Difference between revisions of "Part:BBa K1614007"

Revision as of 19:25, 20 September 2015

HRP-mimicking DNAzyme

A DNAzyme with peroxidase acivity. (Travascio, P., Li, Y., and Sen, D. (1998). DNA-enhanced peroxidase activity of a DNA-aptamer-hemin complex. Chemistry & biology 5, 505-517.) It forms a G-quadruplex structure in which hemin can be incorporated. This enables it to catalyze the fission of hydrogen peroxide to water and a reactive oxygen species (ROS). Thus it can be used to catalyze chemiluminescence and a series of colorimetric reactions, known from the horseraddish peroxidase from Amoracia rusticana. This part can be joined to other functional DNA.

This part was used in different applications: http://2015.igem.org/Team:Heidelberg/Results/Standardization

• We have joined this HRP DNAzyme with aptamers predicted by our software MAWS (http://2015.igem.org/Team:Heidelberg/software/maws) We call the combination an AptaBody; it is able to detect proteins on a Western blot http://2015.igem.org/Team:Heidelberg/Project/AB
• We propose this DNAzyme as multifunctional readout on a Southern or Northern blot http://2015.igem.org/Team:Heidelberg/project/rd
• We used this DNAzyme to validate (http://2015.igem.org/Team:Heidelberg/project/hlpd) both our software tools: MAWS (http://2015.igem.org/Team:Heidelberg/software/maws) and JAWS (http://2015.igem.org/Team:Heidelberg/software/jaws)
• We combined this part with a F8 DNA self-cleaving DNAzyme that is switchable via a aptamer (http://2015.igem.org/Team:Heidelberg/project/hlpd)

The HRP DNAzyme's catalytic activity has been shown to be modulated by hemin concentration, where the optimum is achieved at equimolar concentrations. Further conditions for optimal activity are a pH of 8.5, no special metals are required. note that at non basic pH, and without further solvent or detergent (e.g Triton-X100), hemin will not dissolve in water, which will result in loss of activity. If the DNAzyme-hemin solution produces a brown-red precipitate, hemin is not dissolved and pH should be raised. Optimal concentration of DNAzyme for readout applications was determined to be 1 µM.

Usage and Biology

Sequence and Features