Combining protein and DNA engineering to create bioswitches

结合蛋白质和 DNA 工程来创建生物开关

• 批准号: 10561100
• 负责人: STEWART N LOH
• 金额: $ 43.59万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10561100
• 关键词: Address; Antibodies; BCAR1 gene; Bacillus amyloliquefaciens ribonuclease; Base Sequence; Binding; Biological; Biological Markers; Biology; Biophysics; Biosensor; Bone Marrow Stem Cell; Calcium; Calcium Signaling; Calmodulin; Cause of Death; Cells; Cellular Phone; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Computing Methodologies; Cytomegalovirus; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; Devices; Disease; Disease Marker; Engineering; Enzymes; Exhibits; Eye; Family; Fibronectins; Fluorescence; Fluorescence Resonance Energy Transfer; Genes; Goals; Guide RNA; Human; Human Activities; Immunocompromised Host; Individual; Kinetics; Ligand Binding; Ligands; Mediating; Methodology; Modification; Molecular Conformation; Monitor; Morbidity - disease rate; Nature; Nucleic Acid Binding; Nucleic Acids; Outcome; Output; Pathway interactions; Protein Conformation; Protein Engineering; Protein Family; Proteins; Proteome; RNA; RNA Binding; RNA Sequences; Reaction Time; Regulation; Research; Ribonucleases; Source; System; Technology; Therapeutic; Toxin; Transplant Recipients; Transplantation; Variant; Viral; aptamer; base; combinatorial; cytotoxic; design; experimental study; frontier; link protein; luminescence; monocyte; mortality; nanoluciferase; pathogen; point-of-care detection; prevent; protein folding; protein function; ratiometric; response; scaffold; sensor; simulation; small molecule; tool

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.

项目摘要和相关性 该项目的目标是开发将普通蛋白质转化为配体的机制 激活的构象开关。当发现这种类型的天然蛋白质时，它们的 工程可以带来改变生物学的技术。例如，CRISPR相关蛋白 催化活性通过指导 RNA 的结合而开启，钙调蛋白经历大构象 连接钙后发生变化。将这些蛋白质开发成 DNA 操作工具和荧光钙 传感器分别彻底改变了基因编辑和钙信号研究。目前的提案 提出了这样的问题：“如果可以通过结合其他蛋白质和酶来打开/关闭，还有什么可能？ DNA、RNA 或其他配体？”。 该项目采用生物物理、计算和细胞相结合的方法来开发 将蛋白质功能与配体结合联系起来的一般机制。蛋白质开关的三个家族将是 创建的。第一个是生物传感器，可插入现有的 DNA 工具（例如适体和立足点介导的工具） 链位移发夹）无需对传感器进行任何修改，即可检测 DNA 或 RNA 序列 选择。输出是比率（蓝色/绿色）发光，可以通过手机摄像头检测到。这 第二个家族采用纤连蛋白 3“单体”作为输入域，荧光蛋白作为输出 域提供比率 FRET 响应，或荧光强度大幅增加，当 遇到细胞内靶标。在第三种开关设计中，细菌 RNase 的酶活性为 由巨细胞病毒 (CMV) RNA 启动，杀死受 CMV 感染的人类细胞。最后一个目标解决了 预防移植相关巨细胞病毒疾病的迫切需要。 关联。这项研究将开启人类蛋白质组的生物活性，使其受到潜在的调控 核酸、蛋白质和小分子的结合。模块化设计允许混合和匹配 不同的蛋白质产生具有自然界中未发现的功能的分子。例子包括生物传感器 用于病原体和疾病生物标志物，以及一种杀死病毒感染的人类细胞同时留下的酶 未受感染的细胞未受到伤害。