Phage display selection of small molecule switchable transcription factors

DNA分子中的生物体遗传信息指导细胞如何构建蛋白质，蛋白质是生命的结构构建块和分子机器的关键。细胞中存在许多不同的蛋白质，尽管它们的数量根据细胞生命周期的阶段和对来自其周围环境的信号的响应而变化。揭示蛋白质在将通用干细胞转化为允许多细胞生物体发育的专门组织的过程中的作用是一个例子，这类问题将极大地受益于控制特定蛋白质在特定时刻存在的数量的能力。在这里，我们提出了一种通过添加我们选择的小分子来控制蛋白质生产的方法。蛋白质合成始于被称为转录因子的专门蛋白质与邻近编码蛋白质本身的位置的DNA区域的结合。阻断转录因子与DNA的结合可以阻止由该转录因子靶向的基因编码的蛋白质的合成。我们的假设是，通过设计当它们结合特定的小分子时改变形状的转录因子，我们可以将它们从不能结合DNA的非活性形式转变为结合DNA并开启蛋白质合成的活性形式。我们将解决设计一个转换转录因子的挑战，该因子对选择的小分子做出反应，以在动物中显示有利的特性，包括缺乏毒性和良好的口服吸收。为此，我们选择了一种抗组胺药物，它能与参与炎症反应的蛋白质特异性相互作用，因此副作用很少。由于很难从头开始设计蛋白质，我们将创建编码大量天然转录因子变体的基因，这些变体被选择用于在转录因子中创建一个空腔，我们的小分子可以结合到其中。蛋白质依靠其内部来保持精确的形状，在没有小分子的情况下，我们预测空腔将破坏转录因子的形状，使其不再结合DNA。我们将把我们的转录因子基因放入感染细菌的病毒中，这样细菌就产生了病毒颗粒，这些病毒颗粒将转录因子展示在它们的外部。使用DNA作为诱饵，我们将“钓出”只有当小分子存在时才与DNA结合的病毒颗粒，丢弃剩余的病毒。虽然我们只能分离出极少量的病毒颗粒，但这些病毒颗粒可以通过在感染细菌中复制而扩增。通过多次重复选择和扩增过程，大多数病毒将显示能够响应小分子而转换其DNA结合的转录因子。我们将分析这些转录因子在存在和不存在小分子的情况下与DNA结合的紧密程度，并确定有关其三维结构的信息，以验证我们对其作用模式的假设。我们的最终目标将是将一种新的转录因子放入哺乳动物细胞中，以测试其响应小分子而开启感兴趣基因的能力。这项工作将对参与蛋白质结构和设计以及蛋白质如何与小分子和DNA相互作用的研究人员感兴趣。通过使研究人员能够选择性地打开细胞中的蛋白质，然后监测结果，基因作用的研究将有直接的应用。这将有助于深入了解生长、细胞分化、衰老和疾病进展等过程。更好地了解这些过程，它们可能发生故障的方式以及如何控制它们，将通过加强避免人类健康状况不佳以及改善农业和粮食生产来提高生活质量。

The genetic information of organisms within DNA molecules instructs cells how to construct proteins which are key the structural building blocks and molecular machines of life. Many different proteins are present in a cell, although their amounts vary according to the stage of the lifecycle of the cell and in response to signals from its surroundings. Unravelling the roles of proteins in the processes that convert general purpose stem cells into specialized tissues allowing the development of multicellular organisms is an example of the type of problem that would benefit enormously from an ability to control the quantities of specific proteins present at a particular moment in time. Here we propose a means for controlling production of a protein by addition of a small molecule of our choice. Protein synthesis begins with the binding of specialized proteins known as transcription factors to regions of DNA adjacent to locations coding for proteins themselves. Blocking the transcription factor from binding DNA prevents the synthesis of the protein(s) coded by the genes that are targeted by that transcription factor. Our hypothesis is that by designing transcription factors that change shape when they bind a specific small molecule, we can flip them from an inactive form that is incapable of binding DNA to an active form that binds DNA and switches on protein synthesis. We will address the challenge of designing a switching transcription factor that responds to a small molecule chosen to display favourable properties in animals including lack of toxicity and good oral uptake. For this purpose we have chosen an antihistamine drug that interacts specifically with a protein involved in the inflammatory response and so exhibits few side effects. As it is difficult to design proteins from scratch, we will create genes encoding a large number of variants of a natural transcription factor that have been chosen to create a cavity in the transcription factor into which our small molecule could bind. Proteins rely on their interiors to maintain a precise shape, and in the absence of the small molecule we predict that the cavity will destabilize the shape of the transcription factor so that it no longer binds DNA. We will place our transcription factor genes into a virus that infects bacteria, so that bacteria produce virus particles which display the transcription factor on their exterior. Using DNA as bait, we will 'fish out' virus particles that bind to the DNA only when the small molecule is present, discarding the remaining virus. Although we may isolate only a very small number of virus particles, these can be amplified by replication in infected bacteria. By repeating the process of selection and amplification a number of times the majority of virus will display transcription factors that are able to switch their DNA binding in response to the small molecule. We will analyse how tightly these transcription factors bind DNA in the presence and absence of the small molecule, and determine information about their three dimensional structures to verify our hypothesis on their mode of action. Our final objective will be to place a new transcription factor into mammalian cells to test its ability to switch on a gene of interest in response to the small molecule. This work will be of interest to researchers involved in the structure and design of proteins, and how proteins interact with small molecules and DNA. There will be direct applications for the study of the roles of genes, by enabling researchers to selectively switch on proteins in cells and then monitor the outcome. This will yield insight into processes such as growth, cell differentiation, ageing and disease progression. Better understanding of these processes, the ways in which they can malfunction, and how to control them, will lead to an increase in the quality of life through enhanced avoidance of ill health in humans and improvements to agriculture and food production.