Label-free voltammetric detection of single-nucleotide mismatches recognized by the protein MutS |
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Authors: | Michal Masa?ík Kate?ina Cahová Rene Kizek Emil Pale?ek Miroslav Fojta |
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Institution: | (1) Institute of Biophysics v.v.i., Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno, Czech Republic;(2) Present address: Institute of Pathological Physiology, Faculty of Medicine, Masaryk University, Komenskeho nam. 2, 662 43 Brno, Czech Republic;(3) Present address: Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zemedelska 1, 613 00 Brno, Czech Republic |
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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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Keywords: | Single-nucleotide polymorphism Point mutation Base mismatches MutS Magnetic beads Protein electrochemistry Quartz-crystal microbalance |
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