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.

项目概要 所有神经元都显着不同，现有方法不允许从头可视化 完整大脑中的特定活细胞，无需繁琐的制作转基因动物的任务。该项目将 应对这一巨大挑战：我们的跨学科团队将开发和验证新型纳米探针， 快速（<30 分钟）标记高度异质性细胞群中的特定神经元。对于这些 应用，体外神经元选择 Neuro-SELEX（配体的指数系统进化 富集）将用于生成基于核酸的探针文库。这些基于适体​​的工具 还将作为“拉出”分子构建体来识别与相关的细胞特异性膜蛋白 独特的神经元身份和接线。因此，这项研究将提供广泛的先进技术 纳米工具在单细胞及其隔室水平上破译神经回路的组织。 我们的初步数据表明，Neuro-SELEX 可以生产多用途工具包，以独特的方式 绘制特定神经元或轴突的图谱，无需先验了解其完整的分子多样性 神经系统。这些结果与我们发表的数据一起为三个方面提供了科学前提 概念验证的目标。可以说，Aplysia 是此类技术的一个非常强大的实验模型 发展。首先，为了选择性地标记已识别的神经元和神经胶质细胞，混合荧光适体将 使用化学进化来生成神经元特异性选择。我们将开发高通量 具有成本效益的系统，可大规模制造分子探针，针对每个关键功能 识别出简单记忆形成电路中的神经元。其次，我们将设计荧光探针（例如， 带有荧光团的修饰核酸）用于体内多种神经元细胞类型的多重标记。这 条形码将允许同一电路内的突触前和突触后伙伴同时可视化 实时。此外，这些探针将被化学修饰以将分子构建体自我传递到 数百个靶细胞，无需直接注射、电穿孔或制作转基因动物。 第三，在蛋白质组学实验中，我们将利用这些探针作为特异性结合标签或配体来捕获 并识别模型电路每种神经元类型特异的膜蛋白，包括可能的 突触成分和受体。 这些多功能纳米探针具有高选择性和高通量制造能力，将 足智多谋地测试细胞基因组和复杂神经元表型之间的因果关系。 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 从长远来看，新型荧光标记物和分子报告基因可用于早期诊断和 治疗广泛的神经系统和细胞特异性疾病以及个性化医疗。