Universally-applicable RNA mapping at subcellular and single-base resolution

项目摘要/摘要 在提高RNA鉴定和定量(即转录组图谱)分辨率方面的每一步变化 已经改变了我们对细胞生物学的理解。作为最新的技术革命， 绘制RNA分子空间分布图的能力揭示了亚细胞RNA的重要性 本地化，提高了我们对细胞和发育生物学的理解，并将单个细胞 转录学领域。然而，尽管最近取得了进展，但目前的RNA图谱工具仅限于 具有长的特定序列的分子(主要是&gt；500 nT)。许多其他重要的RNA物种(如剪接 异构体、miRNA和RNA编辑)，具有更短的特定序列基序，仍然无法在原位获得。我们的 目标是使这一下一个分辨率在转录组学方面跃升，即用高转录-RNA作图- 选择性和单碱基敏感性。我们计划通过开发一种名为Smoy-的新工具来实现这一目标- FISH(单核苷酸特异性、最小偏离目标效应和高产量)，它能够在空间上 在单个细胞中映射所有类型的RNA分子。在这个提案中，我们将展示奶油鱼 从技术开发和实例应用测试两个方面进行阐述。更具体地说，对于技术 开发，我们预计将实现一个平台，可以建议健壮和可靠的光滑鱼探头- 设置为任何指定的RNA靶序列，这是一个用户友好的系统，将被广泛访问。要实现 目标，我们将结合磁镊子的独特力量(单分子工具与纳米和 毫秒级分辨率)，以系统地量化核酸杂交的动力学和动力学 各种条件下，以揭示所设计的光滑鱼探针组形成 所需的二级结构，并建立一个机械建模框架来描述和预测 杂交的动态过程。至于示例应用程序测试，我们将使用Smoy-Fish来研究 具有异构体特异性(即与其线性异构体共存)的环状RNA的神秘生物发生过程 这是第一次。我们预计将揭示定量调控环状RNA表达的可能性，并 高效，即为未来的治疗策略奠定基础。总而言之，这项工作将提供下一个- 用于原位RNA鉴定和量化生成工具，使我们能够定位和量化许多重要的， 但目前无法在单个细胞中获得单分子水平的RNA物种，从而有助于开启一个新时代 从基础科学研究到临床应用。

Project Summary/Abstract Each step-change in increasing resolution on RNA identification and quantification (i.e., transcriptome profiling) has been transformative to our understanding of cell biology. As the most recent technological revolution, the ability to map the spatial distribution of RNA molecules has revealed the importance of subcellular RNA localization, advanced our understanding of cell and developmental biology, and transformed the single cell transcriptomics field. However, despite the recent development, current RNA-mapping tools are limited to molecules with long specific sequence (mostly > 500 nt). Many other important RNA species (such as splicing isoforms, miRNA, and RNA editing), with much shorter specific sequence motif, remain inaccessible in situ. Our goal is to make this next resolution jump in profiling transcriptomics, i.e., RNA-mapping with high transcript- selectivity and single base-sensitivity. We plan to achieve the goal by developing a new tool, named SMOOTHY- FISH (Single-nucleotide specificity, Minimum Of Off-Target effects, and High Yield), which enables spatially mapping all types of RNA molecules in individual cells. In this proposal, we will demonstrate SMOOTHY-FISH from two aspects: technique development and one example application test. More specifically, for the technical development, we anticipate achieving a platform that can suggest robust and reliable SMOOTHY-FISH probe- sets to any assigned RNA target sequence, a user-friendly system that will be widely accessible. To achieve the goal, we will incorporate the unique power of magnetic tweezers (a single molecule tool with nanometer and milliseconds resolution) to systemically quantify the dynamics and kinetics of nucleic acid hybridization under various conditions, to reveal how fast and how stable the designed SMOOTHY-FISH probe-sets can form the needed secondary structure, and to build a mechanistic modeling framework to describe and predict the hybridization dynamic process. As for the example application test, we will use SMOOTHY-FISH to study the mystery biogenesis process of circular RNA with isoform-specificity (i.e., co-existence with its linear isoforms) for the first time. We anticipate revealing the potential to modulate circular RNA expression quantitatively and efficiently, i.e., lay a foundation for therapeutic strategies in the future. Together, this work will provide a next- generation tool for in situ RNA identification and quantification, allow us to localize and quantify many important, but currently inaccessible RNA species at the single molecule level in individual cells, thus help open a new era of RNA biology, from basic science research to clinical applications.