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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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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.
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f1: 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.

Mentions: The study involved three 16-nt AT rich ssDNAs as the targets, which contain a cytosine (TC), 5′-methylcytosine (TmC) and 5′-hydromethylcytosine (ThmC) at the 10th nucleotide (5′ → 3′), respectively (Table 1). Their common probe, P, contains a cytosine at the corresponding position, such that when P is hybridized with the three targets, their hybrids P·TC, P·TmC and P·ThmC, form a C-C, C-mC and C-hmC mismatched base-pair respectively. Since Ag+ was tested in the experiments, we could not use KCl buffer due to AgCl precipitation. Therefore, we first tested how the single-stranded DNA (ssDNA) P (Figure 1) interacts with the nanopore in KNO3 solution. Short (<1 ms) and long events in the range of 1–10 ms were easily identified (Figure 1a,b). The residual current also has a wide distribution, with a peak at 17.4 ± 0.84 pA (Figure 1c). Others have previously noted that KNO3 has unknown effects on DNA translocation and some extraordinary long events were seen, with about 10-fold lower occurrence rate constant (Kon) of ssDNA in KNO3 than in the KCl buffer8, as well as in certain cations such as Li+33 and ion liquid34. In order to ensure the ssDNA interactions were excluded, we only considered events longer than 10 ms as the DNA duplexes interact with the nanopore. A control experiment demonstrated that Ag+ itself does not affect the open pore current (Figure 1d.e). The positively charged Ag+ is driven away from the nanopore by the applied voltage.


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)

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

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

f1: 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.
Mentions: The study involved three 16-nt AT rich ssDNAs as the targets, which contain a cytosine (TC), 5′-methylcytosine (TmC) and 5′-hydromethylcytosine (ThmC) at the 10th nucleotide (5′ → 3′), respectively (Table 1). Their common probe, P, contains a cytosine at the corresponding position, such that when P is hybridized with the three targets, their hybrids P·TC, P·TmC and P·ThmC, form a C-C, C-mC and C-hmC mismatched base-pair respectively. Since Ag+ was tested in the experiments, we could not use KCl buffer due to AgCl precipitation. Therefore, we first tested how the single-stranded DNA (ssDNA) P (Figure 1) interacts with the nanopore in KNO3 solution. Short (<1 ms) and long events in the range of 1–10 ms were easily identified (Figure 1a,b). The residual current also has a wide distribution, with a peak at 17.4 ± 0.84 pA (Figure 1c). Others have previously noted that KNO3 has unknown effects on DNA translocation and some extraordinary long events were seen, with about 10-fold lower occurrence rate constant (Kon) of ssDNA in KNO3 than in the KCl buffer8, as well as in certain cations such as Li+33 and ion liquid34. In order to ensure the ssDNA interactions were excluded, we only considered events longer than 10 ms as the DNA duplexes interact with the nanopore. A control experiment demonstrated that Ag+ itself does not affect the open pore current (Figure 1d.e). The positively charged Ag+ is driven away from the nanopore by the applied voltage.

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