Evaluation of MafG interaction with Maf recognition element arrays by surface plasmon resonance imaging technique.
Specific interactions between transcription factors and cis-acting DNA sequence motifs are primary events for the transcriptional regulation. Many regulatory elements appear to diverge from the most optimal recognition sequences. To evaluate affinities of a transcription factor to various suboptimal sequences, we have developed a new detection method based on the surface plasmon resonance (SPR) imaging technique. Transcription factor MafG and its recognition sequence MARE (Maf recognition elements) were adopted to evaluate the new method. We modified DNA immobilization procedure on to the gold chip, so that a double-stranded DNA array was successfully fabricated. We further found that a hydrophilic flexible spacer composed of the poly (ethylene glycol) moiety between DNA and alkanethiol self-assembled monolayers on the surface is effective for preventing nonspecific adsorption and facilitating specific binding of MafG. Multiple interaction profiles between MafG and six of MARE-related sequences were observed by the SPR imaging technique. The kinetic values obtained by SPR imaging showed very good correlation with those obtained from electrophoretic gel mobility shift assays, although absolute values were deviated from each other. These results demonstrate that the double-stranded DNA array fabricated with the modified multistep procedure can be applied for the comprehensive analysis of the transcription factor-DNA interaction.
TOYOBO Co. Ltd. Bio 21 Project, 10-24 Toyo-Cho, Tsuruga, Fukui 914-0047, Japan.
Motoki
M
Kyo
Tae
T
Yamamoto
Hozumi
H
Motohashi
Terue
T
Kamiya
Toshihiro
T
Kuroita
Toshiyuki
T
Tanaka
James Douglas
JD
Engel
Bunsei
B
Kawakami
Masayuki
M
Yamamoto
D001665:Binding Sites; D016384:Consensus Sequence; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006046:Gold / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D051259:MafG Transcription Factor; D008301:Maleimides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D013438:Sulfhydryl Compounds / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C103944:MAFG protein, human; D051259:MafG Transcription Factor; D008301:Maleimides; D012097:Repressor Proteins; D013438:Sulfhydryl Compounds; C071675:sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; D011092:Polyethylene Glycols; D006046:Gold