Part:BBa_K1614007

HRP-mimicking DNAzyme

Notice: Functional DNA

This part is a sequence of a functional ssDNA. It is only active as single-stranded DNA. It can not be cloned into a plasmid. For use order it as a DNA oligo.

A DNAzyme with peroxidase acivity¹. 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.

Characterization - Team Heidelberg 2015: Exploration of the condition of the individual ssDNA G4/Hemin DNAenzyme activity

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.

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.

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.

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

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

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.

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

Characterization - Team Leiden 2020: Improving DNAzyme's activity and pH tolerance by changing primary structure

The optimal pH of this DNAzyme is 8.5 and its activity sharply decreases within the pH range of 8 to 6 (Figure 3)². Therefore, this DNAzyme is not suited for use at low pH conditions. In certain applications, it may be preferred to use peroxidase-mimicking DNAzymes in low-pH conditions. For instance, iGEM team Leiden 2020 needed a low-pH oxidation buffer to decrease the power of a reducing agent that inhibited the GQ-catalyzed oxidation reaction. Li et al. (2016) showed for various peroxidase-mimicking DNAzymes that the addition of an adjacent adenine base at the DNAzyme’s 3’-end enhances its activity at pH 4 to 8 (Figure 3). Here, GEM Leiden 2020 demonstrated that this part (BBa_K1614007) showed no peroxidase-mimicking activity in a pH 6.0 phosphate buffer. However, upon adding an adjacent adenine at the 3’ terminus of its sequence, the peroxidase-mimicking activity was greatly enhanced at pH 6.0. The improved part was added to the Registry as new part BBa_K3343001.

Figure 3. Initial peroxidase-mimicking rates of the original part (BBa_K1614007 / Dz-00 in blue) and the improved part (BBa_K3343001 / Dz-11 in red) in phosphate buffer with pH 3 to 8. Figure adapted from Li et al. (2016)².

Reaction conditions
- The peroxidase-mimicking activity of three DNAzymes was analyzed through TMB oxidation in the presence of 0.045% H₂O₂, 1 µM hemin, 0.47 mg/mL KCl and 0.06 mg/mL TMB.
- The oxidation reaction was performed in 0.1 M phosphate buffer pH 6.0 (1.307 grams of Na₂HPO₄.7H₂O (MW=268.07 g/mol) and 13.126 grams of NaH₂PO₄.H₂P (MW=137.99 g/mol) in 1 L H₂O).
- The concentration of DNAzyme was 0.1 µM.
- Since hemin does not dissolve well in water at low pH, hemin stock (50 µM) was first dissolved in 100% DMSO. TMB stock solution was also made using DMSO. Both stock solutions were diluted to its intended assay concentrations using 0.1 M phosphate buffer (pH 6.0).
- Extensive methods can be found at https://2020.igem.org/Team:Leiden/Experiments

Results
The activities of the original DNAzyme (BBa_K1614007) and improved DNAzyme (BBa_K3343001) were monitored by measuring the absorbance at 650 nm over time. This is the first TMB oxidation product (Figure 4). A third DNAzyme (BBa_K3343000), was included as an extra control, as this is one of the DNAzyme with highest peroxidase-mimicking activity known in literature². This DNAzyme has an intrinsic adenine at the 3’ terminus of its sequence. The three DNAzymes that were tested here can be found in Table 1. A blank with buffer in place of DNAzyme served as negative control.

Figure 4. Oxidation of TMB using H₂O₂, catalyzed by G-quadruplex DNAzyme. Figure adapted from team Leiden 2020’s preprint³.

Table 1. Three DNAzymes used in the comparison experiment.

Registry Part	Description	Sequence
BBa_K1614007	Original part, most widely used DNAzyme	GGGTAGGGCGGGTTGGG
BBa_K3343001	Improved part with additional adenine	GGGTAGGGCGGGTTGGGA
BBa_K3343000	DNAzyme with highest activity known in literature	CTGGGAGGGAGGGAGGGA

The activity of the original part (BBa_K1614007) was similar to the blank, indicating no or very low peroxidase activity at pH 6.0 (Figure 5). The activity of the improved part (BBa_K3343001) was much higher, comparable to DNAzyme BBa_K3343000.

Figure 5. Peroxidase-mimicking activity of the three DNAzymes BBa_K1614007, BBa_K3343001 and BBa_K3343000 in pH 6.0 phosphate buffer. Figure adapted from team Leiden 2020’s preprint³.

Conclusion
In conclusion, the additional adenine at the 3’ end of the DNAzyme greatly improved the DNAzyme peroxidase’s activity at pH 6.0. Without the modification, the older version of the DNAzyme (BBa_K1614007) could not show significant peroxidase activity. The modification that we included to create BBa_K3343001 thus allowed it to retain its catalytic ability at a lower pH range.

References

1. Travascio, P., Li, Y., and Sen, D. DNA-enhanced peroxidase activity of a DNA-aptamer-hemin complex. Chemistry & biology 5, 505-517 (1998).

2. Li, W. et al. Insight into G-quadruplex-hemin DNAzyme/RNAzyme: Adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity. Nucleic Acids Res. 44, 7373–7384 (2016).

3. Van den Brink, M. et al. Rapidemic, a versatile and label-free DNAzyme-based platform for visual nucleic acid detection. bioRxiv 2020.10.14.337808 (2020). doi:10.1101/2020.10.14.337808.

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]