Water Soluble Nanoarrays for Single Cell Proteomics

描述（由申请人提供）：单细胞基因组学的显著进展依赖于聚合酶链反应。目前还没有类似的工具可以在少数细胞或单细胞水平上探测蛋白质多样性和翻译后修饰（PTM）模式的细胞间分布。如果蛋白质捕获阵列可以在分子尺度上合成，那么阵列本身就可以成为适合与单个细胞内容物孵育的试剂。分子阵列可以用于滴定，提供潜在的巨大的动态检测范围，并促进对单个亲本细胞每一代后代的蛋白质含量和PTMs的定量。纳米技术已经为这种系统提供了组件，我们建议整合四种新技术来开发用于蛋白质分析的自组装纳米阵列。首先是利用核酸自组装技术构建纳米密度单分子探针阵列。这些阵列本身就是巨大的分子，在溶液分析中像试剂一样使用，只有在最终读数时才会沉积在表面上。第二个是使用原子力显微镜（AFM）的纳米级读数。AFM每分钟能够读出50万个阵列位置。三是基于核酸适体和多价核酸适体的蛋白质及其PTM变异分子识别元件的开发。由于适体是人工合成的，也是核酸序列，因此单个适体可以通过核酸杂交结合到阵列上的独特位置，无需印刷或光刻。第四个是微流体技术，它允许纳米阵列与裂解或完整的细胞相互作用，并将反应阵列送到预定的位置进行读出。我们的目标是填补蛋白质组学中一个独特的利基：从少量（可能是单个）细胞中平行分析微量蛋白质。我们能制造合适的配体吗？它们能在阵列上工作吗？我们能用原子力显微镜准确地读取阵列吗？我们能合成对翻译后修饰具有高度选择性的配体吗？什么因素导致了阵列的生物降解，我们如何在保持灵敏度和选择性的同时控制它们？在微流体系统中利用蛋白质组学阵列需要什么条件？我们可以采用什么方法将反应阵列的纳升溶液沉积在AFM读出的精确位置？这些问题是本提案的重点。如果成功，这项工作将有可能将核酸多样性与相应的蛋白质PTM多样性在细胞基础上联系起来，首次产生基因组，基因表达，环境和蛋白质翻译后修饰谱之间相互作用的数据：“蛋白质语言”。公共卫生相关性：探测蛋白质多样性的细胞间分布将为了解生物体和许多疾病的发展提供关键信息。我们的目标是开发一种水溶性纳米阵列系统，打开单细胞分析生物化学领域的蛋白质领域，发现新的知识，这将影响我们对细胞如何工作和癌症等病理如何发展的理解。这项新技术将使我们对细胞的系统生物学有更全面的认识。

DESCRIPTION (provided by applicant): Remarkable progress in single cell genomics rests on the polymerase chain reaction. There is no analogous tool to probe the cell-to-cell distribution of protein diversity and posttranslational modification (PTM) patterns at the few- or single-cell level. If protein-capture arrays can be synthesized on a molecular scale, then the arrays themselves become reagents suitable for incubation with the contents of even a single cell. Molecular arrays could be used in titrations, giving a potentially enormous dynamic detection range and facilitating quantification of the protein content and PTMs for each generation of the progeny of a single parent cell. Nanotechnology already provides the components for such a system, and we propose to integrate four new technologies to develop self-assembled nanoarrays for protein analysis. The first is construction of an array of single molecule probes arranged at nanometer density using nucleic acid self-assembly. The arrays are themselves giant molecules and are used like a reagent in a solution analysis, only being deposited onto a surface for a final readout. The second is a nanoscale readout using atomic force microscopy (AFM).The AFM is capable of reading out >30,000 array sites per minute. The third is the development of new molecular recognition elements based on aptamers and multivalent aptamers for proteins and their PTM variants. Because aptamers are synthetic and also nucleic acid sequences, individual aptamers can bind to a unique position on the array through nucleic acid hybridization and require no printing or lithography. The fourth is microfluidic technology to allow the nanoarrays to interact with lysed or intact cells, and to deliver the reacted arrays to predefined locations for readout. Our goal is to fill a unique niche in proteomics: parallel analysis of minute amounts of protein from small numbers of (potentially individual) cells. Can we make suitable ligands and will they work on arrays? Can we read the arrays accurately with AFM? Can we synthesize ligands that are highly selective for posttranslational modifications? What factors cause biodegradation of the arrays, and how can we control them while maintaining sensitivity and selectivity? What conditions are required to exploit the arrays for proteomics in a microfluidic system? What methodologies can we employ to deposit nanoliter solutions of reacted arrays at precise locations for AFM readout? These questions are the focus of this proposal. If successful, this work will make it possible to correlate nucleic acid diversity with the corresponding protein PTM diversity on a cell-by- cell basis, yielding, for the first time, data on the interplay between genome, gene expression, the environment and the spectrum of protein posttranslational modifications: the 'protein language'. PUBLIC HEALTH RELEVANCE: Probing the cell-to-cell distribution of protein diversity will provide critical information for understanding the development of living organisms and many diseases. Our goal is to develop a water soluble nanoarray system to open up to single-cell analysis the protein realm of the biochemical universe, uncovering new knowledge that will impact our understanding of how cells work and of how pathologies like cancer develop. This new technology will afford us a fuller view of the systems biology of the cell.