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

开发适用于所有修饰的下一代基于质谱的从头 RNA 测序

• 批准号: 10581994
• 负责人: Shenglong Zhang
• 金额: $ 67.69万
• 依托单位: NEW YORK INST OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10581994
• 关键词: 2019-nCoV; Adopted; Affect; Algorithms; American; Automation; Base Sequence; COVID-19; Chemicals; Complementary DNA; Complex Mixtures; Defect; Development; Disease; Disease model; Goals; Health; High Fat Diet; High-Throughput Nucleotide Sequencing; Human; Length; Location; Maps; Mass Spectrum Analysis; Metabolic Diseases; Methods; Modification; Mus; Nature; Non-Insulin-Dependent Diabetes Mellitus; Nucleotides; Obesity; Phenylalanine-Specific tRNA; Preparation; Proteomics; RNA; RNA Sequences; Research; Resolution; Ribosomal RNA; Running; Sampling; Series; Site; Small RNA; Techniques; Technology; Time; Tissues; Transfer RNA; Variant; Yeasts; base; empowerment; genome-wide; human disease; insight; instrumentation; malignant breast neoplasm; mouse model; next generation; next generation sequencing; novel; pandemic disease; posttranscriptional; prevent; scale up; stoichiometry; tool; transcriptome; transcriptome sequencing; transcriptomics

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.

项目摘要 具有所有潜水员修饰的RNA序列构成RNA的“真实”信息内容。缺陷 RNA修饰说明> 100种人类疾病，例如乳腺癌，2型糖尿病和肥胖症， 影响数百万美国人。尽管具有重要意义，但RNA的真实序列，即身份和位置 全长RNA中的每个核苷酸构建块（是否修改），仍然是一个神秘的，主要是 由于缺乏直接测序任何核苷酸的一般方法，尤其是修饰的核苷酸 （包括未知的）单核苷酸分辨率。没有现有技术可以对所有修改进行测序 类似于大规模或转录组水平上的真实RNA序列。 RNA修饰研究复杂的是发现> 170种修饰类型，并且 并非所有的核苷酸修饰在其RNA位点被完全修改为100％。他们甚至是 基于NGS的技术无法检测到，这些技术需要将RNA转换为没有任何的CDNA 修改信息。映射RNA修改的工具仅限于一些流行的修改，并且 通常可以一次仅分析一种修改类型。质谱法（MS）当前是唯一的技术 可以表征所有RNA修饰；但是，传统的MS方法丢失了有关 修饰核苷酸的位置和同时存在。 为了解决这些杰出的问题，我们最近开发了一系列新颖的下一代弥撒 基于光谱的测序（NextGen MassSpec-Seq）方法可以直接序列直接序列 没有cDNA的TRNA，可以简单地对所有核苷酸修饰进行序列和量化。为了 该提案的持续时间，我们将进一步开发NextGen MassSpec-Seq，以有效地对TRNA进行序列 不同的细胞甚至疾病状况，使其可扩展到高吞吐量，并扩展其 仅在任何RNA类型上定量地序列和映射所有修改 转录组级别。特别是，我们建议开发用于全长测序的大规模测序的MS TRNA，以及所有潜水核苷酸的修改（AIM 1），使MS能够同时顺序和 量化多个RNA修饰，允许在单核苷酸和化学计量学处进行定量映射 精度（AIM 2），扩展NextGen MassSpec-Seq，并将其与高通量NGS测序相结合 在转录组水平上直接对潜水员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“真实” RNA序列的常规问题，并提供了研究RNA的变革性工具 修改，这将促进对文字后修改功能及其功能的更好理解 与RNA相关疾病和大流行病的相关性。