Difference between revisions of "Part:BBa K1614007"

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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

Characterization

1. Exploration of the condition of the individual ssDNA G4/Hemin DNAenzyme activity

1.1 Determination of reaction quantity and reaction time of hydrogen peroxide in the system

In the reaction process, both Hemin and G4/ Hemin DNAenzyme will carry out enzyme-catalyzed reaction with hydrogen peroxide as the substrate, which will reach the peak due to the constant decrease of substrate concentration during the reaction, and then attenuated due to the characteristics of the reaction. We used ssDNA G4 and Hemin solution with the same initial concentration of 1uM to determine the better conditions for the addition of hydrogen peroxide solution by measuring the reaction time of adding different amounts of hydrogen peroxide system.

Figure 1 The absorbance of the reaction system for different amount of hydrogen peroxide in different time

As shown in the figure (Figure 1), the time interval represented by the highest two points was selected as a good time for the reaction measurement. Under the condition of adding 1ul hydrogen peroxide, the scatter was dense and the reaction time had little influence. Under the condition of adding 3ul hydrogen peroxide, the best reaction time was between 2-5min. Under the condition of adding 5ul hydrogen peroxide, the best reaction time was between 5-7min. Under the condition of adding 7ul hydrogen peroxide, the optimum reaction time was over 10min.

1.2 The activity of reaction for G4/Hemin mimic DNA enzyme turn to time

Figure 2 the relationship between time and the G4/Hemin DNAenzyme. Line green is the average of the four parallel experiments.

It can be seen that there is a peak value in the curve obtained (Figure 2). When 3ul 30% hydrogen peroxide is added in the pre-experiment, the peak value can be obtained to be 2-5min, so the results are consistent with the pre-experiment. Hemin itself has the catalytic effect of hydrogen peroxide, but when the G4 conjugated sequence is introduced and incubated for a period of time, the simulated enzyme formed by the combination of the two has higher enzyme activity and reaches the reaction peak at 2-5min. The results were used as a reference for the selection of reaction conditions.

Sequence and Features