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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

Ag+ stabilizes DNA duplex containing C-C mismatches.(a) The capturing of C-C duplex (ssDNA TC 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.
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f2: Ag+ stabilizes DNA duplex containing C-C mismatches.(a) The capturing of C-C duplex (ssDNA TC 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.

Mentions: The addition of Ag+ increases the stability of dsDNA containing a C-C mismatch, which leads to an increase in the complex's dwell time within the nanopore (Figure 2a,b). We can see that ssDNAs (dwell time <10 ms) and dsDNAs (dwell time >10 ms) were well separated (Figure 2c). For details on the probe screening process, please refer to the supplementary information (Supplementary Note S1). The events with an ending spike35363738 were identified (Figure 2a,b enlarged single current traces), indicating the DNA duplex capturing and dissociation (See Supplementary Note S2 for detailed description). The difference in dwell time provides a key differentiator between C-C and C-Ag-C. In detail, P·TC hybrid (C-C) yielded the dwell time distribution with a peak at 59 ± 5 ms (Figure 2c, blue), while C-Ag-C yielded a dwell time distribution with the first peak at 51 ± 6 ms and the second peak at 384 ± 12 ms (Figure 2c, red). Molecular dynamics (MD) simulations indicate that hydrogen bonds are alternatively formed between N4A-N3B and N3A-N4B atoms (simulations described in details below), and there is a 2.6-fold difference in binding energy bewteen these two conformations. This difference in binding energy could be the reason that we observed two dwell time distributions peaks. This second peak demonstrates dwell times with C-Ag-C that are 6.5-fold longer than those with C-C (Figure 2c). We interpret that the prolonged blocking events are due to the binding of Ag+ to the C-C mismatch in the P·TC hybrid. As reported previously, the binding of Ag+ forms a C-Ag-C bridge base pair that stabilizes the P·TC complex1234, resulting in an extended dwell time under the same holding potential. The Ag+ effect is equivalent to an increase in dsDNA hybridization energy, which was calculated to be 3.8 ± 0.5 kJ·mol−1 using ΔE = RTln(τ+Ag/τ−Ag), where τ−Ag and τ+Ag are block durations before and after the addition of Ag+. We also found a decrease in residual current after the addition of Ag+. They are 41.5 ± 0.4 pA (without Ag+) and 36.8 ± 0.2 pA (with Ag+), respectively (Figure 2d). The change is 4.7 ± 0.45 pA (by error propaganda equation). The hydrated radius of Ag+ is 0.34 nm39, and as a result, the substantial radius of Ag+ in complex with the DNA blocks more current flow. Thus it is reasonable to see a deeper current blockage for DNA with Ag+.


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)

Ag+ stabilizes DNA duplex containing C-C mismatches.(a) The capturing of C-C duplex (ssDNA TC 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Ag+ stabilizes DNA duplex containing C-C mismatches.(a) The capturing of C-C duplex (ssDNA TC 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.
Mentions: The addition of Ag+ increases the stability of dsDNA containing a C-C mismatch, which leads to an increase in the complex's dwell time within the nanopore (Figure 2a,b). We can see that ssDNAs (dwell time <10 ms) and dsDNAs (dwell time >10 ms) were well separated (Figure 2c). For details on the probe screening process, please refer to the supplementary information (Supplementary Note S1). The events with an ending spike35363738 were identified (Figure 2a,b enlarged single current traces), indicating the DNA duplex capturing and dissociation (See Supplementary Note S2 for detailed description). The difference in dwell time provides a key differentiator between C-C and C-Ag-C. In detail, P·TC hybrid (C-C) yielded the dwell time distribution with a peak at 59 ± 5 ms (Figure 2c, blue), while C-Ag-C yielded a dwell time distribution with the first peak at 51 ± 6 ms and the second peak at 384 ± 12 ms (Figure 2c, red). Molecular dynamics (MD) simulations indicate that hydrogen bonds are alternatively formed between N4A-N3B and N3A-N4B atoms (simulations described in details below), and there is a 2.6-fold difference in binding energy bewteen these two conformations. This difference in binding energy could be the reason that we observed two dwell time distributions peaks. This second peak demonstrates dwell times with C-Ag-C that are 6.5-fold longer than those with C-C (Figure 2c). We interpret that the prolonged blocking events are due to the binding of Ag+ to the C-C mismatch in the P·TC hybrid. As reported previously, the binding of Ag+ forms a C-Ag-C bridge base pair that stabilizes the P·TC complex1234, resulting in an extended dwell time under the same holding potential. The Ag+ effect is equivalent to an increase in dsDNA hybridization energy, which was calculated to be 3.8 ± 0.5 kJ·mol−1 using ΔE = RTln(τ+Ag/τ−Ag), where τ−Ag and τ+Ag are block durations before and after the addition of Ag+. We also found a decrease in residual current after the addition of Ag+. They are 41.5 ± 0.4 pA (without Ag+) and 36.8 ± 0.2 pA (with Ag+), respectively (Figure 2d). The change is 4.7 ± 0.45 pA (by error propaganda equation). The hydrated radius of Ag+ is 0.34 nm39, and as a result, the substantial radius of Ag+ in complex with the DNA blocks more current flow. Thus it is reasonable to see a deeper current blockage for DNA with Ag+.

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