Large-Scale, Quantitative Protein Affinity Assays on a High-Throughput DNA Sequencing Chip

在高通量 DNA 测序芯片上进行大规模定量蛋白质亲和力测定

基本信息

批准号：
10007027
负责人：
Curtis Layton
金额：
$ 24.78万
依托单位：
PROTILLION BIOSCIENCES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2020-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10007027
关键词：
Adopted Affinity Amino Acids Antibodies Avidity Behavior Binding Binding Proteins Biochemical Biological Biological Assay Biological Sciences Biophysics Cells ChIP-seq Collaborations Cryoelectron Microscopy Crystallization DNA Sequence DNA biosynthesis DNA sequencing Data Data Set Dependence Detection Development Devices Diagnostic Directed Molecular Evolution Engineering Enzyme Tests Epitopes Family Fluorescence Generations Genetic Transcription Goals HIV HIV Infections Hand Heart High-Throughput DNA Sequencing Human Biology ITIM Immobilization In Situ Individual Informatics Infrastructure Kinetics Lead Length Letters Libraries Ligands Location Maps Measurement Measures Methods Mutation Mutation Analysis Nucleic Acids PD-1 blockade Peptide Library Peptide antibodies Peptides Pharmaceutical Preparations Protein Analysis Protein Array Protein Engineering Protein Family Proteins Protocols documentation Publishing RNA Reagent Sodium Chloride Specialist Structure Surface Plasmon Resonance T-Lymphocyte Techniques Technology Temperature Tertiary Protein Structure Testing Therapeutic Therapeutic Intervention Thermodynamics Time Translations Universities Variant Work X-Ray Crystallography base biophysical properties cancer immunotherapy expectation experimental study fluorescence imaging high throughput analysis improved insight instrumentation nanobodies preference protein biomarkers protein function protein protein interaction therapeutic target

项目摘要

Abstract The high-throughput DNA sequencing revolution has brought a match between the scale of nucleic acid quantification and the complexity of human biology. However, despite expectation that similarly-widespread and scalable methods for probing protein function would emerge, most protein functional assays and mutational analyses remain largely low to medium throughput. While multiplexed protein methods exist, they tend to be employed only by a small number of “specialists” because 1) they have not reached the immense scalability of DNA sequencing methods, and 2) they are technically challenging to implement. Protillion is commercializing a platform for high-throughput protein analysis built directly on the ubiquitous Illumina sequencer, demonstrating a path to bringing protein functional characterization into the high-throughput era. This protein mapping platform (Prot-MaP) enables the generation of tens of millions of clusters of immobilized proteins directly on DNA sequencing flow cell through efficient tethered in-situ transcription and translation. Fluorescence-based protein functional assays can then be performed directly on this protein array, with results quantified by highly sensitive fluorescence imaging. Prot-MaP enables the generation of immense mutational datasets for both peptides and full-length functional proteins, allowing high-throughput analysis of mutational effects based on direct biophysical observations of protein function. Unlike directed evolution methods this platform allows for direct, quantitative measurements of the function of entire protein libraries, and a shorter time-to-result in a single experiment on automated, widely distributed instrumentation. We will deploy the Prot- MaP platform in two different modes – one using small, ~15 amino acid peptides as affinity reagents, and another displaying larger, ~115 amino acid nanobodies, to characterize the biophysical interactions between T Cell immunoreceptor with Ig and ITIM domains, or TIGIT protein. TIGIT is an ideal example target, as it is highly soluble, commercially available, and has diagnostic (for detecting HIV progression) and therapeutic (in combination with PD1 blockade in cancer immunotherapy) application potential. We plan to analyze our high- throughput data sets 1) to understand the benefits afforded pre-selection of peptides compared to rational engineering of peptide libraries, 2) to identify clades of peptides and nanobodies that form different families of binders, 3) to test the identification of these distinct families that bind distinct epitopes on TIGIT experimentally, and 4) assess the salt and temperature dependence of binding, 5) examine the dependence of specific key peptides to binding using structural information. The identification of nanobodies and peptides that bind distinct regions of TIGIT will open the door to creation of both sandwich-style affinity assays, as well as high affinity ligands (via avidity) for potential therapeutic application. Overall, this work will create and demonstrate a platform for large-scale biochemical characterization and analysis of protein-protein interactions.

抽象的高通量DNA测序但是，人类生物学的量化和复杂性。以及可探测蛋白质功能的可扩展方法，大多数蛋白质功能分析和突变分析在很大程度上保持了多种蛋白质方法。倾向于仅由少数“专家”雇用，因为1）他们尚未达到巨大 DNA测序方法的可扩展性，2）它们在技术上具有挑战性商业化直接基于无处不在的Illumina的高含量蛋白分析的平台音序器，展示了将蛋白质功能表征带入高吞吐量时代的途径。该蛋白质映射平台（Prot-MAP）使数千万固定的簇产生直接在DNA测序流细胞上有效的蛋白质基于荧光的蛋白质功能测定可以直接在Theis蛋白阵列上，结果结果通过高度敏感的荧光成像量化。肽和全长功能蛋白的数据集，允许对突变的高齿分析基于直接的蛋白质功能的直接生物物理观察。平台允许对整个蛋白质库的功能进行直接定量的测量，并且更短对自动化的，willy分布式仪器的单个实验的时间。以两种不同模式的地图平台 - 一种使用小，约15个氨基酸作为亲和力试剂另一个显示较大的〜115个氨基酸纳米型，以表征t之间的生物物理相互作用具有IG和ITIM结构域的细胞免疫受体，或Tigit蛋白。高度可溶，可商购，并具有诊断性（用于检测HIV进度）和治疗性（IN）在癌症免疫疗法中的PD1结合）应用潜力。吞吐量数据集1）了解肽指南针的预选为理性的预选肽库的工程，2）确定形成不同家族的肽和纳米生物的进化枝 3）测试这些不同家族的识别，这些家族对Tigit经验的不同表位， 4）评估结合的盐和温度依赖性，5）检查特定键的依赖性使用结构信息结合的肽。 Tigit区域将为创建三明治风格的亲和力测定法和高亲和力开放配体（通过自然）用于潜在的治疗应用。大规模生化表征和蛋白质 - 蛋白质相互作用的分析的平台。