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

总结。 信使RNA和非编码RNA的复杂时空动力学几乎影响到 细胞功能。除了作为DNA和蛋白质之间的中心中介外，RNAs还调节 基因在多个水平上的表达，在表观遗传调节和基因组组织中发挥作用，并作为 组装和整合大分子络合物的物理支架，具有重要的意义 正常发育，以及疾病病因学。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，用于可视化活细胞中RNA分子的强大工具 是非常有限的。最广泛使用的RNA标签系统包括添加24个MS2茎环和 48个MS2结合蛋白分子与GFP的结合，增加了1300个核苷酸和2.6个丙二醛 一种感兴趣的RNA。虽然这个系统显示了诱人的基因步骤一瞥 表达调控，也许并不令人惊讶，它也被证明扰乱了mRNA的加工，剪接， 本土化和腐朽。因此，迫切需要健壮的、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图景 生物学。在这项工作中，我们将通过生成一套不同的基于核糖开关的RNA标签和 相应的荧光探针可同时对活体单个RNA分子进行多色成像 哺乳动物细胞。我们的方法建立在实验室的初步工作基础上，这些实验室利用了自然界的一种 适配子，钴胺(Cbl)结合的核糖开关，作为RNA标签与一系列Cbl连接的荧光团结合 以诱导荧光开启，从而照亮感兴趣的RNA。我们把这个新的RNA标签平台称为 Riboglow，并证明了其在活的哺乳动物细胞中显示mRNA和小的U1 SnRNA的能力。而当 与其他染料结合适配子和黄金标准相比，Riboglow的性能令人印象深刻 24xMS2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，显著提高了性能 (目标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，即 核糖开关序列的多样性和体外选择以设计优化的适体-连接物对的能力， 具有增强荧光开启功能的RNA/探针组合，以及支持 用光谱不同的探针同时检测多个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Riboglow 2.0(目标2)的强大和系统的流水线。 我们将定义体外生化和生物物理性质、细胞对比度和单分子敏感性， 演示在不同的细胞检测中标记不同RNA的功能，并确保将细胞毒性降至最低 和RNA函数的摄动。将目标1和目标2集成到设计工程师的迭代周期中- Characterize将为不同的生物应用提供一个强大的Riboglow工具箱。

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.