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: 10707393
• 负责人: STEWART N LOH
• 金额: $ 40.72万
• 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/10707393
• 关键词: Acceleration; 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; Family; Fibronectins; Fluorescence; Fluorescence Resonance Energy Transfer; Genes; Goals; Guide RNA; Human; Human Activities; Immunocompromised Host; Individual; Kinetics; Ligand Binding; Ligands; Ligation; Mediating; Methodology; Modification; Molecular Conformation; Monitor; Morbidity - disease rate; Nature; Nucleic Acid Binding; Nucleic Acids; Outcome; Output; Pathogen detection; 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; combinatorial; cytotoxic; design; experimental study; frontier; link protein; luminescence; monocyte; mortality; nanoluciferase; nucleic acid binding protein; 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.

项目摘要及相关性

项目成果

Adaptable, Turn-On Monobody (ATOM) Fluorescent Biosensors for Multiplexed Detection in Cells.

用于细胞多重检测的适应性强的开启单体 (ATOM) 荧光生物传感器。

• DOI: 10.1101/2023.03.28.534597
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Sekhon,Harsimranjit;Ha,Jeung-Hoi;Presti,MariaF;Procopio,SpencerB;Mirsky,PaigeO;John,AnnaM;Loh,StewartN
• 通讯作者: Loh,StewartN

Enhancing response of a protein conformational switch by using two disordered ligand binding domains.

通过使用两个无序的配体结合域增强蛋白质构象转换的响应。

• DOI: 10.3389/fmolb.2023.1114756
• 发表时间: 2023
• 期刊: Frontiers in molecular biosciences
• 影响因子: 5