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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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Related in: MedlinePlus

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.
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f3: 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.

Mentions: The addition of Ag+ also increases the stability of dsDNA containing an mC-C mismatch (probe P is hybridized with the target TmC, their hybrid P·TmC forms a single C-mC mismatch), though the increase in dwell time is less than those for C-C (Figure 3a,b). We found that P·TmC yielded a dwell time distribution peaked at 69 ± 6 ms (Figure 3c, blue), while P·TmC with Ag+ yielded a peak at 92 ± 10 ms (Figure 3c, red), which represents a 1.3-fold increase in dwell time, corresponding to a 0.53 ± 0.07 kJ·mol−1 increase of the energy for dsDNA dehybridization. This energy increase is lower than the 3.8 kJ·mol−1 for dsDNA containing a C-C mismatched base pair bound with Ag+, suggesting that the effect of Ag+ on stabilization of dsDNA with a C-mC mismatch is much weaker than that with a C-C 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)

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

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

f3: 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.
Mentions: The addition of Ag+ also increases the stability of dsDNA containing an mC-C mismatch (probe P is hybridized with the target TmC, their hybrid P·TmC forms a single C-mC mismatch), though the increase in dwell time is less than those for C-C (Figure 3a,b). We found that P·TmC yielded a dwell time distribution peaked at 69 ± 6 ms (Figure 3c, blue), while P·TmC with Ag+ yielded a peak at 92 ± 10 ms (Figure 3c, red), which represents a 1.3-fold increase in dwell time, corresponding to a 0.53 ± 0.07 kJ·mol−1 increase of the energy for dsDNA dehybridization. This energy increase is lower than the 3.8 kJ·mol−1 for dsDNA containing a C-C mismatched base pair bound with Ag+, suggesting that the effect of Ag+ on stabilization of dsDNA with a C-mC mismatch is much weaker than that with a C-C 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