Combining protein and DNA engineering to create bioswitches

结合蛋白质和 DNA 工程来创建生物开关

基本信息

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
项目类别：
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

项目摘要

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当发现这种类型的天然存在的蛋白质时，它们的构象开关被激活。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。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是三个蛋白质开关家族。第一个是插入现有 DNA 工具（例如适体和立足点介导）的生物传感器。链位移发夹）无需对传感器进行任何修改，即可检测 DNA 或 RNA 序列输出是比率（蓝色/绿色）发光，可以通过手机摄像头检测到。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。预防移植相关巨细胞病毒疾病的迫切需要。相关性。这项研究将开启人类蛋白质组的生物活性的潜在调控。核酸、蛋白质和小分子的结合允许混合和匹配。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。