DNAzymes as Catalysts for l-Tyrosine and Amyloid β Oxidation

Abstract

Single-stranded deoxyribonucleic acids have an enormous potential for catalysis by applying tailored sequences of nucleotides for individual reaction conditions and substrates. If such a sequence is guanine-rich, it may arrange into a three-dimensional structure called G-quadruplex and give rise to a catalytically active DNA molecule, a DNAzyme, upon addition of hemin. Here, we present a DNAzyme-mediated reaction, which is the oxidation of l-tyrosine toward dityrosine by hydrogen peroxide. With an optimal stoichiometry between DNA and hemin of 1:10, we report an activity of 101.2 ± 3.5 μUnits (μU) of the artificial DNAzyme Dz-00 compared to 33.0 ± 1.8 μU of free hemin. Exemplarily, DNAzymes may take part in neurodegeneration caused by amyloid beta (Aβ) aggregation due to l-tyrosine oxidation. We show that the natural, human genome-derived DNAzyme In1-sp is able to oxidize Aβ peptides with a 4.6% higher yield and a 33.3% higher velocity of the reaction compared to free hemin. As the artificial DNAzyme Dz-00 is even able to catalyze Aβ peptide oxidation with a 64.2% higher yield and 337.1% higher velocity, an in-depth screening of human genome-derived DNAzymes may identify further candidates with similarly high catalytic activity in Aβ peptide oxidation.

Figure 1

Absorption spectra after the formation of catalytically active Dz-00 (green) as well as hemin (red) and G-quadruplex (blue) for comparison.

Figure 2

Fluorescence spectra of l-tyrosine (green) before and after oxidization to dityrosine (red). The fluorescence spectra showed a strong increase in fluorescence from 380 to 470 nm during l-tyrosine oxidation.

Figure 3

Time-dependent l-tyrosine oxidation by 100 nM free hemin (yellow) and 100 nM Dz-00 (blue). Dz-00 was supplemented by equimolar free hemin. The reaction contained 250 μM l-tyrosine and was started by adding 250 μM H₂O₂. No reaction was observed, when Dz-00 was mixed with l-tyrosine in the absence of H₂O₂ (black data points). Error bars indicate standard deviations of three independent measurements.

Figure 4

Stoichiometry test of Dz-00 (blue) and free hemin (yellow). Stoichiometries of 1:1, 1:2, 1:5, 1:10, and 1:20 of DNA (fixed to 100 nM) to hemin were tested to identify the ratio that gave the highest activity. The concentrations of l-tyrosine and H₂O₂ used in the experiments were set to 250 μM.

Figure 5

Influence on activity by modifying the sequence of Dz-00. The fluorescence fold increases of different sequences applied to the reaction are depicted.

Figure 6

Oxidation of Aβ 1–40. Comparison of Dz-00 (blue), In1-sp (red), and free hemin (yellow) as well as a control (black). DNAzymes were supplemented with hemin by a molar stoichiometry of 1:10. The starting concentration of Aβ and H₂O₂ was 125 μM.

Figure 7

HPSEC chromatograms of nonoxidized Aβ 1–40 (A) and Dz-00-oxidized Aβ 1–40 (B). (A) Chromatogram indicates nonoxidized Aβ in size exclusion chromatography excited at 250 nm and emission measured at 305 nm (black) and 405 nm (red), respectively. (B) Dz-00-oxidized Aβ 1–40 excited at 250 nm and emission measured at 305 nm (black) and 405 nm (red), respectively.