Recording neural activities onto DNA

描述（由申请人提供）：神经记录的进展对于理解大脑和开发大脑疾病的治疗方法至关重要。目前的神经记录最多只能捕捉几百个相互作用的神经元。记录的神经元数量相对较少，因为目前的神经记录设备，如电极、放大器、激光和照相机，都是宏观的。我们的研究目标是在分子尺度上创造神经记录器，通过将神经活动记录到DNA上，就像分子自动收报带一样。该装置将由一种经过改造的DNA聚合酶组成，它可以廉价地合成并容易地传递到神经元中，在那里，它将把每个神经元活动的时间动态记录到局部DNA分子上，之后可以通过越来越便宜的基因组测序技术对其进行分析。我们研究的长期目标是实现范式转变，使记录仪器自由，易于使用，并可扩展到任意数量的神经元。我们将通过三个管道获得纳米级记录设备：(1)聚合酶设计管道。我们将在不同的DNA聚合酶中寻找一种聚合酶，当离子浓度增加时，这种聚合酶会产生许多复制错误，因此当神经元活跃时。我们将使用定向蛋白工程来添加离子敏感结构域。最后，我们将利用高通量蛋白质定向进化，生产具有理想性能的聚合酶。(2)模板设计流水线。我们将设计并传递一个工程DNA模板到待复制的细胞。我们将利用转染，这是可行的，但在一些神经科学实验中可能不方便，后来转向病毒模板传递方法，这可能更简单。(3)统计管道。得到的DNA序列需要转换回具有神经生物学意义的信号。这种转换需要精确，对诸如生物聚合酶噪声和纠错等各种问题具有鲁棒性。这种方法是创新的，因为它重新定义了使用分子工程记录的概念，从而生产出一种比同类设备小几个数量级的设备，而且可以说比同类设备更通用。这项提议的研究意义重大，因为它允许一系列新的电生理实验。该方法将补充其他新兴方法，这些方法有望导致基于大型数据集的神经科学，例如连接组学。由此产生的技术将易于使用且价格低廉，并且有望允许同时记录潜在任意数量的神经元，其时间精度可与现有最先进的钙成像相媲美。它承诺大量增加神经数据和全新的方法来深入研究大脑的工作方式和如何治疗大脑疾病。

DESCRIPTION (provided by applicant): Progress in neural recording is critical to understanding the brain and developing treatments for brain disorders. Current neural recordings can, at best, capture a few hundred interacting neurons. The number of recorded neurons is relatively small because current neural recording devices, such as electrodes, amplifiers, lasers, and cameras, are macroscopic. The objective of our research is to create neural recorders at the molecular scale, by writing neural activities onto DNA, like a molecular ticker tape. The device will consist of an engineered DNAP polymerase that can be cheaply synthesized and easily delivered to neurons, where it will write the temporal dynamics of activity of each neuron onto local DNA molecules, which can later be analyzed via increasingly cheap genome sequencing technologies. The long term goal of our research is to enable a paradigm shift, making recording instrumentation-free, easy to use, and scalable to arbitrary numbers of neurons. We will obtain the nanoscale recording device using three pipelines: (1) Polymerase design pipeline. We will search through different DNA polymerases to find a polymerase that makes many replication mistakes when ion concentrations increase, and thus when neurons are active. We will use directed protein engineering to add ion-sensitive domains. Lastly we will use high throughput protein directed evolution, to produce a polymerase with desirable properties. (2) Template design pipeline. We will design and deliver an engineered DNA template to the cell to be copied. We will utilize transfection, which is feasible but might not be convenient in some neuroscientific experiments, moving later towards viral template delivery methods, which may be simpler. (3) Statistics pipeline. The resulting DNA sequences need to be converted back into signals of neurobiological meaning. Such conversion needs to be precise, robust to various problems such as biological polymerase noise, and error-correcting. The approach is innovative, because it reinvents the concept of recording using molecular engineering to produce a device that is orders of magnitude smaller and arguably more versatile than comparable devices. The proposed research is significant, because it allows a whole range of new electrophysiological experiments. The approach will complement other emerging approaches that promise to lead to large dataset based neuroscience, e.g. connectomics. The resulting technique will be easyto- use and inexpensive, yet will promise to allow recording simultaneously from potentially arbitrary numbers of neurons, with temporal precision comparable to existing state-of-the-art calcium imaging. It promises massively increased amounts of neural data and entirely new approaches to asking deep questions about the way the brain works and how to cure disease of the brain.