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

J 2007

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

MASAŘÍK, Michal, K. CAHOVÁ, René KIZEK, Emil PALEČEK, Miroslav FOJTA et. al.

Basic information

Original name

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

Name in Czech

Voltametrická detekce změn jednoho nukletidu pomoci MutS proteinu bez pouziti znacek

Authors

MASAŘÍK, Michal (203 Czech Republic), K. CAHOVÁ (203 Czech Republic), René KIZEK (203 Czech Republic, guarantor), Emil PALEČEK (203 Czech Republic) and Miroslav FOJTA (203 Czech Republic)

Edition

ANALYTICAL AND BIOANALYTICAL CHEMISTRY, HEIDELBERG, GERMANY, SPRINGER-VERLAG HEIDELBERG, 2007, 1618-2642

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

Genetics and molecular biology

Country of publisher

Germany

Confidentiality degree

není předmětem státního či obchodního tajemství

Impact factor

Impact factor: 2.867

RIV identification code

RIV/00216224:14110/07:00022108

Organization unit

Faculty of Medicine

UT WoS

000245292200032

Keywords in English

voltammetric detection;single nucleotide mismatches; MutS protein

Abstract

ORIG CZ

V originále

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-mu L 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 mu L 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.

In Czech

Voltametrická detekce změn jednoho nukletidu pomoci MutS proteinu bez pouziti znacek

Links

LC06035, research and development project

Name: Centrum biofyzikální chemie, bioelektrochemie a bioanalýzy. Nové nástroje pro genomiku, proteomiku a biomedicínu.

Investor: Ministry of Education, Youth and Sports of the CR, Centre of Biophysical Chemistry, Bioelectrochemistry and Bioanalysis. New Tools for Genomics, Proteomics and Biomedicine

Citovat

MASAŘÍK, Michal, K. CAHOVÁ, René KIZEK, Emil PALEČEK and Miroslav FOJTA. Label-free voltammetric detection of single nucleotide mismatches recognized by MutS protein. ANALYTICAL AND BIOANALYTICAL CHEMISTRY. HEIDELBERG, GERMANY: SPRINGER-VERLAG HEIDELBERG, 2007, vol. 388, No 1, p. 259-270. ISSN 1618-2642.

@article{718630,
   author = {Masařík, Michal and Cahová, K. and Kizek, René and Paleček, Emil and Fojta, Miroslav},
   article_location = {HEIDELBERG, GERMANY},
   article_number = {1},
   keywords = {voltammetric detection;single nucleotide mismatches; MutS protein},
   language = {eng},
   issn = {1618-2642},
   journal = {ANALYTICAL AND BIOANALYTICAL CHEMISTRY},
   title = {Label-free voltammetric detection of single nucleotide mismatches recognized by MutS protein},
   volume = {388},
   year = {2007}
}

TY  - JOUR
ID  - 718630
AU  - Masařík, Michal - Cahová, K. - Kizek, René - Paleček, Emil - Fojta, Miroslav
PY  - 2007
TI  - Label-free voltammetric detection of single nucleotide mismatches recognized by MutS protein
JF  - ANALYTICAL AND BIOANALYTICAL CHEMISTRY
VL  - 388
IS  - 1
SP  - 259-270
EP  - 259-270
PB  - SPRINGER-VERLAG HEIDELBERG
SN  - 16182642
KW  - voltammetric detection;single nucleotide mismatches
KW  - MutS protein
N2  - 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-mu L 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 mu L 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.
ER  -

MASAŘÍK, Michal, K. CAHOVÁ, René KIZEK, Emil PALEČEK and Miroslav FOJTA. Label-free voltammetric detection of single nucleotide mismatches recognized by MutS protein. \textit{ANALYTICAL AND BIOANALYTICAL CHEMISTRY}. HEIDELBERG, GERMANY: SPRINGER-VERLAG HEIDELBERG, 2007, vol.~388, No~1, p.~259-270. ISSN~1618-2642.

Detailed Information on Publication Record