Part:BBa_K3343000

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, also called EAD2 or EAD2+3’A, is one of the peroxidase-mimicking G-quadruplex DNAzymes with highest activity known in literature¹. It has an intrinsic adenine at the 3’ terminus of its sequence, which is known to be of great importance to high peroxidase-mimicking activity of G-quadruplex structures. Here, we compare the activity of this DNAzyme with that of two other DNAzymes (BBa_K3343001 and BBa_K1614007) and report the molar maximum TMB conversion rate (k_cat) and substrate affinity (K_m) of this part.

The text and figures were adapted from team Leiden 2020’s scientific preprint².

Usage and biology

Guanine-quadruplex (GQ) sequences were known to have peroxidase-like activity when associated with hemin¹. This allowed GQ-hemin complexes to be used as a catalyst for peroxidation reactions as an alternative to horseradish peroxidases^1,3. Due to their enzyme-like ability, GQ-hemin complexes are often referred to as GQ DNAzymes. Just like horseradish peroxidases, GQ-hemin complexes can catalyze the color-changing oxidation reaction of 3, 3’, 5, 5’-tetramethylbenzedine (TMB) in the presence of peroxide (H₂O₂) (Figure 1). Alternatively, GQ-hemin complexes can oxidize 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), luminol and tyramine in the presence of H₂O₂, accompanied by a color change, chemiluminescence and fluorescence emission, respectively.

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

Characterization

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

Different GQ sequences may have different enzyme-like activity. We compared EAD2+3’A with one of the most widely used DNAzymes among scientists (BBa_K1614007)¹ and a DNAzyme highly similar to BBa_K1614007 but with an additional adenine base at the 3’ terminus (BBa_K3343001). We identified EAD2+3’A as the most potent DNAzyme in the used reaction conditions (phosphate buffer pH 6.0) (Figure 2).

Figure 2. Peroxidase-mimicking activity of the three DNAzymes BBa_K1614007 (5’ GGGTAGGGCGGGTTGGG), BBa_K3343001 (5’ GGGTAGGGCGGGTTGGGA) and EAD2+3’A (BBa_K3343000, 5’ CTGGGAGGGAGGGAGGGA).

Characterization of EAD2+3’A catalytic activity was performed based on the DNAzyme’s ability to oxidize TMB in the presence of excess amount of peroxide and different concentrations of TMB. All reactions were performed in a pH 6.0 phosphate buffer. The rate of TMB oxidation was then monitored based on oxidized TMB’s absorbance at 650 nm wavelength (molar absorption coefficient is 39,000 M_-1 cm_-1)⁴. Kinetic parameters of the DNAzyme were described using the Michaelis-Menten equation (Eq. 1), with k_cat and K_m representing the molar maximum TMB conversion rate and TMB affinity of EAD2+3’A, respectively. The values of k_cat and K_m were calculated using multiple regression analysis by simultaneously minimizing the error of all measurement-simulation pairs. The approximated kinetic parameters of EAD2+3’A against TMB are shown in Figure 3, Figure 4, and Table 1.

The K_m value of the DNAzyme suggested that a TMB concentration of 0.06 mg/mL was sufficient in order to reach 90% of the reaction’s maximum initial rate k_cat. Further increase in TMB concentration may thus be not as significant in further increasing maximum reaction rate. Based on this result, a TMB concentration of 0.06 mg/mL was selected to be optimal for future assays and analyses.

Figure 3. TMB oxidation by EAD2+3’A DNAzyme. Kinetic parameters of the DNAzyme were measured based on its ability to catalyze TMB oxidation reaction at different TMB concentrations. In the figure, data points represent initial oxidation rate measured from each experimental replicate while the curve represents model-predicted kinetics at each given TMB concentration. Rate kinetics of EAD2+3’A were assumed to follow Michaelis-Menten kinetics. The initial rate of each individual replicates was calculated based on differences in oxidized TMB absorbance value at 650 nm for the first minute of the observation. The resulting model could sufficiently describe each individual replicate measurement (Figure 4).

Figure 4. Predicted TMB oxidation kinetics by EAD2+3’A could sufficiently describe reaction rates of each individual reactions. The values above each figure represented TMB concentration used in each observation. Observations obtained from each experimental replicate were represented as data points. Reaction rate between each two observed time points were assumed to be constant and resulted to a linear increase in A650 value. Lines shown in the figure represent the simulated increase in A650 value over the observation period at a given TMB concentration, as predicted by the fitted kinetic model, and by using the average of each measurement replicates at time point zero as starting point of the simulation.

Conclusion

We compared this part, a peroxidase-mimicking DNAzyme named EAD2+3’A, with two other DNAzymes and showed that this part has highest activity in phosphate buffer pH 6.0. In addition, we determined the molar maximum TMB conversion rate (k_cat) and substrate affinity (K_m) of EAD2+3’A, which were 3.971 x 104 min^-1 MDNAzyme^-1 and 6.798 x 10-3 g L^-1, respectively. The parameter values were implemented in team Leiden 2020’s empirical modelto assess the performance of the oxidation reaction and guide future optimization of our technique.

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. 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.

3. Yang, X. et al. Characterization of G-quadruplex/hemin peroxidase: Substrate specificity and inactivation kinetics. Chem. - A Eur. J. 17, 14475–14484 (2011).

4. Liu, Y., Zhu, G., Yang, J., Yuan, A. & Shen, X. Peroxidase-like catalytic activity of Ag3PO4 nanocrystals prepared by a colloidal route. PLoS One 9, e109158 (2014).

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]