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Label-free voltammetric detection of single-nucleotide mismatches recognized by the protein MutS

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Abstract

MutS, a protein involved in DNA mismatch repair, recognizes mispaired and unpaired bases in duplex DNA. We have previously used MutS in an electrochemical double-surface technique (DST) for in-vitro detection of point mutations in DNA. The DST involved binding of unlabeled MutS to DNA heteroduplexes at the surface of magnetic beads followed by a highly sensitive electrochemical determination of the protein by measurement of a catalytic protein signal (peak H) at mercury electrodes. Detection of MutS using a peak resulting from oxidation of tyrosine and tryptophan residues of the protein at a carbon-paste electrode (CPE) was also possible but was approximately three orders of magnitude less sensitive. In this work we present an optimized technique for ex-situ voltammetric determination of MutS at a CPE. Choice of optimum experimental conditions (pH of supporting electrolyte, square-wave voltammetry settings, etc.) resulted in substantial improvement of the sensitivity of the assay, enabling detection of approximately 140 pg (1.6 fmol protein monomer) MutS in a 5-μL sample. The sensitivity was increased further by acid hydrolysis of the protein before measurement. The hydrolyzed protein was detectable down to 5 pg (approx. 56 amol) MutS in 5 μL solution. By using the DST combined with determination of the bound unlabeled MutS at the CPE we demonstrated selective interactions of the protein with single-base mismatches and discrimination among different base mispairs in 30-mer or 95-mer DNA duplexes. In agreement with previous studies, binding of the protein to the 30-mer substrates followed the trend G:T>>C:A>A:A>C:T>homoduplex. The electrochemical data were confirmed by use of an independent technique—a quartz-crystal microbalance for real-time monitoring of MutS interactions with DNA duplexes containing different base mispairs. By using the electrochemical DST a G:T mismatch was detectable in up to 1000-fold excess of homoduplex DNA.

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Acknowledgements

This work was supported by the GACR (grants 203/04/1325 to M.F and 301/07/0490 to E.P.), the Ministry of Industry and Trade of the CR (1H-PK/42), the Ministry of Education, Youth and Sports of the CR (LC06035), and by Institutional Research Plan No. AVOZ50040507.

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Author notes

  1. Michal Masařík

    Present address: Institute of Pathological Physiology, Faculty of Medicine, Masaryk University, Komenskeho nam. 2, 662 43, Brno, Czech Republic

  2. Rene Kizek

    Present address: Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zemedelska 1, 613 00, Brno, Czech Republic

Authors and Affiliations

  1. Institute of Biophysics v.v.i., Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65, Brno, Czech Republic

    Michal Masařík, Kateřina Cahová, Rene Kizek, Emil Paleček & Miroslav Fojta

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  1. Michal Masařík
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  2. Kateřina Cahová
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  4. Emil Paleček
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Correspondence to Emil Paleček or Miroslav Fojta.

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Masařík, M., Cahová, K., Kizek, R. et al. Label-free voltammetric detection of single-nucleotide mismatches recognized by the protein MutS. Anal Bioanal Chem 388, 259–270 (2007). https://doi.org/10.1007/s00216-007-1181-7

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  • DOI: https://doi.org/10.1007/s00216-007-1181-7

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