DNA

Part:BBa_K3343001

Designed by: Marijn van den Brink   Group: iGEM20_Leiden   (2020-09-08)


DNAzyme with peroxidase activity

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.


This part is an improved version of BBa_K1614007, the most widely used peroxidase-mimicking DNAzyme1. By adding an additional adenine residue at the 3’ end of BBa_K1614007, its activity and tolerance for pH lower than its optimal pH of 8.5 were enhanced substantially. Our results (iGEM Leiden 2020) have demonstrated a large increase in activity for BBa_K3343001 (+3A) as compared to BBa_K1614007 (-3A) in pH 6 phosphate buffer. Nevertheless, further research into a wider range of buffers and different pH would contribute to the characterization of this new part.


Usage and biology

This part is an 18-nt long sequence of single-stranded DNA, which folds into a G-quadruplex (G4) structure and binds hemin(iron(III)-protoporphyrin IX)1. In this structure, it acquires peroxidase-mimicking activity, through which it can oxidize substrates like 3, 3′, 5, 5′-tetramethylbenzidine (TMB), 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), luminol and tyramine in the presence of hydrogen peroxide (H2O2) (Figure 1). The oxidation is accompanied with a color change for TMB and ABTS, chemiluminescence for luminol and fluorescence emission for tyramine. Due to their small size, easy manipulation and easy synthesis, G-quadruplex DNAzymes have been exploited as reporters in biosensors and other biomolecular machines, where they replace traditional enzymatic reporters, such as horseradish peroxidase (HRP)2].

The optimal pH of the original part (BBa_K1614007) is approximately 8.51,3. Its activity sharply decreases within the pH range of 8 to 61. However, for some applications a high peroxidase activity at pH lower than 8 is needed. In the project of iGEM Leiden 2020, a lower pH was required to decrease the reducing power of dithiothreitol (DTT) in the oxidation reaction mix. DTT is a reducing agent, which inhibits the accumulation of oxidized product. In this project, it could not be prevented that DTT was present in the oxidation reaction mix and thus the pH had to be decreased to reduce its activity.


Figure 1. Oxidation of TMB using H2O2, catalyzed by G-quadruplex DNAzyme.

Figure 1. Oxidation of TMB using H2O2, catalyzed by G-quadruplex DNAzyme. Figure adapted from team Leiden 2020’s preprint3.


Characterization

Here, we compare the activity of the original DNAzyme (BBa_K1614007) and its improved part (BBa_K3343001) with an additional adenine residue (+3A). Based on previous findings by Li et al. (2016) that suggest that the adenine acts as a potential distal ligand in the G4–hemin complex, we hypothesize that this extra adenine residue increases the peroxidase-mimicking activity at pH 6.01.


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


The activity of the DNAzymes was monitored by measuring the absorbance at 650 nm over time. This is the first oxidation product (Figure 1). A third DNAzyme (BBa_K3343000) was included as an extra control, as this is one of the DNAzymes with highest peroxidase-mimicking activity known in literature1. This DNAzyme has an intrinsic 3’ adenine. The three DNAzymes that were tested can be found in Table 1. A blank with buffer in place of DNAzyme served as negative control.


Table 1. Three DNAzymes used in our 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


Results

The activity of the original part (BBa_K1614007) was similar to the blank, indicating no or very low peroxidase-mimicking activity at pH 6.0 (Figure 2). The activity of the improved part (BBa_K3343001) was much higher, comparable to DNAzyme BBa_K3343000.


Figure 2. Peroxidase-mimicking activity of the three DNAzymes BBa_K1614007, BBa_K3343001 and BBa_K3343000 in pH 6.0 phosphate buffer.

Figure 2. 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. 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).

2. Kosman, J. & Juskowiak, B. Peroxidase-mimicking DNAzymes for biosensing applications: A review. Analytica Chimica Acta 707, 7–17 (2011).

3. http://parts.igem.org/Part:BBa_K1614007

4. 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]


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