Neuron-SELEX: Development of neuron-specific nanoscale toolkits for single-cell recognition

Neuron-SELEX：开发用于单细胞识别的神经元特异性纳米级工具包

• 批准号: 10657633
• 负责人: LEONID L MOROZ
• 金额: $ 38.51万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10657633
• 关键词: Ablation; Animals; Antibodies; Aplysia; Axon; Bar Codes; Benzene; Binding; Brain; Cells; Chemical Evolution; Chemicals; Complex; DNA; Data; Diagnostic; Disease; Electroporation; Etiology; Evolution; Experimental Designs; Experimental Models; Fine needle aspiration biopsy; Flare; Fluorescent Probes; Genome; Genomics; Glutamates; Heterogeneity; Hybrids; In Situ Hybridization; In Vitro; Infrastructure; Injections; Interneurons; Knowledge; Label; Libraries; Ligands; Maps; Mass Spectrum Analysis; Membrane; Membrane Proteins; Memory; Micelles; Modeling; Molecular; Molecular Probes; Molecular Target; Motor Neurons; Nervous System; Neuroglia; Neurologic; Neurons; Nociceptors; Nucleic Acids; Organ; Phenotype; Population; Process; Proteomics; Publishing; Reporter; Research; Synapses; System; Technology; Testing; Time; Transgenic Animals; Viral; Visualization; analog; aptamer; base; biomarker discovery; cell type; cholinergic; cost efficient; design; experimental study; fabrication; fluorophore; in vivo; molecular manufacturing; multidisciplinary; nano; nanoprobe; nanoscale; nanotool; neural; neural circuit; neuron development; neuropathology; new technology; novel; nucleic acid analog; nucleobase; optogenetics; personalized medicine; postsynaptic; postsynaptic neurons; presynaptic; presynaptic neurons; receptor; single-cell RNA sequencing; technology development; tool; virtual

Project Summary All neurons are remarkably different and existing approaches do not allow de novo visualization of specific living cells in intact brains, without laborious tasks of making transgenic animals. The project will address this grand challenge: our interdisciplinary team will develop and validate novel nanoscopic probes, to rapidly (<30 min) label specific neurons within highly heterogeneous cell populations. For these applications, in vitro neuronal selection Neuro-SELEX (systematic evolution of ligands by exponential enrichment) will be used to generate libraries of nucleic acid-based probes. These aptamer-based tools will also serve as “pull-out” molecular constructs to identify cell-specific membrane proteins associated with unique neuronal identity and wiring. As a result, this research will provide a broad spectrum of advanced nanotools to decipher the organization of neural circuits at the level of single cells and their compartments. Our preliminary data indicate that the Neuro-SELEX can produce multipurpose toolkits to uniquely map specific neurons or axons without a priori knowledge about their molecular diversity in the intact nervous system. These results, together with our published data, provide the scientific premise for three proof-of-the-concept aims. Arguably, Aplysia is a very powerful experimental model for such technology development. First, to selectively label identified neurons and glial cells, hybrid fluorescent aptamers will be generated using chemical evolution for neuron-specific selection. We will develop a high-throughput cost-efficient system to manufacture molecular probes at a large scale, targeting each key, functionally identified, neuron within a simple-memory forming circuit. Second, we will design fluorescent probes (e.g., modified nucleic acids with fluorophores) for multiplex labeling of several neuronal cell types in vivo. This bar-coding would allow simultaneous visualization of pre- and postsynaptic partners within the same circuit in real time. Furthermore, these probes will be chemically modified to self-deliver molecular constructs into hundreds of target cells without the needs of direct injection, electroporation or making transgenic animals. Third, in proteomic experiments, we will utilize these probes as specific binding tags or ligands to capture and identify membrane proteins specific for each neuronal type of the model circuit including possible synaptic components and receptors. These versatile nanoprobes, with high selectivity and high-throughput fabrication capabilities, will be resourceful to test causality relationships between cellular genomes and complex neuronal phenotypes. Technologies and infrastructure should be applicable to virtually all animal cell types and organs. In perspective, novel fluorescent markers and molecular reporters can be used in early diagnostics and therapy for a broad spectrum of neurological and cell-specific disorders as well as in personalized medicine.

项目摘要 所有神经元都非常不同，现有方法不允许从头可视化 完整大脑中特定的活细胞，没有生产转基因动物的实验室任务。该项目将 应对这个大挑战：我们的跨学科团队将发展并验证新颖的纳米镜问题， 在高度异质细胞群体中快速（<30分钟）的特定神经元。为此 应用，体外神经选择神经螺旋（通过指数对配体的系统演变 富集将用于产生基于核酸的问题的文库。这些基于APATMER的工具 还将用作“拉出”分子构建体，以识别与细胞特异性的膜蛋白 独特的神经元身份和接线。结果，这项研究将提供广泛的高级 纳米方在单个细胞及其隔室的水平上破译神经回路的组织。 我们的初步数据表明神经 - 塞雷克斯可以产生多功能工具包以唯一 绘制特定的神经元或轴突，没有关于其完整分子多样性的先验知识 神经系统。这些结果以及我们已发布的数据提供了三个的科学前提 概念证明的目的。可以说，Aplysia是此类技术的非常强大的实验模型 发展。首先，为了有选择地标记确定的神经元和神经胶质细胞，杂交荧光体适体将 可以使用化学演化进行神经特异性选择产生。我们将开发高通量 具有成本效益的系统，以大规模制造分子问题，针对每个钥匙，功能 在简单的内存形成电路中识别出神经元。其次，我们将设计荧光探针（例如， 用荧光团修饰的核酸）用于体内几种神经元细胞类型的多次标记。这 条形码将允许在同一电路中简单地可视化前和突触后伴侣 实时。此外，这些问题将在化学上修改为自托分子构建体 数百个目标细胞无需直接注射，电穿孔或制造转基因动物。 第三，在蛋白质组学实验中，我们将使用这些问题作为特定的结合标签或配体来捕获 并确定针对模型电路的每种神经元类型的膜蛋白 突触组件和接收器。 这些具有高选择性和高通量制造能力的多功能纳米探针将 足智多谋测试细胞基因组与复杂神经元表型之间的休闲关系。 技术和基础设施应适用于几乎所有动物细胞类型和器官。在 透视图，新颖的荧光标记和分子记者可用于早期诊断和 多种神经和细胞特异性疾病以及个性化医学的治疗。