Development of Next-Generation Mass Spectrometry-based de novo RNA Sequencing for all Modifications

项目摘要 具有所有不同修饰的RNA序列构成RNA的“真实”信息内容。缺陷 RNA修饰导致超过100种人类疾病，如乳腺癌、2型糖尿病和肥胖症， 影响着数百万美国人尽管其重要性，RNA的真实序列，即，身份和位置 一个完整长度的RNA中的每一个核苷酸组成部分（修饰或未修饰）的结构，仍然是一个谜，主要是 由于缺乏直接测序任何核苷酸，特别是修饰的核苷酸的通用方法 （包括未知的）单核苷酸分辨率。现有技术无法对所有修改进行排序 同时以大规模或转录组水平解折叠真实RNA序列。 使RNA修饰研究复杂化的是，已经发现了>170种修饰类型， 并非所有的核苷酸修饰都在其RNA位点完全修饰至100%。他们甚至 基于NGS的技术无法检测到，这需要将RNA转化为没有任何 修改信息。用于绘制RNA修饰图的工具仅限于少数流行的修饰， 通常一次只能分析一种修改类型。质谱法（MS）是目前唯一的技术 可以表征所有的RNA修饰;然而，常规的MS方法丢失了关于RNA修饰的信息。 修饰的核苷酸的位置和共存。 为了解决这些突出的问题，我们最近开发了一系列新的下一代质量 基于光谱的测序（NextGen MassSpec-Seq）方法，可以直接从头测序 不含cDNA的tRNA，可以同时对所有核苷酸修饰进行测序和定量。为 在此提议期间，我们将进一步开发NextGen MassSpec-Seq，以有效地对tRNA进行测序， 不同的细胞甚至疾病条件，使其可扩展到高通量，并扩大其 应用于同时对任何RNA类型上的所有修饰进行定量测序和作图， 转录组水平。具体地说，我们建议开发MS用于大规模的全长DNA从头测序。 tRNA与所有不同的核苷酸修饰（Aim 1）一起，使MS能够同时测序和 定量多个RNA修饰，允许在单核苷酸和化学计量的定量映射， 精确度（目标2），扩大NextGen MassSpec-Seq并将其与高通量NGS测序联合收割机结合， 在转录组水平上对不同的RNA修饰进行直接测序（Aim 3）。我们的工具将解决一个长期- 如何揭示“真正的”RNA序列，并为研究RNA提供一个变革性的工具， 修饰，这将促进更好地理解转录后修饰的功能及其 与RNA相关疾病和流行病的关系。

PROJECT SUMMARY An RNA sequence with all its diverse modifications constitutes ‘true’ information content of the RNA. Defects in RNA modifications account for >100 human diseases, such as breast cancer, type-2 diabetes and obesity, affecting millions of Americans. Despite its significance, the true sequence of a RNA, i.e., identity and location of each and every nucleotide building block (modified or not) within a full-length RNA, remains a mystery, mainly because of the lack of a general method to directly sequence any nucleotide, especially modified nucleotides (including unknown ones) at single-nucleotide resolution. No existing technology can sequence all modifications simultaneously to unfold the true RNA sequences at a large scale or the transcriptomic level. What complicates RNA modification studies is that >170 modification types have been discovered, and not all of nucleotide modifications are modified completely to 100% at their RNA sites. They are even undetectable by NGS-based technologies, which require the conversion of RNA to cDNAs that do not have any modification information. Tools to map RNA modifications are limited only to a few popular modifications, and can usually analyze only one modification type at a time. Mass spectrometry (MS) is currently the only technique that can characterize all RNA modifications; however, conventional MS methods lose information regarding the location and co-occurrence of modified nucleotides. To resolve these outstanding issues, we have recently developed a series of novel next generation mass spectrometry-based sequencing (NextGen MassSpec-Seq) approaches that can de novo directly sequence tRNAs without a cDNA and can sequence and quantify all nucleotide modifications simultaneously. For the duration of this proposal, we will further develop NextGen MassSpec-Seq to sequence tRNAs efficiently in different cellular and even disease conditions, make it scalable toward high throughput, and expand its application to simultaneously sequence and map all modifications quantitatively on any RNA type and at the transcriptomic level. Specifically, we propose to develop MS for large-scale de novo sequencing of full-length tRNAs, together with all diverse nucleotide modifications (Aim 1), empower MS to simultaneously sequence and quantify multiple RNA modifications, allowing quantitative mapping at single nucleotide and stoichiometric precision (Aim 2), scale up NextGen MassSpec-Seq and combine it with high-throughput NGS sequencing for direct sequencing of diverse RNA modifications at the transcriptomic level (Aim 3). Our tool will address a long- standing issue of how to reveal the ‘true” RNA sequences and provide a transformative tool for studying RNA modifications, which will promote better understanding of functions of post-transcriptional modifications and their correlations to RNA-related diseases and pandemics.