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起着核心作用，反过来又充当信使， 催化剂、信号分子等等。这些作用中的许多需要RNA折叠成特定的结构。 结构，并且给定的RNA种类通常可以采用多种结构来调节性质 例如蛋白质相互作用、配体结合和催化活性。Widom实验室的研究 重点开发和应用新的光谱方法来研究RNA结构， 动力学我们结合联合收割机体，单分子和超快光谱，以获得一个 RNA折叠和相互作用在长度尺度上的全面图片，从Angiotensin到 微米和皮秒的时间尺度。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， 切除不编码蛋白质的RNA片段，并通过核糖开关结合配体， 调节细菌中基因的表达。 单分子测量提供了一个独特的窗口， RNA折叠的动态性质，超快光谱探测非常快速的过程， 无法通过其他方式访问。然而，这些是显著低通量的技术， 通常一次仅对单个RNA序列进行测量。一个完全 如果这些技术可以应用于一个新的科学问题， 高通量的方式。我们将通过制定一个进程， 对数百个不同的序列进行单分子荧光测量， 同步将制备RNA序列文库，其在以下位点含有随机碱基： 并且混合物将经受单分子荧光测量， 真实的监控结构重组和对刺激的反应。每一个分子 原位测序以确定什么序列产生其单分子信号。 我们在未来5年的目标是优化这种方法，并用它来回答问题， 只能通过高通量方法来解决。我们将研究 关键序列对前体mRNA剪接的影响，包括微调 剪接和保守序列，当被破坏时导致疾病。我们还将测量 折叠热力学和动力学的数百个RNA序列，并使用结果， 对RNA结构预测算法进行基准测试和改进。这项研究计划将大大 提高单分子荧光测量的通量， 在广阔的序列空间中快速获得生物物理见解。

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.