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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

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 Å3) 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.
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f5: 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 Å3) 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.

Mentions: Molecular dynamics (MD) simulations of DNA duplexes containing these mismatches reveal how Ag+ may bind to the mismatches, as well as different coordination configurations between the mismatched bases (Supplementary Note S4 for simulation description). As shown in Figure 5a, a DNA duplex, with the same sequence as that in experiment was solvated in an electrolyte. The C-C base pairing was formed by the hydrogen bond between the N3 atom of one cytosine base (in the strand A) and the N4 atom of the other cytosine base (in the strand B) (Figure 5b). Besides the conformation shown in Figure 5b, another possible paring was formed by the hydrogen bond between N4A and N3B atoms (Supplementary Movie S1). The distances between N3 and N4 atoms of different bases, as shown in Figure 5d, indicate that hydrogen bonds are alternatively formed between N4A and N3B atoms and between N3A and N4B atoms. This type of pairing results in the formation of a binding site for a cation (Figure 5b). During the simulation, K+ ions were found in the binding site and the mean residence time for K+ was about 10 ns (Supplementary Movie S2). As confirmed in an independent MD simulation (Supplementary Figure s5, Movie S3), Ag+ can also enter the binding site and further stabilize the paring between mismatched C-C bases. Correspondingly, these simulations also indicate that the dwell time of the duplex with a Ag+ is longer (Figure 2c) due to the enhanced stability.


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)

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 Å3) 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: 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 Å3) 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.
Mentions: Molecular dynamics (MD) simulations of DNA duplexes containing these mismatches reveal how Ag+ may bind to the mismatches, as well as different coordination configurations between the mismatched bases (Supplementary Note S4 for simulation description). As shown in Figure 5a, a DNA duplex, with the same sequence as that in experiment was solvated in an electrolyte. The C-C base pairing was formed by the hydrogen bond between the N3 atom of one cytosine base (in the strand A) and the N4 atom of the other cytosine base (in the strand B) (Figure 5b). Besides the conformation shown in Figure 5b, another possible paring was formed by the hydrogen bond between N4A and N3B atoms (Supplementary Movie S1). The distances between N3 and N4 atoms of different bases, as shown in Figure 5d, indicate that hydrogen bonds are alternatively formed between N4A and N3B atoms and between N3A and N4B atoms. This type of pairing results in the formation of a binding site for a cation (Figure 5b). During the simulation, K+ ions were found in the binding site and the mean residence time for K+ was about 10 ns (Supplementary Movie S2). As confirmed in an independent MD simulation (Supplementary Figure s5, Movie S3), Ag+ can also enter the binding site and further stabilize the paring between mismatched C-C bases. Correspondingly, these simulations also indicate that the dwell time of the duplex with a Ag+ is longer (Figure 2c) due to the enhanced stability.

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