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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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Discrimination of mismatch DNA related to SNP by CT kinetics in DNAs. Bold letters indicate the SNP sequences. Adenine consecutive sequence is attached to SNP sequence for charge injection. (a, 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 of M1-C and M2-C (red), M1-T and M2-T (black), respectively. The smooth curves superimposed on the curves (green) 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 32 laser shots.
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Figure 5: Discrimination of mismatch DNA related to SNP by CT kinetics in DNAs. Bold letters indicate the SNP sequences. Adenine consecutive sequence is attached to SNP sequence for charge injection. (a, 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 of M1-C and M2-C (red), M1-T and M2-T (black), respectively. The smooth curves superimposed on the curves (green) 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 32 laser shots.

Mentions: For the kinetic discrimination of a mismatch sequence, the sequences related to a SNP were chosen as a model system. We investigated whether the information about the SNP sequence is transformed into the CT kinetics in DNA. The sequences used in this study are shown in Figure 5. DNAs modified with NI and PTZ at the 5′ end which have an A consecutive sequence and a part of the coding sequences for the nuclear LIM interactor-interacting factor 2 and DNA-binding domain T-box 22, carrying SNP rs12392145 (M1) and rs1965744 (M2) C to T substitution, were employed in this study. These SNPs were chosen as a model for the kinetic SNP discrimination because the rate constants determined in this study were able to adapt to these sequences. First, the time-resolved transient absorption measurement and kinetic modeling of M1-C and M1-T were carried out. Of special interest, the decreased kinetics of the CT were observed for the M1-T containing mismatch, in agreement with the kinetic modeling by the rate constants for the CT. We then examined M2-C and M2-T using the transient absorption method and kinetic modeling. Interestingly, the CT kinetics for the M2-T containing mismatch sequence are increased, consistent with the kinetic modeling by the rate constants. These results suggest that the CT kinetics are sensitive to the position of the mismatch incorporation.Figure 5.


Kinetics of charge transfer in DNA containing a mismatch.

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

Discrimination of mismatch DNA related to SNP by CT kinetics in DNAs. Bold letters indicate the SNP sequences. Adenine consecutive sequence is attached to SNP sequence for charge injection. (a, 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 of M1-C and M2-C (red), M1-T and M2-T (black), respectively. The smooth curves superimposed on the curves (green) 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 32 laser shots.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Discrimination of mismatch DNA related to SNP by CT kinetics in DNAs. Bold letters indicate the SNP sequences. Adenine consecutive sequence is attached to SNP sequence for charge injection. (a, 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 of M1-C and M2-C (red), M1-T and M2-T (black), respectively. The smooth curves superimposed on the curves (green) 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 32 laser shots.
Mentions: For the kinetic discrimination of a mismatch sequence, the sequences related to a SNP were chosen as a model system. We investigated whether the information about the SNP sequence is transformed into the CT kinetics in DNA. The sequences used in this study are shown in Figure 5. DNAs modified with NI and PTZ at the 5′ end which have an A consecutive sequence and a part of the coding sequences for the nuclear LIM interactor-interacting factor 2 and DNA-binding domain T-box 22, carrying SNP rs12392145 (M1) and rs1965744 (M2) C to T substitution, were employed in this study. These SNPs were chosen as a model for the kinetic SNP discrimination because the rate constants determined in this study were able to adapt to these sequences. First, the time-resolved transient absorption measurement and kinetic modeling of M1-C and M1-T were carried out. Of special interest, the decreased kinetics of the CT were observed for the M1-T containing mismatch, in agreement with the kinetic modeling by the rate constants for the CT. We then examined M2-C and M2-T using the transient absorption method and kinetic modeling. Interestingly, the CT kinetics for the M2-T containing mismatch sequence are increased, consistent with the kinetic modeling by the rate constants. These results suggest that the CT kinetics are sensitive to the position of the mismatch incorporation.Figure 5.

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