Riboglow: a robust multi-color riboswitch-based platform for imaging RNA in living cells

摘要 信使RNA和非编码RNA的复杂时空动力学几乎影响了基因表达的所有方面。 细胞功能除了作为DNA和蛋白质之间的中间媒介外，RNA还调节 基因在多个水平上表达，在表观遗传调控和基因组组织中发挥作用，并作为 物理支架组装和整合大分子复合物，具有重要意义， 正常发育，以及疾病的病因。然而，尽管RNA在生物学和生长中的重要性 复杂和动态定位模式的证据，活细胞中RNA分子可视化的强大工具 是非常有限的。最广泛使用的RNA标记系统涉及添加24个MS 2茎环， 48个分子的MS 2结合蛋白与GFP融合，添加> 1300个核苷酸和> 2.6 MDa， 感兴趣的RNA。虽然这个系统已经揭示了基因的各个步骤的诱人一瞥， 表达调控，也许并不令人惊讶，它也已被证明扰乱mRNA加工，剪接， 定位和衰变。因此，迫切需要一个强大的，互补的，和最小的干扰， 可视化活细胞中单个RNA分子的工具，以绘制复杂和不断演变的RNA景观 生物学在这项工作中，我们将通过产生一套不同的基于核糖开关的RNA标签来满足这一需求， 相应的荧光探针，用于活体中单个RNA分子的同时多色成像， 哺乳动物细胞我们的方法建立在我们实验室的初步工作基础上， 适体，钴胺素（Cbl）结合核糖开关作为RNA标签，结合一系列Cbl连接的荧光团 以诱导荧光开启，从而照亮感兴趣的RNA。我们把这个新的RNA标签平台 Riboglow，并证明了其能够可视化mRNA和小U1 snRNA在活的哺乳动物细胞。而 与其他染料结合适体和金标准相比，Riboglow的性能令人印象深刻 24 xMS 2系统，还有很大的改进空间。在本提案中，我们将创建Riboglow 2.0， 通过系统地优化RNA/探针平台的模块， (Aim 1）。在三个独立的子目标中，我们将利用核糖开关结构基序的模块化性质， 核糖开关序列的多样性和体外选择的能力以工程化优化的适体-接头对， 具有增强的荧光开启的RNA/探针组合和正交适体/探针对， 用光谱不同的探针同时检测多种RNA。在我们的第二个目标中，我们将创建一个 用于表征、验证和基准测试Riboglow 2.0（Aim 2）的强大而系统的管道。 我们将定义体外生物化学和生物物理特性，细胞对比度和单分子敏感性， 在不同的细胞分析中展示标记不同RNA的功能，并确保最小的细胞毒性 和RNA功能的干扰。将目标1和目标2整合到设计-工程师- 将导致一个功能强大的Riboglow工具箱，用于各种生物应用。

SUMMARY. The complex spatiotemporal dynamics of messenger RNAs and non-coding RNAs affect virtually all aspects of cellular function. In addition to serving as the central intermediary between DNA and proteins, RNAs regulate gene expression at multiple levels, play roles in epigenetic regulation and genome organization, and serve as physical scaffolds to assemble and integrate macromolecular complexes, with important implications for normal development, as well as disease etiology. Yet, despite the importance of RNA in biology and growing evidence of complex and dynamic localization patterns, robust tools for visualizing RNA molecules in live cells are highly limited. The most widely used RNA tagging system involves addition of 24 MS2 stem loops and binding of 48 molecules of the MS2 binding protein fused to GFP, adding > 1300 nucleotides and > 2.6 MDa to an RNA of interest. While this system has revealed tantalizing glimpses at the individual steps of gene expression regulation, perhaps not surprisingly, it has also been shown to perturb mRNA processing, splicing, localization, and decay. Thus, there is a pressing need for robust, complementary, and minimally perturbing tools to visualize individual RNA molecules in living cells to map the complex and evolving landscape of RNA biology. In this work, we will meet this need by generating a suite of diverse riboswitch-based RNA tags and corresponding fluorescent probes for simultaneous, multi-color imaging of individual RNA molecules in live mammalian cells. Our approach builds on preliminary work from our labs that exploits one of nature’s aptamers, the cobalamin (Cbl)-binding riboswitch as an RNA tag that binds a series of Cbl-linked fluorophores to induce fluorescence turn-on, thus lighting up the RNA of interest. We called this new RNA tagging platform Riboglow and demonstrated its ability to visualize mRNA and small U1 snRNA in live mammalian cells. While the performance of Riboglow was impressive compared to other dye binding aptamers and the gold standard 24xMS2 system, there is significant room for improvement. In this proposal, we will create Riboglow 2.0, with dramatically improved properties by systematically optimizing modules of the RNA/probe platform (Aim 1). In three independent subaims, we will exploit the modular nature of riboswitch structural motifs, the diversity of riboswitch sequences and power of in vitro selection to engineer optimized aptamer-linker pairs, RNA/probe combinations with enhanced fluorescence turn-on, and orthogonal aptamer/probe pairs to enable simultaneous detection of multiple RNAs with spectrally distinct probes. In our second aim, we will create a robust and systematic pipeline for characterizing, validating and benchmarking Riboglow 2.0 (Aim 2). We will define in vitro biochemical and biophysical properties, cellular contrast and single molecule sensitivity, demonstrate functionality for tagging different RNAs in diverse cellular assays, and ensure minimal cytotoxicity and perturbation of RNA function. Integration of Aim 1 and Aim 2 into an iterative cycle of design-engineer- characterize will result in a powerful Riboglow toolbox for diverse biological applications.