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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

基本信息

批准号：
10471341
负责人：
LEONID L MOROZ
金额：
$ 40.54万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
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/10471341
关键词：
Ablation Address Animals Antibodies Aplysia Axon Bar Codes Benzene Binding Brain Cells Chemical Evolution Chemicals Complex DNA Data Diagnostic Disease Electroporation Etiology Evolution 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 structure 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 fluorophore in vivo molecular manufacturing multidisciplinary nanoprobe nanoscale nanotool neural circuit neuron development neuropathology new technology novel nucleic acid analog nucleobase optogenetics personalized medicine postsynaptic postsynaptic neurons receptor relating to nervous system single-cell RNA sequencing technology development tool virtual

项目摘要

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.

项目总结