High-throughput structure probing of RNA based on specific ligation of cyclo-phosphate ends combined with deep sequencing

与蛋白质类似，RNA分子折叠成多种复杂的结构，以完成它们在细胞中的任务。虽然rRNA、tRNA、核酶、核糖开关和终止子中的高度限定的折叠长期以来是众所周知的，但在mRNA中也发现了越来越多的限定结构，其与稳定性特别是可翻译性相关。因此，确定细胞内所有转录物的结构以更好地理解其功能和调控是非常有趣的。虽然体外方法来表征RNA结构是简单的，并建立良好的，在体内的调查是相当费力。目前，只有少数几种可靠的方法可用，如细胞SHAPE，DMS-Seq及其变体SHAPE-MaP和DMS-MaPseq。这些方法基于RT终止信号或RT期间产生的特定错误特征的读出。缺点是RT终止也由自发终止或稳定的RNA二级结构产生，并且并非所有单链特异性化学修饰都导致RT特征。我们希望建立一种新的方法，将连接位置读取为单链区域。通过特异性连接酶将由单链RNA中的铅切割诱导的2 '3'-环磷酸和5 'OH末端融合到衔接子上，并通过深度测序鉴定。基于RT的cDNA终止未注册。通过分析两个产生的RNA末端的连接产物实现背景信号的进一步降低。在实验方法的同时，我们将开发一种精确的生物信息学工具，用于单链信号的定性和定量识别，以尽可能准确地实现RNA结构。由于直接测量某个位置的结构需要高的读段覆盖率（每个位置10-15个读段），我们还将开发沿沿着小间隔聚集记录信号的方法，以便即使在较低的覆盖率下也能得出可靠的结论。大肠杆菌，因为最近公布的调查显示高度矛盾的结果。此外，我们还想分析在低温环境下，mRNA结构是如何被调控以实现高效翻译的。总之，我们想开发一种新的RNA结构的高通量分析方法，以解决有关RNA折叠对稳定性、翻译效率及其在不同细胞系统中的调控的影响等重要问题。用于优化连接酶的表达质粒将被保藏在非盈利性载体保藏中心addgene，以便科学界能够获得这种方法。

Similar to proteins, RNA molecules fold up into manifold and complex structures in order to fulfill their tasks in the cell. While highly defined folds in rRNAs, tRNAs, ribozymes, riboswitches and terminators are well-known for a long time, an increasing amount of defined structures are also found in mRNAs, correlating with stability and especially translatability. Hence, it is of great interest to identify the structures of all transcripts within a cell to better understand their functionality and regulation. While in vitro approaches to characterize RNA structures are straightforward and well established, the in vivo investigation is rather laborious. Currently, there are only a few robust methods available, like in cell SHAPE, DMS-Seq and their variants SHAPE-MaP und DMS-MaPseq. These approaches are based on the read-out of RT stop signals or specific error signatures generated during RT. A disadvantage is that RT stops are also generated by spontaneous termination or stable RNA secondary structures and that not all single-strand specific chemical modifications lead to RT signatures. We want to establish a novel method that reads ligation positions as single stranded regions. 2’3’-cyclo-phosphate and 5’OH ends induced by lead cleavage in single-stranded RNA are fused to adapters by specific ligases and identified by deep sequencing. RT-based cDNA terminations are not registered. A further reduction of the background signal is achieved by the analysis of ligation products of both generated RNA ends. In parallel to the experimental approach, we will develop a precise bioinformatic tool for qualitative and quantitative recognition of single-strand signals to achieve RNA structures as accurate as possible. As a direct measure of the structure at a certain position requires a high read coverage (10-15 reads per position), we will also develop methods that aggregate the recorded signal along small intervals so that reliable conclusions can be drawn even with lower coverage.Combined with ribosome profiling analyses, we will use this novel orthogonal strategy to clarify how mRNA structure correlates with translational efficiency in E. coli, as recently published investigations show highly conflictive results. In addition, we want to analyze in psychrophilic microorganisms how mRNA structures are regulated at extremely low temperatures to allow efficient translation.Taken together, we want to develop a novel high throughput analysis of RNA structures to address important questions concerning the impact of RNA folding on stability, translational efficiency and their regulation in different cellular systems. Expression plasmids for the optimized ligases will be deposited in the non-profit vector collection addgene, so that the scientific community has access to this method.