Combining protein and DNA engineering to create bioswitches

项目摘要和相关性 该项目的目标是开发普通蛋白质可以转化为配体的机制- 激活构象转换当这种类型的天然蛋白质被发现时， 工程学可以产生改变生物学的技术。例如，CRISPR相关蛋白 催化活性通过引导RNA的结合而开启，并且钙调蛋白经历大的构象变化。 在结扎钙后的变化。将这些蛋白质开发成DNA操纵工具和荧光钙 传感器，分别彻底改变了基因编辑和钙信号的研究。现时的建议 提出这样的问题：“如果其他蛋白质和酶可以通过结合来打开/关闭，还有什么可能呢 DNA、RNA或其他配体？” 拟议的项目采用生物物理，计算和细胞相结合的方法来开发一种 蛋白质功能与配体结合的一般机制。三个蛋白质开关家族将被 创造第一种是插入现有DNA工具（如适体和立足点介导）的生物传感器 链置换发夹），而不对传感器进行任何修饰，以检测 选择输出为比率（蓝色/绿色）发光，可由手机摄像头检测。的 第二个家族采用纤连蛋白3“单体”作为输入结构域，荧光蛋白作为输出 结构域提供比率FRET响应，或荧光强度的大幅增加，当 遇到细胞内目标在第三种开关设计中，细菌RNA酶的酶活性是 由巨细胞病毒（CMV）RNA打开，杀死CMV感染的人类细胞。最后一个目标是解决 迫切需要预防与移植相关的CMV疾病。 本案无关这项研究将开放人类蛋白质组的生物活性，通过 核酸、蛋白质和小分子的结合。模块化设计允许混合和匹配 不同的蛋白质，以产生分子的功能，没有发现在自然界中。例子包括生物传感器 病原体和疾病的生物标志物，以及一种酶，杀死病毒感染的人体细胞，同时离开 未受感染的细胞毫发无伤

Project Summary and Relevance The goal of this project is to develop mechanisms by which ordinary proteins can be turned into ligand- activated conformational switches. When naturally-occurring proteins of this type are discovered, their engineering can result in technologies that transform biology. For example, CRISPR-associated protein catalytic activity is switched on by binding of guide RNA, and calmodulin undergoes a large conformational change upon ligating calcium. Developing these proteins into DNA manipulation tools and fluorescent calcium sensors, respectively have revolutionized gene editing and the study of calcium signaling. The current proposal asks the question, “what else is possible if other proteins and enzymes can be made to switch on/off by binding of DNA, RNA, or other ligands?”. The proposed project takes a combined biophysical, computational, and cellular approach to develop a general mechanism for linking protein function to ligand binding. Three families of protein switches will be created. The first is a biosensor that plugs into existing DNA tools (such as aptamers and toehold-mediated strand displacement hairpins) without any modification to the sensor, to detect a DNA or RNA sequence of choice. The output is ratiometric (blue/green) luminescence that can be detected by cell phone camera. The second family employs fibronectin 3 ‘monobodies’ as the input domains and fluorescent proteins as the output domains to provide a ratiometric FRET response, or large increase in fluorescence intensity, when encountering an intracellular target. In the third switch design, the enzymatic activity of a bacterial RNase is turned on by cytomegalovirus (CMV) RNA to kill CMV-infected human cells. This last aim addresses the pressing need of preventing transplant-related CMV disease. Relevance. This study will open the biological activity of the human proteome to potential regulation by binding of nucleic acids, proteins, and small molecules. The modular design allows mixing and matching of different proteins to generate molecules with functionalities not found in nature. Examples include biosensors for pathogens and disease biomarkers, and an enzyme that kills virally-infected human cells while leaving uninfected cells unharmed.