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

• 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
• 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

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.

摘要 高通量DNA测序革命带来了与核酸规模相匹配的 量化和人类生物学的复杂性。然而，尽管有类似普遍的预期 探索蛋白质功能的可扩展方法将会出现，大多数蛋白质功能分析和 突变分析在很大程度上仍然是低到中等的吞吐量。虽然存在多重蛋白质方法，但它们 倾向于只受雇于一小部分“专家”，因为1)他们还没有达到 DNA测序方法的可扩展性，以及2)它们的实施在技术上具有挑战性。普罗的利翁是 将直接建立在无处不在的Illumina上的高通量蛋白质分析平台商业化 测序仪，展示了一条将蛋白质功能表征带入高通量时代的途径。 这个蛋白质图谱平台(PROT-MAP)能够生成数千万簇的固定化蛋白质 蛋白质直接对DNA进行测序，通过高效的拴系原位转录和翻译流动细胞。 然后可以直接在该蛋白质阵列上进行基于荧光的蛋白质功能分析，结果是 通过高灵敏的荧光成像进行量化。Prot-MAP使巨大突变的产生成为可能 多肽和全长功能蛋白的数据集，允许高通量分析突变 基于对蛋白质功能的直接生物物理观察的效果。与定向进化方法不同，该方法 平台允许对整个蛋白质文库的功能进行直接、定量的测量，并提供更短的 在自动化、分布广泛的仪器上进行单一实验所需的时间。我们将部署这个防护罩- 两种不同模式的MAP平台-一种使用~15个氨基酸小肽作为亲和试剂，以及 另一种展示了更大的，~115个氨基酸纳米体，以表征T细胞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。TIGIT是一个理想的示例目标，因为它 高度溶解，商业上可获得，具有诊断(用于检测HIV进展)和治疗(在 结合PD1阻断在癌症免疫治疗中的应用潜力。我们计划分析我们的高潮- 吞吐量数据集1)以了解与Rational相比预选多肽所带来的好处 多肽文库工程，2)识别形成不同家族的多肽和纳米体的分支 结合剂，3)为了在实验上测试结合TIGIT上不同表位的这些不同家族的鉴定， 4)评估结合的盐度和温度依赖性，5)检验特定键的依赖性 利用结构信息与多肽结合。结合不同基因的纳米体和多肽的鉴定 TIGIT的区域将为创建夹心式亲和分析以及高亲和力打开大门 潜在治疗应用的配体(通过亲和力)。总体而言，这项工作将创建并演示一个 大规模生化表征和分析蛋白质-蛋白质相互作用的平台。