Development of DNA-Labeled Antibody Mimetics as Low-Cost, High-Performance Reagents for Single Cell Analysis of Membrane Proteins

开发 DNA 标记抗体模拟物作为膜蛋白单细胞分析的低成本、高性能试剂

• 批准号: 10321090
• 负责人: Richard John Suderman
• 金额: $ 25.25万
• 依托单位: NECTAGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321090
• 关键词: Adopted; Adoption; Animals; Antibodies; Antigens; Automobile Driving; Bacteria; Binding; Binding Sites; Biological; Cell Surface Proteins; Cell surface; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Chemistry; Communities; Complex; Custom; Cysteine; DNA; Data; Detection; Development; Equipment; Escherichia coli; Event; Generations; Genomics; Immune; Immunize; Individual; Label; Length; Libraries; Measurement; Measures; Membrane Proteins; Messenger RNA; Noise; Oligonucleotides; Performance; Phage Display; Population; Population Heterogeneity; Procedures; Protein Analysis; Proteins; Publications; Publishing; RNA analysis; Reaction; Reagent; Research; Signal Transduction; Site; Technology; Testing; Variant; Work; antibody detection; base; cost; cost estimate; design; experimental study; genome-wide; genome-wide analysis; mimetics; monomer; multiple omics; nanobodies; next generation sequencing; novel strategies; scale up; single cell analysis; single-cell RNA sequencing; success; tool; transcriptome sequencing

Averaging signals over a population of cells often misses biologically relevant variation among single cells. New approaches are emerging with the power to identify differences between individual cells that are not detectable in studies of entire populations. One approach, called single‐cell RNA‐seq, has rapidly been adopted in the scientific community. Single‐cell RNA‐seq is now increasingly performed simultaneously with the measurement of protein levels. Recent technologies use DNA‐labeled antibodies to measure levels of 10‐100 proteins in the context of a genome‐wide analysis. However, these technologies have not yet been widely adopted because of the cost and effort required and the relatively small number of proteins that can be measured. Significant cost and effort are required to prepare DNA‐labeled antibodies which have limited commercial availability. Furthermore, results with antibodies are sometimes imprecise because antibodies have multiple binding sites for their targets and multiple conjugation sites for their DNA‐label. We propose to develop a technology platform optimized for DNA‐based detection of proteins in single‐cell experiments. The core of our platform is a potent and selective single domain antibody mimetic called a nanoCLAMP that can be customized for specific applications and produced inexpensively. For single‐cell applications, nanoCLAMPs have the potential for higher performance and lower cost than traditional antibodies. Unlike traditional antibodies, binding events are straightforward to interpret because nanoCLAMPs are monomers with a single target binding site and a single site for DNA‐labeling. nanoCLAMPs are also economical to develop and use because they can be isolated without the need to immunize animals and can be produced in bacteria at high levels. This work proposes 3 specific aims: 1. Generate a test set of 5‐10 nanoCLAMPs against cell surface proteins. 2. Optimize DNA labeling and cell‐binding conditions. 3. Compare the performance of nanoCLAMPs to traditional antibodies in single‐cell experiments and assess the feasibility of scale‐up. Successful completion of these aims will establish the feasibility of a technology platform with the potential to accelerate the adoption of simultaneous single‐cell RNA and protein analyses.

对细胞群上的信号进行平均通常会遗漏单细胞之间的生物相关变化。新 新出现的方法能够识别个体细胞之间无法检测到的差异， 在整个人群的研究中。一种称为单细胞RNA测序的方法已迅速被采用， 科学界。单细胞RNA测序现在越来越多地与测序同时进行 测量蛋白质水平。最近的技术使用DNA标记的抗体来测量10 - 100 蛋白质在全基因组分析的背景下。然而，这些技术尚未广泛应用。 由于所需的成本和努力以及可测量的蛋白质数量相对较少，因此采用了这种方法。 需要大量的成本和努力来制备具有有限商业价值的DNA标记的抗体。 空房的此外，抗体的结果有时是不精确的，因为抗体具有多个 其靶点的结合位点和其DNA标记的多个缀合位点。 我们建议开发一种优化的技术平台，用于基于DNA的单细胞蛋白质检测。 实验我们平台的核心是一种有效的和选择性的单域抗体模拟物，称为 nanoCLAMP可针对特定应用进行定制，生产成本低廉。对于单细胞 应用，nanoCLAMP具有比传统的更高的性能和更低的成本的潜力。 抗体的与传统抗体不同，结合事件可以直接解释，因为nanoCLAMP 是具有单个靶结合位点和单个DNA标记位点的单体。nanoCLAMP还 因为它们可以在不需要免疫动物的情况下被分离，并且可以 在细菌中大量产生。这项工作提出了三个具体目标： 1.生成针对细胞表面蛋白的5 - 10个nanoCLAMP的测试集。 2.优化DNA标记和细胞结合条件。 3.在单细胞实验中比较nanoCLAMP与传统抗体的性能，并评估 扩大规模的可行性。 成功完成这些目标将确立一个技术平台的可行性， 加速采用同时进行单细胞RNA和蛋白质分析。