High-Throughput Droplet-Scale Functional Screening of DNA-Encoded Combinatorial Libraries

DNA 编码组合文库的高通量液滴规模功能筛选

• 批准号: 10004373
• 负责人: Brian M Paegel
• 金额: $ 19.39万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10004373
• 关键词: Academia; Architecture; Automation; Bacteria; Biochemical; Biological Assay; Biological Process; Cathepsins; Cell physiology; Cleaved cell; Code; Collection; Complement; Consumption; DNA; Data; Devices; Dose; Drug Industry; Equipment; Exhibits; FDA approved; Face; Fluorescence; Frequencies; Goals; Government; HIV-1 protease; High-Throughput Nucleotide Sequencing; In Vitro; Incubated; Individual; Industrialization; Informatics; Infrastructure; Label; Laboratories; Libraries; Location; Logistics; Microfluidics; Microscopy; Miniaturization; Mission; Modeling; Molecular; Molecular Bank; Multi-Drug Resistance; Nelfinavir; Optics; Output; Pepstatins; Peptide Hydrolases; Phase; Phenotype; Process; Protease Inhibitor; Proteome; PubChem; Quality Control; Reaction; Reagent; Renin; Resistance; Resources; Robotics; Ships; Solid; Sorting - Cell Movement; Source; Structure; Structure-Activity Relationship; Technology; Translating; Triage; United States National Institutes of Health; Validation; Viral; Viral Cell Proliferation; Virus; base; combinatorial; cost; data acquisition; diphenyl; drug discovery; genome sequencing; high throughput screening; human disease; human pathogen; inhibitor/antagonist; instrument; instrumentation; member; miniaturize; mutant; new therapeutic target; novel therapeutics; operation; pathogen; portability; programs; prototype; reconstitution; response; screening; small molecule; square foot; statine; tool

Project Summary / Abstract The NIH established the Molecular Libraries Program (MLP) and its network of high-throughput screening (HTS) centers to discover probes – highly selective small molecules that modulate cellular function – within the proteomes of humans and pathogens. Probes are not only tools for studying biological function to validate new drug targets, but are also potential leads for new therapeutics. Though successful in its mission to provide HTS resources to academia and having generated hundreds of probes, the MLP is sunsetting and its operational screening centers face the logistic and financial issues that industrial HTS centers have battled for decades (large facilities, costly robotic handling and optical screening equipment upkeep, static compound libraries). Proteome-wide and pathogen-wide probe discovery remains a compelling goal well within reach thanks to distributed and economical genome sequencing technology, which inspires this proposal to develop a similarly distributable molecular screening platform. Combining droplet-scale microfluidic miniaturization and automation with consumable DNA-encoded solid-phase compound libraries comprises a proposed bid to reconstitute the operations of a HTS center in a single benchtop instrument. The device loads compound library beads into picoliter-scale droplets of assay reagent, photochemically cleaves the compound from the bead, incubates the dosed droplets, reads the fluorescence of the incubated droplets, and sorts droplets exhibiting a desired assay fluorescence profile for collection and high-throughput sequencing. The instrument will (1) screen a million compound library in ~6 h, (2) require several square feet of space, (3) consume ~100 µL of assay reagent, and (4) generate dose-response screening data, resulting in massively parallel pan-library structure-activity relationship profiles. This technology will distribute molecular screening, moving it into academic, industrial and government laboratories nationwide, and on a cost scale that will enable discovery of thousands of probes annually.

项目摘要 /摘要 NIH建立了分子图书馆计划（MLP）及其高通量筛选网络 （HTS）发现问题的中心 - 在调节细胞功能的高度选择性的小分子 - 内部 人类和病原体的蛋白质组织。探针不仅是研究生物功能以验证的工具 新药物靶标，但也是新疗法的潜在潜在客户。虽然成功地提供了 HTS资源用于学术界并产生了数百个问题，MLP正在日落及其 运营筛查中心面临着工业HTS中心争夺的物流和财务问题 数十年（大型设施，昂贵的机器人处理和光学筛选设备维护，静态化合物 图书馆）。全蛋白质组和整个病原体的探针发现仍然是一个令人信服的目标 感谢分布式和经济基因组测序技术，这激发了这一建议 类似的分布分子筛选平台。结合液滴尺度的微流体小型化 和使用易于DNA编码的固相化合物库的自动化，包括提议的竞标 重建单个台式仪器中HTS中心的操作。设备加载化合物 图书馆珠子进入测定试剂的Picoliter-Scale-Scale-Scale-Scale-Scales滴，光化学从 珠子，孵育剂量的液滴，读取孵化的液滴的荧光，并分类液滴 表现出所需的测定荧光曲线，用于收集和高通量测序。乐器 （1）屏幕在约6 h，（2）需要几平方英尺的空间，（3）消耗〜100 µL测定试剂，（4）产生剂量反应筛选数据，导致大量平行泛曲线 结构活性关系曲线。该技术将分发分子筛选，将其移至 全国各地的学术，工业和政府实验室，并以成本量表为基础，可以发现 每年数千个问题。