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

基本信息

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
项目类别：
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

项目摘要

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.

对细胞群的信号进行平均常常会忽略单个细胞之间的生物学相关变化。正在出现的方法能够识别单个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 测序的方法已被迅速采用。科学界现在越来越多地与单细胞 RNA 测序同时进行。最近的技术使用 DNA 标记的抗体来测量 10-100 的水平。然而，这些技术尚未得到广泛应用。由于所需的成本和精力以及可测量的蛋白质数量相对较少而被采用。制备 DNA 标记抗体需要大量成本和精力，但商业化有限此外，抗体的结果有时不精确，因为抗体有多种。其靶标的结合位点和 DNA 标记的多个缀合位点。我们建议开发一个针对单细胞中基于 DNA 的蛋白质检测进行优化的技术平台我们平台的核心是一种有效的、选择性的单域抗体模拟物，称为“a”。 nanoCLAMP 可针对特定应用进行定制，并且生产成本低廉，适用于单细胞。与传统应用相比，nanoCLAMP 具有更高的性能和更低的成本的潜力与传统抗体不同，由于 nanoCLAMP，结合事件很容易解释。是具有单个靶结合位点和单个 DNA 标记位点的单体。开发和使用经济，因为无需对动物进行免疫即可分离它们，并且可以这项工作提出了 3 个具体目标： 1. 生成针对细胞表面蛋白的 5-10 个 nanoCLAMP 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建立一个具有潜力的技术平台的可行性加速同步单细胞 RNA 和蛋白质分析的采用。