzyme is stemmed, so that no hemin could bind and there is no peroxidase activity.
+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.
-By binding of ketamin to the aptamer, F8 is activated and cleaves the stemming part, so the HRP mimicking DNAzyme is activated, peroxidase activity could be used for different readout techniques.
+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.