Insight into G-quadruplex-hemin DNAzyme/RNAzyme: adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity

Abstract

G-quadruplex (G4) with stacked G-tetrads structure is able to bind hemin (iron (III)-protoporphyrin IX) to form a unique type of DNAzyme/RNAzyme with peroxidase-mimicking activity, which has been widely employed in multidisciplinary fields. However, its further applications are hampered by its relatively weak activity compared with protein enzymes. Herein, we report a unique intramolecular enhancement effect of the adjacent adenine (EnEAA) at 3' end of G4 core sequences that significantly improves the activity of G4 DNAzymes. Through detailed investigations of the EnEAA, the added 3' adenine was proved to accelerate the compound I formation in catalytic cycle and thus improve the G4 DNAzyme activity. EnEAA was found to be highly dependent on the unprotonated state of the N1 of adenine, substantiating that adenine might function as a general acid-base catalyst. Further adenine analogs analysis supported that both N1 and exocyclic 6-amino groups in adenine played key role in the catalysis. Moreover, we proved that EnEAA was generally applicable for various parallel G-quadruplex structures and even G4 RNAzyme. Our studies implied that adenine might act analogously as the distal histidine in protein peroxidases, which shed light on the fundamental understanding and rational design of G4 DNAzyme/RNAzyme catalysts with enhanced functions.

Figure 1.

Effect of different adjacent nucleobases on G4 DNAzyme activity. (A–C) Represent the absorption spectra, the dynamic change of the concentration and the photographs of the peroxidation product (ABTS•–), respectively. The symbol ‘∼’ represents the sequence of Dz-00 (5′-GGGTAGGGCGGGTTGGG-3′). For other DNAzyme variants, one of four nucleotide bases (A, T, G or C) is added at 3′ (Dz-11 to -14) or 5′ end of Dz-00 (Dz-15 to -18), respectively.

Figure 2.

(A) Schematic structure of Dz-11-hemin complex (top), the chemical structures of hemin (middle) and G-tetrad (bottom). (B–F) The influences of different factors on EnEAA effect: (B) the different length of poly-T spacer between G4 core sequence and adenine base, (C) poly-A at 3′ terminal of G4 core sequence, (D) G4 structure with intramolecular duplex for restricting the orientation of adenine, (E) G4 structure with the proximate adenine nucleotide provided by the complementary strand and (F) the formation of A•T base pair. (G) The structure of A•T base pair. Error bar represents the standard deviation of triplicate measurements.

Figure 3.

(A) Proposed peroxidation cycle facilitated by G-quadruplex DNAzyme for the ABTS²⁻-H₂O₂ reaction. (B) Decay kinetics of the G4 DNAzymes’ absorbance at 404 nm in the presence of H₂O₂ (2 mM). (C and D) Represent the time-dependent adsorption spectra change in visible region to probe the formation of compound I-like intermediate in the reaction process of the Dz-00-hemin (C) and Dz-11-hemin (D) with H₂O₂ (2 mM), respectively. The direction of absorbance change is indicated by the arrow.

Figure 4.

Plots of the initial peroxidation rates of Dz-00 (tetragonum) and Dz-11 (triangle) DNAzymes as a function of pH values in MES solution (A) and in phosphate solution (B).

Figure 5.

Molecular models of Dz-00-hemin (A and B) and Dz-11-hemin (C and D) complexes, respectively. A, C: side view, B and D: top view. Molecular docking studies were performed by AutoDock and the docking structures were prepared by Chimera 1.10.2.

Figure 6.

(A and E) The catalytic activities of DNAzymes with various purine derivatives at 3′ terminal of Dz-00 in pH 5.1. (B and F) The structures of purine derivatives. Their relative pK_a values are shown in brackets. (C and D) Plots of the initial peroxidation rates of AP (C) and DAP (D) modified DNAzymes as a function of pH values in MES solution. Error bar represents the standard deviation of triplicate measurements.

Figure 7.

Optimized structures of purine derivatives with H₂O₂ at B3LYP/aug-cc-pVDZ and MP2/aug-cc-pVDZ level, where DE is the binding energy of the complex binding with H₂O₂ and v_H-Oα is the H–Oα (in N1–H–Oα) stretching vibration of H₂O₂. (A): purine (P); (B): adenine (6-aminopurine, A), (C): 2-aminopurine (AP) and (D): 2,6-diaminopurine (DAP).

Scheme 1.

The proposed mechanism of adenine-mediated acceleration of G4 peroxidase catalysis.

Figure 8.

Catalytic rates (upper) and ratio (v_Dz-11/v_Dz-00, below) of Dz-00 and Dz-11 with different buffer species. [DNA] = 100 nM, [hemin] = 100 nM, [buffer] = 25 mM at pH 5.1. Error bar represents the standard deviation of triplicate measurements.