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Kinetics of charge transfer in DNA containing a mismatch.

Osakada Y, Kawai K, Fujitsuka M, Majima T - Nucleic Acids Res. (2008)

Bottom Line: While the single-base mismatch would significantly affect the CT in DNA, the kinetic basis for the drastic decrease in the CT efficiency through DNA containing mismatches still remains unclear.We assumed that further elucidating of the kinetics in mismatched sequences can lead to the discrimination of the DNA single-base mismatch based on the kinetics.In this study, we investigated the detailed kinetics of the CT through DNA containing mismatches and tried to discriminate a mismatch sequence based on the kinetics of the CT in DNA containing a mismatch.

View Article: PubMed Central - PubMed

Affiliation: The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki Osaka 567-0047, Japan.

ABSTRACT
Charge transfer (CT) in DNA offers a unique approach for the detection of a single-base mismatch in a DNA molecule. While the single-base mismatch would significantly affect the CT in DNA, the kinetic basis for the drastic decrease in the CT efficiency through DNA containing mismatches still remains unclear. Recently, we determined the rate constants of the CT through the fully matched DNA, and we can now estimate the CT rate constant for a certain fully matched sequence. We assumed that further elucidating of the kinetics in mismatched sequences can lead to the discrimination of the DNA single-base mismatch based on the kinetics. In this study, we investigated the detailed kinetics of the CT through DNA containing mismatches and tried to discriminate a mismatch sequence based on the kinetics of the CT in DNA containing a mismatch.

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Strategy and results of kinetic SNP typing using ssDNA probe. (a) Schematic representation of ssDNA probe. (b) Time profiles of the transient absorption of PTZ•+ at 520 nm during the 355 nm-laser flash photolysis of Ar-saturated aqueous solution containing 100 mM NaCl, 20 mM sodium phosphate (pH 7.0) at a strand concentration of 50 μM at 20°C. The smooth curves superimposed on the curves are the fit derived from the kinetic model using kht values depicted in Table 1 and Supplementary Table S1. The represented profiles were obtained from the accumulation of 64 laser shots.
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Figure 6: Strategy and results of kinetic SNP typing using ssDNA probe. (a) Schematic representation of ssDNA probe. (b) Time profiles of the transient absorption of PTZ•+ at 520 nm during the 355 nm-laser flash photolysis of Ar-saturated aqueous solution containing 100 mM NaCl, 20 mM sodium phosphate (pH 7.0) at a strand concentration of 50 μM at 20°C. The smooth curves superimposed on the curves are the fit derived from the kinetic model using kht values depicted in Table 1 and Supplementary Table S1. The represented profiles were obtained from the accumulation of 64 laser shots.

Mentions: To further support the kinetic SNP discrimination, we prepared ssDNA doubly modified with NI and PTZ at both ends which enables investigation of the CT without direct modification of the target strand (Figure 6). We performed the time-resolved transient absorption measurements with the 13-mer ssDNA probe, 5′-NI-AAATACAGTCTCT-PTZ-3′. The DNA sequence was designed on the basis of the antisense sequence of the COL25A1, which is a brain-specific membrane-bound collagen related to Alzheimer's disease, carrying the SNP C to T substitution (refSNP ID; rs1963939, allele position 387) (35). We assumed that the kinetics of the CT through the wild-type or target SNP sequence is predictable by kinetic modeling depending on the rate constants as described above. We first tested the rs1963939-C sequence. The time-resolved transient absorption measurement after hybridization with the strand 3′-TTTATGTCAGAGA-5′ was carried out. The experiment was carried out at 20°C, and we modeled the kinetics of the CT through the rs1963939-C sequence. Kinetic modeling showed that the PTZ•+ approached the maximum value within a time of <10 μs (Figure 6, green curve under the red data). In agreement with the kinetic modeling, we observed the maximum PTZ•+ formation within a time of <10 μs. We then tested rs1963939-T. We examined the transient absorption measurement using the target sequence, 3′-TTTATGTTAGAGA-5′, in which a C base replaced a T base of a short COL25A1 fragment, corresponding to a C387T SNP. In the case of the rs1963939-T sequences, we also first modeled the kinetics of the CT through the SNP sequence. Based on the kinetic modeling, the approaching time of about 40 μs was estimated as represented by the green curve under the black data. We measured the PTZ•+ with an approaching time of ∼40 μs from the transient absorption measurement. The result is slightly different than the expected values from the kinetic modeling using the rate constants obtained in this study and those reported in the literature (22). This is probably because the CT through the DNA containing mismatches as well as the fully matched DNA is affected by the sequences or the motions of both neighboring base pairs of the mismatch as mentioned above. These results suggest that the kinetic discrimination using the ssDNA doubly modified probe is possible, though determining the kinetic parameters of the mismatch in other combinations of neighboring base pairs is required for further precision.Figure 6.


