Chemical probing of RNA tertiary structure in a whole transcriptome at single-atom resolution

RNA（核糖核酸）现在被广泛认为是几乎所有细胞活动的关键参与者，然而关于大多数RNA分子的三维结构的信息有限。这提出了一个挑战，因为众所周知，RNA分子的生物学功能取决于它的结构。为了了解细胞中大量RNA分子的结构和功能之间的关系，需要新的高通量实验方法来确定结构。该项目将开发一种新的实验方法，可以在一次实验中提供细胞中所有RNA分子三维折叠的详细信息。这种新方法结合了典型的活性氧分子羟基自由基对RNA结构的化学探测和高通量测序分析，从而可以同时监测细胞中所有RNA分子的结构。该提案将为研究生和本科生提供教育和培训机会，包括STEM领域中代表性不足的群体。该项目的目的是开发一种新的方法，在一个实验中获得细胞中所有RNA的三级结构信息。该实验方法将羟基自由基化学探测实验应用于高通量测序分析。一项关键的创新将是合成一套新型的“捕获和释放”小分子探针，这些探针将用于捕获羟基自由基切割RNA的主要产物，这是一条由5'-醛部分终止的链。羟基自由基裂解和醛捕获和释放将以流动形式进行，以简化方法。该方法将通过将其应用于已知结构的RNA分子来开发和验证。经过验证的方法将用于在体外和活细胞中绘制整个转录组的三级结构，用于酵母和人类细胞。该项目得到了生物科学理事会分子和细胞生物科学部分子生物物理组的支持。

RNA (ribonucleic acid) is now widely appreciated to be a key participant in nearly all of the activities of the cell, yet only limited information on the three-dimensional structures of most RNA molecules are available. This presents a challenge, because it is known that the biological function of an RNA molecule depends on its structure. To understand the relationship between structure and function for the large number of RNA molecules that populate the cell, new high-throughput experimental approaches for structure determination are needed. This project will develop a new experimental method that can provide detailed information on the three-dimensional folding of all the RNA molecules in a cell, in one experiment. The new method combines chemical probing of RNA structure by the hydroxyl radical, the quintessential Reactive Oxygen Species, with analysis by high-throughput sequencing, so that the structures of all RNA molecules in a cell can be monitored simultaneously. This proposal will provide educational and training opportunities for graduate and undergraduate students including underrepresented groups in STEM fields. The aim of this project is to develop a new method for obtaining tertiary structural information for all of the RNA in a cell, in one experiment. The experimental approach will adapt the hydroxyl radical chemical probing experiment for analysis by high-throughput sequencing. A key innovation will be the synthesis of a novel set of "catch and release" small molecule probes that will be used to capture the major product of hydroxyl radical cleavage of RNA, a strand terminated by a 5'-aldehyde moiety. Hydroxyl radical cleavage and aldehyde catch and release will be performed in flow format, to streamline the method. The method will be developed and validated by applying it to RNA molecules of known structure. The validated method will then be used to map the tertiary structure of the entire transcriptome, in vitro and in living cells, for yeast and human cells. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.