Masarík Michal, Cahová Katerina, Kizek Rene, Palecek Emil, Fojta Miroslav
Institute of Biophysics v.v.i., Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65, Brno, Czech Republic.
Anal Bioanal Chem. 2007 May;388(1):259-70. doi: 10.1007/s00216-007-1181-7. Epub 2007 Feb 28.
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-microL 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 microL 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.
MutS是一种参与DNA错配修复的蛋白质，可识别双链DNA中的错配和未配对碱基。我们之前曾在电化学双表面技术（DST）中使用MutS对DNA中的点突变进行体外检测。DST包括将未标记的MutS与磁珠表面的DNA异源双链体结合，然后通过在汞电极上测量催化蛋白信号（峰H）对蛋白质进行高灵敏度的电化学测定。使用蛋白质酪氨酸和色氨酸残基在碳糊电极（CPE）上氧化产生的峰来检测MutS也是可行的，但灵敏度大约低三个数量级。在这项工作中，我们提出了一种用于在CPE上异位伏安法测定MutS的优化技术。选择最佳实验条件（支持电解质的pH值、方波伏安法设置等）可大幅提高检测灵敏度，能够在5微升样品中检测到约140皮克（1.6飞摩尔蛋白质单体）的MutS。在测量前对蛋白质进行酸水解可进一步提高灵敏度。水解后的蛋白质在5微升溶液中低至5皮克（约56阿托摩尔）的MutS也可检测到。通过将DST与在CPE上测定结合的未标记MutS相结合，我们证明了该蛋白质与单碱基错配的选择性相互作用，以及在30聚体或95聚体DNA双链体中对不同碱基错配的区分。与之前的研究一致，该蛋白质与30聚体底物的结合遵循G:T>>C:A>A:A>C:T>同源双链体的趋势。通过使用一种独立技术——石英晶体微天平实时监测MutS与含有不同碱基错配的DNA双链体的相互作用，证实了电化学数据。通过使用电化学DST，在高达1000倍过量的同源双链体DNA中可检测到G:T错配。
