Mapping the sequence landscape of RNA structure, dynamics and protein interactions using high-throughput single-molecule FRET

项目摘要 RNA在几乎所有的生物过程中都扮演着中心角色，反过来又充当信使， 催化剂、信号分子等等。这些角色中的许多都需要RNA折叠成特定的 结构，并且给定的RNA物种通常可以采用多种结构来调节特性 如蛋白质相互作用、配体结合和催化活性。Widom实验室中的研究 重点开发和应用新的光谱方法来研究RNA结构和 动力学。我们将体谱、单分子光谱和超快光谱相结合，以获得 RNA折叠和相互作用的全景图-从Angstroms到 微米和时间尺度--短至皮秒。我们目前正在使用这些方法来研究 前信使RNA(Pre-mRNA)剪接过程中的RNA-蛋白质相互作用 不编码蛋白质的RNA片段被切除，并通过核糖开关结合配体， 它调节细菌中的基因表达。 单分子测量提供了了解异质和非均质的独特窗口 RNA折叠的动态性质，超快光谱探测非常快速的过程 无法通过其他方式到达的。然而，这些都是显著的低吞吐量技术， 通常一次只对一个RNA序列进行测量。完全是一个 如果这些技术可以应用于一种 高吞吐量方式。我们将通过开发一个进程来实现这一目标 对数百种不同的序列进行单分子荧光测量 同时。将准备一个RNA序列文库，该文库包含以下位置的随机碱基 兴趣和混合物将受到单分子荧光测量，以 实时监测结构重组和对刺激的反应。然后每个分子都会 进行原位测序，以确定是什么序列产生了它的单分子信号。 我们未来5年的目标是优化这种方法，并用它来回答以下问题 只能通过高吞吐量方法来解决。我们将调查其机理。 关键序列对前信使核糖核酸剪接的影响，包括微调的可变序列 剪接和保守的序列，当被破坏时会导致疾病。我们还将测量 数百个RNA序列的折叠热力学和动力学，并使用结果来 对RNA结构预测算法进行基准测试和改进。这项研究计划将极大地 提高单分子荧光测量的吞吐量，使详细的 生物物理洞察力将在巨大的序列空间中快速实现。

Project Summary RNA plays a central role in nearly every biological process, acting in turn as a messenger, catalyst, signaling molecule and more. Many of these roles require RNA to fold into specific structures, and a given RNA species can often adopt multiple structures that modulate properties such as protein interactions, ligand binding and catalytic activity. Research in the Widom Lab focuses on developing and applying new spectroscopic methods to study RNA structure and dynamics. We combine bulk, single-molecule and ultrafast spectroscopy in order to obtain a comprehensive picture of RNA folding and interactions over length-scales from Angstroms to microns and time-scales as short as picoseconds. We are currently using these methods to study RNA-protein interactions during pre-messenger RNA (pre-mRNA) splicing, the process in which segments of RNA that do not code for protein are excised, and ligand binding by riboswitches, which regulate gene expression in bacteria. Single-molecule measurements offer a unique window into the heterogeneous and dynamic nature of RNA folding, and ultrafast spectroscopy probes very rapid processes that are inaccessible by other means. However, these are notably low-throughput techniques, with measurements typically being performed on only a single RNA sequence at a time. An entirely new class of scientific questions could be addressed if these techniques could be applied in a high-throughput manner. We will bring this goal to realization by developing a process for performing single-molecule fluorescence measurements on hundreds of different sequences simultaneously. A library of RNA sequences will be prepared containing random bases at sites of interest and the mixture will be subjected to single-molecule fluorescence measurements to monitor structural rearrangements and response to stimuli in real time. Each molecule will then be sequenced in situ in order to determine what sequence gave rise to its single-molecule signal. Our goals for the next 5 years are to optimize this method and to use it to answer questions that can only be addressed via high-throughput approaches. We will investigate the mechanistic impacts of key sequences on pre-mRNA splicing, including variable sequences that fine-tune splicing and conserved sequences that lead to disease when disrupted. We will also measure the folding thermodynamics and kinetics of hundreds of RNA sequences and use the results to benchmark and improve RNA structure prediction algorithms. This research program will greatly increase the throughput of single-molecule fluorescence measurements, enabling detailed biophysical insights to be achieved rapidly across a vast sequence space.