Single molecule investigation of Ag+ interactions with single cytosine-, methylcytosine- and hydroxymethylcytosine-cytosine mismatches in a nanopore

Abstract

Both cytosine-Ag-cytosine interactions and cytosine modifications in a DNA duplex have attracted great interest for research. Cytosine (C) modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) are associated with tumorigenesis. However, a method for directly discriminating C, mC and hmC bases without labeling, modification and amplification is still missing. Additionally, the nature of coordination of Ag(+) with cytosine-cytosine (C-C) mismatches is not clearly understood. Utilizing the alpha-hemolysin nanopore, we show that in the presence of Ag(+), duplex stability is most increased for the cytosine-cytosine (C-C) pair, followed by the cytosine-methylcytosine (C-mC) pair, and the cytosine-hydroxymethylcytosine (C-hmC) pair, which has no observable Ag(+) induced stabilization. Molecular dynamics simulations reveal that the hydrogen-bond-mediated paring of a C-C mismatch results in a binding site for Ag(+). Cytosine modifications (such as mC and hmC) disrupted the hydrogen bond, resulting in disruption of the Ag(+) binding site. Our experimental method provides a novel platform to study the metal ion-DNA interactions and could also serve as a direct detection method for nucleobase modifications.

Figure 1. The ssDNA P interacts with the nanopore.

(a) The representative current trace recorded at 150 mV. Two types of events were identified: a1: spike-like current profile which last about 200 us and a2: rectangular-like current profile which last about 1 to 10 ms. (b) The histogram of the dwell time in Log form. The long events (>10⁰ = 1 ms) were easily identified. (c) The histogram of residual currents when the ssDNA P interacts with the nanopore. The nanoporecurrent traces of the empty pore (d) and with the addition of 50 uM AgNO₃ (e) recorded at 150 mV in 1 M KNO₃.

Figure 2. Ag⁺ stabilizes DNA duplex containing C-C mismatches.

(a) The capturing of C-C duplex (ssDNA T_C hybridized with P) in the nanopore. (b) The capturing of C-C duplex with the addition of Ag⁺. (c) The histogram of the dwell time in Log form. The C-C generated a single peak of 59 ± 5 ms (blue). The C-Ag-C generated two peaks of 51 ± 6 ms and 384 ± 12 ms (red), which increased the dwell time by 6.5 fold compared to the C-C duplex. (d) The histogram of residual currents. The C-C generated a single peak of 41.5 ± 0.4 pA (blue); The C-Ag-C generated a peak of 36.8 ± 0.2 pA. The difference was 4.7 ± 0.45 pA between C-C and C-Ag-C. The red circles indicate the capturing of DNA duplexes. The enlarged single traces in a and b demonstrated the DNA duplex dissociation signature with an ending spike. Recordings were made at 150 mV.

Figure 3. Weak interaction of Ag⁺ with a DNA duplex containing mC-C mismatches.

The representative current traces of mC-C (a) and mC-Ag-C (b) capturing. (c) The histogram of the dwell time in Log form. The mC-C generated a single peak of 69 ± 6 ms (blue). The mC-Ag-C generated a single peak of 92 ± 10 ms (red), which increased the dwell time by 1.3 fold. (d) The histogram of residual currents. The mC-C generated a single peak of 37.4 ± 0.7 pA (blue). The mC-Ag-C generated two peaks of 33.9 ± 0.8 pA and 38.1 ± 0.8 pA (red). The difference was 3.5 ± 1.1 pA between mC-C and the first peak of mC-Ag-C. The red circles indicate the capturing of DNA duplexes. Recordings were made at 150 mV.

Figure 4. No observable interaction of Ag⁺ with a DNA duplex containing hmC-C mismatches.

The representative current traces of hmC-C (a) and hmC-Ag-C (b) capturing. (c) The histogram of the dwell time in Log form. The hmC-C generated a single peak of 19.6 ± 1 ms (blue). The hmC-Ag-C generated a single peak of 17.3 ± 1 ms (red). (d) The histogram of residual currents. The hmC-C generated a single peak of 36.3 ± 0.95 pA (blue); The hmC-Ag-C generated a single peak of 36.2 ± 0.71 pA (red). The difference was 0.1 ± 1.19 pA. The red circles indicate the capturing of DNA duplexes. Recordings were made at 150 mV.

Figure 5. Molecular dynamics simulations of DNA duplex containing C-C, mC-C and hmC-C mismatches.

(a) (LBQ is the creator of figure 5a). Side-view of the simulation system. The DNA duplex is in the “stick” presentation and two backbones are illustrated as yellow and green belts respectively. Potassium ions that neutralize the entire simulation system are shown as tan balls. Water in a cubic box (78.5 × 78.5 × 78.5 ų) is shown transparently. (b) A snap-shot of pairing between two cytosine bases. The dashed circle highlights the binding site for a cation. (c) A snap-shot of hmC-C pairing before the pairing was broken. (d–f) Time-dependent distances between the N3 atom of one base and the N4 atom of the other base, in C-C(d), mC-C(e) and hmC-C(f) mismatches.