Evaluation of MafG interaction with Maf recognition element arrays by surface plasmon resonance imaging technique
Evaluation of MafG interaction with Maf recognition element arrays by surface plasmon resonance imaging technique
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DOI：
10.1111/j.1356-9597.2004.00711.x
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发表时间：
2004-02-01
期刊：
影响因子：
2.1
通讯作者：
Yamamoto, M
中科院分区：
文献类型：
--
作者：
Kyo, M;Yamamoto, T;Yamamoto, M
转录因子与顺式作用DNA序列基序之间的特异性相互作用是转录调控的主要事件。许多调控元件似乎偏离最佳识别序列。为了评估转录因子对各种次优序列的亲和力，我们开发了一种基于表面等离子体共振（SPR）成像技术的新检测方法。采用转录因子Maf G及其识别序列MARE（Maf识别元件）对新方法进行评价。我们改进了DNA在金芯片上的固定方法，成功地制备了双链DNA阵列。我们进一步发现，由聚（乙二醇）组成的亲水性柔性间隔区之间的DNA和链烷乙醇的表面上的自组装单分子膜是有效的，以防止非特异性吸附和促进特异性结合的MafG。通过SPR成像技术观察到MafG与6个MARE相关序列之间的多个相互作用谱。通过SPR成像获得的动力学值显示出非常好的相关性与从电泳凝胶迁移率变动测定，虽然绝对值彼此偏离。这些结果表明，用改进的多步法制备的双链DNA阵列可以用于转录因子-DNA相互作用的综合分析。
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.