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

项目总结/摘要 在RNA鉴定和定量上增加分辨率的每一步变化（即，转录组分析） 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 定位，推进了我们对细胞和发育生物学的理解，并改变了单细胞 转录组学领域。然而，尽管最近的发展，目前的RNA作图工具仅限于 具有长特异性序列的分子（大多数> 500 nt）。许多其他重要的RNA种类（如剪接 异构体、miRNA和RNA编辑），具有短得多的特异性序列基序，在原位仍然不可接近。我们 我们的目标是在转录组学分析中实现下一个分辨率的飞跃，即，高转录水平的RNA图谱- 选择性和单碱基敏感性。我们计划通过开发一个名为SMOOTHY的新工具来实现这一目标- FISH（单核苷酸特异性，脱靶效应最小化和高产率），这使得空间 绘制单个细胞中所有类型的RNA分子。在本提案中，我们将演示SMOOTHY-FISH 从技术开发和实例应用测试两个方面进行了分析。更具体地说，对于技术 开发，我们预计实现一个平台，可以建议强大和可靠的SMOOTHY-FISH探针- 设置到任何指定的RNA靶序列，这是一个用户友好的系统，将被广泛访问。实现 目标，我们将结合独特的权力磁镊子（单分子工具，纳米和 毫秒分辨率）来系统地量化在微环境下核酸杂交的动力学和动力学。 各种条件下，揭示如何快速和如何稳定的设计光滑鱼探针集可以形成 所需的二级结构，并建立一个机制建模框架来描述和预测 杂交动态过程至于示例应用程序测试，我们将使用SMOOTHY-FISH来研究 具有同种型特异性的环状RNA的神秘生物发生过程（即，与其线性异构体共存） 第一次我们期望揭示定量调节环状RNA表达的潜力， 有效地，即，为今后的治疗策略奠定基础。总之，这项工作将提供一个下一个- 用于原位RNA鉴定和定量的生成工具，使我们能够定位和定量许多重要的， 但目前无法在单个细胞的单分子水平上获得的RNA种类， 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.