Kinetics of charge transfer in DNA containing a mismatch.

Osakada Y, Kawai K, Fujitsuka M, Majima T - Nucleic Acids Res. (2008)

Strategy and results of kinetic SNP typing using ssDNA probe. (a) Schematic representation of ssDNA probe. (b) Time profiles of the transient absorption of PTZ•+ at 520 nm during the 355 nm-laser flash photolysis of Ar-saturated aqueous solution containing 100 mM NaCl, 20 mM sodium phosphate (pH 7.0) at a strand concentration of 50 μM at 20°C. The smooth curves superimposed on the curves are the fit derived from the kinetic model using kht values depicted in Table 1 and Supplementary Table S1. The represented profiles were obtained from the accumulation of 64 laser shots.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553589&req=5

Figure 6: Strategy and results of kinetic SNP typing using ssDNA probe. (a) Schematic representation of ssDNA probe. (b) Time profiles of the transient absorption of PTZ•+ at 520 nm during the 355 nm-laser flash photolysis of Ar-saturated aqueous solution containing 100 mM NaCl, 20 mM sodium phosphate (pH 7.0) at a strand concentration of 50 μM at 20°C. The smooth curves superimposed on the curves are the fit derived from the kinetic model using kht values depicted in Table 1 and Supplementary Table S1. The represented profiles were obtained from the accumulation of 64 laser shots.
Mentions: To further support the kinetic SNP discrimination, we prepared ssDNA doubly modified with NI and PTZ at both ends which enables investigation of the CT without direct modification of the target strand (Figure 6). We performed the time-resolved transient absorption measurements with the 13-mer ssDNA probe, 5′-NI-AAATACAGTCTCT-PTZ-3′. The DNA sequence was designed on the basis of the antisense sequence of the COL25A1, which is a brain-specific membrane-bound collagen related to Alzheimer's disease, carrying the SNP C to T substitution (refSNP ID; rs1963939, allele position 387) (35). We assumed that the kinetics of the CT through the wild-type or target SNP sequence is predictable by kinetic modeling depending on the rate constants as described above. We first tested the rs1963939-C sequence. The time-resolved transient absorption measurement after hybridization with the strand 3′-TTTATGTCAGAGA-5′ was carried out. The experiment was carried out at 20°C, and we modeled the kinetics of the CT through the rs1963939-C sequence. Kinetic modeling showed that the PTZ•+ approached the maximum value within a time of <10 μs (Figure 6, green curve under the red data). In agreement with the kinetic modeling, we observed the maximum PTZ•+ formation within a time of <10 μs. We then tested rs1963939-T. We examined the transient absorption measurement using the target sequence, 3′-TTTATGTTAGAGA-5′, in which a C base replaced a T base of a short COL25A1 fragment, corresponding to a C387T SNP. In the case of the rs1963939-T sequences, we also first modeled the kinetics of the CT through the SNP sequence. Based on the kinetic modeling, the approaching time of about 40 μs was estimated as represented by the green curve under the black data. We measured the PTZ•+ with an approaching time of ∼40 μs from the transient absorption measurement. The result is slightly different than the expected values from the kinetic modeling using the rate constants obtained in this study and those reported in the literature (22). This is probably because the CT through the DNA containing mismatches as well as the fully matched DNA is affected by the sequences or the motions of both neighboring base pairs of the mismatch as mentioned above. These results suggest that the kinetic discrimination using the ssDNA doubly modified probe is possible, though determining the kinetic parameters of the mismatch in other combinations of neighboring base pairs is required for further precision.Figure 6.

Bottom Line: While the single-base mismatch would significantly affect the CT in DNA, the kinetic basis for the drastic decrease in the CT efficiency through DNA containing mismatches still remains unclear.We assumed that further elucidating of the kinetics in mismatched sequences can lead to the discrimination of the DNA single-base mismatch based on the kinetics.In this study, we investigated the detailed kinetics of the CT through DNA containing mismatches and tried to discriminate a mismatch sequence based on the kinetics of the CT in DNA containing a mismatch.

View Article: PubMed Central - PubMed

Affiliation: The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki Osaka 567-0047, Japan.

ABSTRACT
Charge transfer (CT) in DNA offers a unique approach for the detection of a single-base mismatch in a DNA molecule. While the single-base mismatch would significantly affect the CT in DNA, the kinetic basis for the drastic decrease in the CT efficiency through DNA containing mismatches still remains unclear. Recently, we determined the rate constants of the CT through the fully matched DNA, and we can now estimate the CT rate constant for a certain fully matched sequence. We assumed that further elucidating of the kinetics in mismatched sequences can lead to the discrimination of the DNA single-base mismatch based on the kinetics. In this study, we investigated the detailed kinetics of the CT through DNA containing mismatches and tried to discriminate a mismatch sequence based on the kinetics of the CT in DNA containing a mismatch.

Show MeSH