A droplet microfluidics approach to measuring protein aggregation

测量蛋白质聚集的液滴微流体方法

• 批准号: 9905475
• 负责人: MARC I DIAMOND
• 金额: $ 20.25万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-15 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9905475
• 关键词: Alzheimer' s Disease; Amino Acid Sequence; Amino Acids; Amyloid; Amyloid Fibrils; Amyloid fibers; Amyloidosis; Biochemical; Biochemistry; Biological Assay; Biology; C-terminal; Cell Survival; Cloning; DNA amplification; DNA cassette; Dependence; Diagnostic; Disease; Encapsulated; Exhibits; Familial Dementias; Fluorescence; Genes; Genetic Transcription; Goals; Heparin; In Vitro; Individual; Induced Mutation; Investigation; Knowledge; Length; Libraries; Liquid substance; Maps; Measurement; Measures; Methods; Microfluidics; Mutation; Neurodegenerative Disorders; Oils; Peptides; Phase; Play; Point Mutation; Post-Translational Protein Processing; Process; Proteins; Role; Specific qualifier value; Stream; Structure; Surveys; Syndrome; System; Tauopathies; Techniques; Testing; Therapeutic; Translations; Variant; Work; amyloid formation; aqueous; base; biophysical techniques; cofactor; design; early onset; imaging agent; improved; in vivo; novel; protein aggregation; screening; self assembly; tau Proteins; tau aggregation; tau mutation; tool

Protein aggregation plays an important role in biology and disease. For example, a variety of syndromes termed amyloid diseases involve the self-assembly of proteins into elongated amyloid fibers. A critical first step in understanding the forces that underlie these processes is to define which proteins can self-associate – more specifically, to identify the sequence determinants of aggregation. This is not feasible with current methods, which are low-throughput and can only be used to study a very limited number of sequences. Furthermore, isolating the contribution of protein sequence independent from the surrounding cellular milieu requires measurement in biochemically defined conditions. We propose to develop a droplet microfluidics-based assay to measure protein aggregation. Droplet microfluidics is a technique that generates and manipulates picoliter- sized aqueous droplets, embedded in a stream of fluorinated oil that effectively isolates the droplets from one another. These droplets can be generated at a rate of up to 5000 hertz, and individual genes can be encapsulated in them, enabling the analysis of large libraries. We describe an approach to observe protein self-assembly in droplets and to sequence the genes that give rise to aggregation-prone peptides. We will apply this approach to two sequence libraries comprising variants of the Alzheimer’s associated protein, Tau. If successful, this system could be applied to identify other sequences that self-associate, or sequences or cofactors that modulate amyloid formation in a large variety of disease-associated proteins, as well as in other sorts of aggregation phenomena such as liquid-liquid phase separation.

蛋白质聚集在生物学和疾病中起着重要的作用。例如，各种各样的综合症