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，现有的方法不允许从头开始可视化 在完整的大脑中获得特定的活细胞，而不需要费力地制造转基因动物。该项目将 解决这一重大挑战：我们的跨学科团队将开发和验证新型纳米探测器， 快速(&lt；30分钟)标记高度异质细胞群中的特定神经元。为了这些 应用，体外神经元选择Neuro-SELEX(指数配基的系统进化 浓缩)将被用来产生基于核酸的探针库。这些基于适体的工具 也将作为“拉出”的分子结构来识别细胞特异性膜蛋白与 独一无二的神经元识别和连接。因此，这项研究将提供广泛的高级 在单个细胞及其隔间水平上破译神经回路组织的纳米工具。 我们的初步数据表明，Neuro-SELEX可以生产出独特的多用途工具包 在没有先验知识的情况下绘制特定神经元或轴突的完整分子多样性图 神经系统。这些结果，加上我们公布的数据，为以下三个问题提供了科学前提 概念验证目标。可以说，海兔是这种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，杂交荧光适体将 是通过化学进化产生的，用于神经元特定的选择。我们将开发一种高吞吐量的 经济高效的系统，用于大规模制造分子探针，在功能上针对每个键 识别，神经元内的一个简单的记忆形成电路。第二，我们将设计荧光探针(例如， 带有荧光团的修饰核酸)，用于体内几种神经细胞类型的多重标记。这 条形码将允许在同一电路中同时显示突触前和突触后的伙伴 实时的。此外，这些探针将经过化学修饰，以自我传递分子结构到 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定的结合标签或配体来捕获 并识别模型电路的每种神经元类型特有的膜蛋白，包括可能的 突触组件和受体。 这些多功能的纳米探测器，具有高选择性和高通量制造能力，将 善于测试细胞基因组和复杂神经元表型之间的因果关系。 技术和基础设施应该适用于几乎所有动物细胞类型和器官。在……里面 透视法、新型荧光标记和分子报告器可用于早期诊断和 治疗范围广泛的神经性和细胞特异性疾病，以及个性化医学。