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

Wang Y, Luan BQ, Yang Z, Zhang X, Ritzo B, Gates K, Gu LQ - Sci Rep (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Engineering and Dalton Cardiovascular Research Center University of Missouri, Columbia, MO 65211, USA [2].

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.

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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.
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f4: 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.

Mentions: We also measured the effect of Ag+ on the dsDNA containing a C-hmC mismatched base pair (probe P is hybridized with the target ThmC, their hybrid P·ThmC forms a single C-hmC mismatch). The addition of Ag+ does not appear to affect the stability of dsDNA containing an hmC-C mismatch, though dwell time is lower than those for C-C and mC-C mismatches (Figure 4). We found that P·ThmC yielded a dwell time distribution which is very similar to that of P·ThmC with Ag+ (Figure 4a,b,c). The hmC-C yielded a dwell time distribution peaked at 19.6 ± 1 ms (Figure 4c, blue), while hmC-Ag-C yielded a peak at 17.3 ± 1 ms (Figure 4c, red). For residual current, P·ThmC yielded a peak at 36.3 ± 0.95 pA and P·ThmC with Ag+ yielded a similar peak at 36.2 ± 0.71 pA (Figure 4d). The difference was 0.1 ± 1.19 pA. Overall, these data demonstrate that hmC-C mismatches are less stable than mC-C or C-C mismatches. Therefore, the presence of Ag+ seems to have little effect on the C-hmC mismatch.


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

Wang Y, Luan BQ, Yang Z, Zhang X, Ritzo B, Gates K, Gu LQ - Sci Rep (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126007&req=5

f4: 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.
Mentions: We also measured the effect of Ag+ on the dsDNA containing a C-hmC mismatched base pair (probe P is hybridized with the target ThmC, their hybrid P·ThmC forms a single C-hmC mismatch). The addition of Ag+ does not appear to affect the stability of dsDNA containing an hmC-C mismatch, though dwell time is lower than those for C-C and mC-C mismatches (Figure 4). We found that P·ThmC yielded a dwell time distribution which is very similar to that of P·ThmC with Ag+ (Figure 4a,b,c). The hmC-C yielded a dwell time distribution peaked at 19.6 ± 1 ms (Figure 4c, blue), while hmC-Ag-C yielded a peak at 17.3 ± 1 ms (Figure 4c, red). For residual current, P·ThmC yielded a peak at 36.3 ± 0.95 pA and P·ThmC with Ag+ yielded a similar peak at 36.2 ± 0.71 pA (Figure 4d). The difference was 0.1 ± 1.19 pA. Overall, these data demonstrate that hmC-C mismatches are less stable than mC-C or C-C mismatches. Therefore, the presence of Ag+ seems to have little effect on the C-hmC mismatch.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Engineering and Dalton Cardiovascular Research Center University of Missouri, Columbia, MO 65211, USA [2].

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.

Show MeSH
Related in: MedlinePlus