Insight into an Oxidative DNA-Cleaving DNAzyme: Multiple Cofactors, the Catalytic Core Map and a Highly Efficient Variant

Abstract

An oxidative DNA-cleaving DNAzyme (PL) employs a double-cofactor model "X/Cu²⁺" for catalysis. Herein, we verified that reduced nicotinamide adenine dinucleotide (NADH), flavin mononucleotide, cysteine, dithiothreitol, catechol, resorcinol, hydroquinone, phloroglucinol, o-phenylenediamine, 3,3',5,5'-tetramethylbenzidine, and hydroxylamine acted as cofactor X. According to their structural similarities or fluorescence property, we further confirmed that reduced nicotinamide adenine dinucleotide phosphate (NADPH), 2-mercaptoethanol, dopamine, chlorogenic acid, resveratrol, and 5-carboxyfluorescein also functioned as cofactor X. Superoxide anions might be the commonality behind these cofactors. We subsequently determined the conservative change of individual nucleotides in the catalytic core under four different cofactor X. The nucleotides A4 and C5 are highly conserved, whereas the conservative levels of other nucleotides are dependent on the types of cofactor X. Moreover, we observed that the minor change in the PL's secondary structure affects electrophoretic mobility. Finally, we characterized a highly efficient variant T3G and converted its double-cofactor NADH/Cu²⁺ to sole-cofactor NADH.

Keywords: Biocatalysis; Biochemistry; Chemistry.

Graphical abstract

Figure 1

The Sequence and Secondary Structure of the PL DNAzyme The green letters correspond to the nucleotides within the catalytic core. The red arrowhead and the blue asterisks, respectively, indicate the major and minor sites of DNA cleavage. L and R designate stem-loop structures, where lines indicate Watson-Crick base pairs and dots represent triple helix interactions.

Figure 2

Chemical Structures of Small Compounds and Their Effects on PL Catalysis (A) Thirty types of chemical compounds and their structures. (B) Effects of 30 types of chemical compounds on PL. PL (0.5 μM) was incubated with 100 μM compound at 23°C for 1 h in a mixture containing 100 μM Cu²⁺ (or none), 300 mM NaCl, and 50 mM Tris-HCl (7.0). The reaction system containing VB₂ or FMN needs to be performed in white light. (C) Chemical structures of some cofactor X of PL.

Figure 3

Analysis of Single-Site Variants within the Catalytic Core of PL (A) Heatmaps summarizing the cleavage yield (%) for single-site variants under four groups of cofactors. Colored blocks with asterisks indicate the original PL nucleotides. (B) Relative drift intensity (RDI) of the nucleotides within the core of PL. (C) Single-site mutation affects electrophoretic mobility.

Figure 4

T3G Catalysis in the Presence of Different Cofactors (A) The effects of 30 types of chemical compounds on T3G. (B) Comparison of T3G and PL by treatment with eight kinds of cofactors. T3G and PL were incubated with 100 μM cofactor at 23°C for 1 h in a mixture containing 100 μM Cu²⁺, 300 mM NaCl, and 50 mM Tris-HCl (pH 7.0). E and P represent the PL DNAzyme and cleavage fragments, respectively.

Figure 5

Enzymatic Characterization of T3G (A) pH-dependent activity of T3G in the presence of NADH/Cu²⁺ and GSH/Cu²⁺. Reactions were conducted at 50 mM pH buffer (pH 5.0–9.0). (B) The effects of NaCl and KCl on T3G function. Reactions were conducted with 1–1,000 mM NaCl (KCl). (C) The effect of temperature on T3G activity. Reactions were conducted with 1 M NaCl within 8–40°C. (D) The effects of components (NADH/Cu²⁺, GSH/Cu²⁺, NADH, GSH, Cu²⁺-1M NaCl, Cu²⁺-0.2 M NaCl) of reaction solution on T3G activity. Refer to “Transparent Methods” for the specific composition and concentration of cofactors used in Figure 5D. (E) The effects of NADH and GSH on T3G activity in the presence of 100 μM Gu²⁺. (F) The effects of Cu²⁺ on T3G activity in the presence of either 100 μM NADH or 100 μM GSH. The error bars represented the standard deviations from three repeated measurements.

Figure 6

The Effects of Divalent Metal Ions on T3G Activity T3G was incubated with 100 μM NADH (or GSH) and 100 μM metal ions at 23°C for 1 h in a mixture containing 1 (or 0.2) M NaCl and 50 mM MES (pH 6.4). Fe²⁺ led to DNA aggregation in gel wells and affected DNA migration. CF indicates NADH and GSH. The error bars represented the standard deviations from three repeated measurements.