RNA Scaffolds for Cell Specific Multiplexed Neural Observation

遗传编码的分子工具正在对整个神经科学产生影响。然而，每一个基因 编码的分子工具是蛋白质工程的壮举，需要大量的努力来设计和优化。一 吸引人的想法是，有可能创造出少量的模块化蛋白质构建块， 然后可以按照模式进行组织，并通过搭建来实现用户可编程的功能， 在RNA链上。我们最近从Pumilio同源结构域（PumHD）蛋白中获得了4 构建块，每个构建块偏好RNA的4种碱基中的一种，使得对于细胞中的任何感兴趣的RNA， 可以创建由定义序列中的4个构建块组成的链以结合到该RNA（带来 沿着与该链融合的任何蛋白质有效载荷）。我们建议将这种基于Pumilio（或“Pum”）的 简称）的战略，以创造RNA支架分子工具，解决两个主要类别的问题， 神经科学-基因表达靶向哺乳动物大脑中的特定细胞类型， 特异性，而不需要转基因动物（目标1），以及成像多个独立的能力， 在没有串扰的情况下，单个神经元内的生理活性的荧光报告子（Aim 2）。我们将 在小鼠（目标1，2）和非遗传模型生物体中评估和优化这些支架分子工具 （猕猴），旨在开辟神经科学实验的新前沿（目标3）。我们的补助金很快- paced，4年的赠款，汇集了神经技术开发专家（博伊登），灵长类动物 系统神经科学（Desimone）和核酸技术（Adamala）。我们将免费分享所有工具， 神经科学界，以广泛加速神经科学的进步。

Genetically encoded molecular tools are having impact throughout neuroscience. However, each genetically encoded molecular tool is a feat of protein engineering, requiring extensive effort to design and optimize. One appealing thought is that it might be possible to create a small number of modular protein building blocks, which then could be organized in patterns, and achieve user-programmable functions, by being scaffolded together on strands of RNA. We recently derived from the Pumilio homology domain (PumHD) protein, 4 building blocks, each of which prefers one of the 4 bases of RNA, so that for any RNA of interest in a cell, a chain made out of the 4 building blocks in a defined sequence can be created to bind to that RNA (bringing along any protein payloads that are fused to that chain). We propose to adapt this Pumilio based (or “Pum” for short) strategy to create RNA-scaffolded molecular tools that address two major classes of problem in neuroscience – the targeting of gene expression to specific cell types in the mammalian brain, with high specificity, without requiring transgenic animals (Aim 1), and the ability to image multiple independent fluorescent reporters of physiological activity within individual neurons without crosstalk (Aim 2). We will assess, and optimize, these scaffolded molecular tools in mice (Aims 1, 2) and a non-genetic model organism (macaques), aiming to open up new frontiers of neuroscience experimentation (Aim 3). Our grant is a fast- paced, 4-year grant, which brings together experts in neurotechnology development (Boyden), primate systems neuroscience (Desimone), and nucleic acid technology (Adamala). We will share all tools freely with the neuroscience community, in order to broadly accelerate neuroscience progress.