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.

抽象的 高通量DNA测序革命使核酸的规模匹配 人类生物学的数量和复杂性。但是，目的地期望与众不同 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低至中等吞吐量。尽管存在多重蛋白质方法，但它们 倾向于仅由少数“专家”担任雇员，因为1）他们尚未达到巨大 DNA测序方法的可伸缩性，以及2）它们在技术上挑战了。 Protillion是 商业化直接基于无处不在的Illumina的高通量蛋白分析的平台 音序器，展示了将蛋白质功能表征带入高通量时代的途径。 该蛋白质映射平台（Prot-MAP）使数千万固定的簇产生 蛋白质通过有效的原位转录和翻译直接在DNA测序流中。 然后可以直接在该蛋白阵列上进行基于荧光的蛋白质功能测定，结果结果 通过高度敏感的荧光成像量化。 Prot-Map可以产生巨大的突变 肽和全长功能蛋白的数据集，可以进行突变的高通量分析 基于蛋白质功能的直接生物物理观察结果。与定向的进化方法不同 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 单个实验的自动化，广泛分布的仪器的时间。我们将部署Prot- 以两种不同模式的地图平台 - 一种使用小约15个氨基酸胡椒粉作为亲和力试剂，而 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结构域或Tigit蛋白的细胞免疫受体。 Tigit是一个理想的示例目标，因为它是 高度固体，可商购，并具有诊断性（用于检测HIV进展）和治疗（在 在癌症免疫疗法中结合PD1阻滞）应用潜力。我们计划分析我们的高 吞吐量数据集1）与理性相比，了解Petides预先选择的好处 Pepperide图书馆的工程，2）识别构成不同家族的辣椒和纳米生物的进化枝 粘合剂，3）测试这些不同家族的鉴定，这些家族在实验上结合了在Tigit上结合不同的表位， 4）评估结合的盐和温度依赖性，5）检查特定钥匙的依赖性 使用结构信息肽结合的肽。结合不同的纳米生物和胡椒粉的识别 Tigit地区将为建立三明治风格的亲和力测定和高亲和力打开大门 配体（通过自然）进行潜在的治疗应用。总体而言，这项工作将创建并证明 大规模生化表征和蛋白质蛋白质相互作用的分析平台。