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

项目摘要 所有的神经元都是非常不同的，现有的方法不允许从头可视化 在完整的大脑中的特定的活细胞，而不需要费力的任务，使转基因动物。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针， 快速（<30分钟）标记高度异质细胞群中的特异性神经元。为这些 Neuro-SELEX（通过指数配体的系统进化， 富集）将用于产生基于核酸的探针的文库。这些基于适体的工具 也将作为“拉出”分子构建体，以鉴定与以下相关的细胞特异性膜蛋白： 独特的神经元特性和连接因此，这项研究将提供广泛的先进的 纳米工具来破译神经回路的组织在单细胞和它们的隔间的水平。 我们的初步数据表明，Neuro-SELEX可以生产多用途工具包， 映射特定的神经元或轴突，而不需要关于它们的分子多样性的先验知识， 神经系统这些结果，加上我们发表的数据，为三个方面提供了科学前提。 概念验证目标。可以说，Aesthesia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，混合荧光适体将 是通过化学进化产生的神经元特异性选择。我们将开发一种高通量的 大规模生产分子探针的成本效益高的系统，针对每个关键，功能 一个简单的记忆形成电路中的神经元。其次，我们将设计荧光探针（例如， 具有荧光团的修饰的核酸）用于体内多种神经元细胞类型的多重标记。这 条形码可以同时显示同一回路中的突触前和突触后伙伴 在真实的时间里。此外，这些探针将被化学修饰以将分子构建体自递送到细胞中。 数百个靶细胞，而无需直接注射、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 并识别对模型电路的每种神经元类型特异的膜蛋白， 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， 要善于测试细胞基因组和复杂神经元表型之间的因果关系。 技术和基础设施应适用于几乎所有动物细胞类型和器官。在 因此，新的荧光标记物和分子报告物可用于早期诊断， 用于治疗广泛的神经和细胞特异性疾病以及个性化医疗。

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.