Turn-off colorimetric sensor for sequence-specific recognition of single-stranded DNA based upon Y-shaped DNA structure

Abstract

A novel turn-off colorimetric sensor for sequence-specific recognition of single-stranded DNA (ssDNA) was established by combining Y-shaped DNA duplex and G-quadruplex-hemin DNAzyme. A G-rich single-stranded DNA (Oligo-1) displays peroxidase mimicking catalytic activity due to the specific binding with hemin in the presence of K⁺, which was able to catalyze the oxidation of colorless 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS^2-) by H₂O₂ to generate green ABTS•^- radical for colorimetric assay. Oligonucleotide 2 (Oligo-2) was partly complementary with Oligo-1 and the target DNA. Upon addition of target DNA, Oligo-1, Oligo-2 and target DNA can hybridize with each other to form Y-shaped DNA duplex. The DNAzyme sequence of Oligo-1 was partly caged into Y-shaped DNA duplex, resulting in the inactivation of the DNAzyme and a sharp decrease of the absorbance of the oxidation product of ABTS^2-. Under the optimum condition, the absorbance decreased linearly with the concentration of target DNA over the range of 1.0-250 nM and the detection limit was 0.95 nM (3σ/slope) Moreover, satisfied result was obtained for the discrimination of single-base or two-base mismatched DNA.

Figure 1

Schematic illustration for sequence-specific recognition of single-stranded DNA based upon Y-shaped DNA duplex and G-quadruplex-hemin DNAzyme.

Figure 2

Typical photograph and absorption spectra of ABTS²⁻ and H₂O₂ mixture catalytically oxidized by G-quadruplex-hemin DNAzyme under different conditions. (a) Oligo-1; (b) a + Oligo-2; (c) b + 500 nM target DNA. Experimental conditions: 1.5 μM Oligo-1, 1.5 μM Oligo-2, 15 μM hemin, 40 mM ABTS²⁻ and 25 mM H₂O₂. 10 mM Tris-HCl buffer (pH 7.0, 150 mM KCl) was used.

Figure 3

(A) CD spectroscopy of DNA molecule under different conditions. (a) Oligo-1; (b) a + Oligo-2; (c) b + target DNA. (B) Fluorescent validation of the formation of G-quadruplex through ThT fluorescence. (a) ThT; (b) a + Oligo-1; (c) b + Oligo-2; (d) c + target DNA.

Figure 4

Effect of the sconcentration of (A) Oligo-1, (B) Oligo-2, (C) KCl, (D) hemin, (E) ABTS²⁻ and (F) H₂O₂ on ΔA.

Figure 5

Effect of different sequence of assistant probe on ΔA. Experimental conditions: 1.5 μM signal probe, 150 mM KCl, 15 μM hemin, 40 mM ABTS²⁻, 25 mM H₂O₂ and1.5 μM assistant probe. The concentration of Oligo-3 was 200 nM. 10 mM Tris-HCl buffer (pH 7.0, 150 mM KCl) was used.

Figure 6

Calibration curve of the assay. Experimental conditions: 1.5 μM signal probe, 1.5 μM assisstant probe, 15 μM hemin, 40 mM ABTS²⁻ and 25 mM H₂O₂. 10 mM Tris-HCl buffer (pH 7.0, 150 mM KCl) was used in the experiment.

Figure 7

Sequence selectivity of the assay toward (A) Y-shaped DNA duplex and (B) linear DNA duplex. Experimental conditions: 1.5 μM Oligo-1, 1.5 μM Oligo-2, 15 μM hemin, 40 mM ABTS²⁻ and 25 mM H₂O₂. The concentration of Oligo-3, Oligo-4, Oligo-5 and block DNA was 200 nM. 10 mM Tris-HCl buffer (pH 7.0, 150 mM KCl) was used.