Electrochemical direct detection of DNA deamination catalyzed by APOBEC3G

Abstract

APOBEC3G catalyzes deamination of cytosines in HIV-1 genome, and restricts the HIV-1 infection. Here, we propose a picomole-scale assay for the detection of DNA deamination catalyzed by APOBEC3G. Our results show the suitability of the developed method for a time course analysis of enzyme-catalyzed DNA modifications.

Fig. 1

(a) The chemical structure of the ferrocene/isoquinoline conjugate-connected DNA probe. (b) Schematic representation of (left) complementary and (right) mismatched DNA hybrids on the electrode. In general, a complementary hybrid indicates a slower structural fluctuation than a mismatched hybrid.

Fig. 2

Optimization of a pulse potential frequency for the square-wave voltammetry. (a) Frequency (f) dependence of a peak current derived from a complementary, 15-CCC/Fc-GGG (red), and mismatched hybrids, 15-CCU/Fc-GGG (green) and 15-CUU/Fc-GGG (blue). (b) The normalized peak current derived from a complementary (15-CCC/Fc-GGG), mismatched hybrids (15-CCU/Fc-GGG), and probe only (Fc-GGG). The measurements were performed at 40 Hz, the optimized frequency for the probe Fc-GGG. The values denote the mean ± S.D. derived from five measurements.

Fig. 3

Relationship between the normalized peak current and the relative amount of target DNA. The peak current was measured using a probe DNA Fc-GAA with a mixture of 15-CCU and 15-CUU at a ratio of 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100, respectively. The values denote the mean ± S.D. derived from five measurements.

Fig. 4

Trace of A3G-catalyzed deamination. The normalized peak current was measured with probe DNAs, (a) Fc-GGG, (b) Fc-GGA, (c) Fc-GAA, (d) Fc-AGG, (e) Fc-GAG, (f) Fc-AAA, and (g) Fc-AGA. The values denote the mean ± S.D. derived from three measurements.