化学遗传学是一种强大的科学策略,它利用小的生物活性分子作为实验工具来解开生物过程。自然界中存在的生物活性化合物代表了可用于剖析生物系统功能的结构的巨大多样性。一旦对天然或合成化合物的生物活性进行了严格评估,确定其分子靶标和作用方式仍然存在挑战。这通常是一个耗时耗力的过程。为了促进这项任务,我们决定实施酵母三杂交(Y3H)技术作为一个通用的实验平台,扫描整个拟南芥蛋白质组小信号分子的目标。Y3H技术基于酵母双杂交系统,可以直接克隆体内与合成杂合配体相互作用的蛋白质,其包含与甲氨蝶呤(Mtx)共价连接的感兴趣的生物活性分子。在酵母核中,杂合配体连接两个融合蛋白:与融合到DNA结合域(在酵母菌株中编码)的二氢叶酸还原酶结合的Mtx部分,和生物活性分子部分结合其与DNA激活域(在cDNA表达载体上编码)融合的潜在蛋白质靶标。在cDNA文库筛选过程中,这个三元的形成,转录激活复合物导致酵母细胞中的报告基因激活,从而允许选择感兴趣的小生物活性分子的推定靶标。在这里,我们介绍了构建和应用Y3H平台的策略和实验细节,包括不同杂化配体的化学合成,构建合适的cDNA文库,酵母菌株的选择,和适当的筛选条件。基于获得的结果和现有文献,我们讨论了Y3H方法用于识别小生物活性分子靶标的观点和局限性。
Chemical genetics is a powerful scientific strategy that utilizes small bioactive molecules as experimental tools to unravel biological processes. Bioactive compounds occurring in nature represent an enormous diversity of structures that can be used to dissect functions of biological systems. Once the bioactivity of a natural or synthetic compound has been critically evaluated the challenge remains to identify its molecular target and mode of action, which usually is a time-consuming and labor-intensive process. To facilitate this task, we decided to implement the yeast three-hybrid (Y3H) technology as a general experimental platform to scan the whole Arabidopsis proteome for targets of small signaling molecules. The Y3H technology is based on the yeast two-hybrid system and allows direct cloning of proteins that interact in vivo with a synthetic hybrid ligand, which comprises the biologically active molecule of interest covalently linked to methotrexate (Mtx). In yeast nucleus the hybrid ligand connects two fusion proteins: the Mtx part binding to dihydrofolate reductase fused to a DNA-binding domain (encoded in the yeast strain), and the bioactive molecule part binding to its potential protein target fused to a DNA-activating domain (encoded on a cDNA expression vector). During cDNA library screening, the formation of this ternary, transcriptional activator complex leads to reporter gene activation in yeast cells, and thereby allows selection of the putative targets of small bioactive molecules of interest. Here we present the strategy and experimental details for construction and application of a Y3H platform, including chemical synthesis of different hybrid ligands, construction of suitable cDNA libraries, the choice of yeast strains, and appropriate screening conditions. Based on the results obtained and the current literature we discuss the perspectives and limitations of the Y3H approach for identifying targets of small bioactive molecules.