LC/MS-Based Direct RNA Sequencing with Concomitant Capability to Sequence Multiple Base Modifications at Single-base Resolution

基于 LC/MS 的直接 RNA 测序，同时能够以单碱基分辨率对多个碱基修饰进行测序

• 批准号: 10217648
• 负责人: Shenglong Zhang
• 金额: $ 35万
• 依托单位: NEW YORK INST OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-03 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217648
• 关键词: 16S ribosomal RNA sequencing; Address; Affect; American; Communities; Complementary DNA; Complex; Databases; Development; Diabetes Mellitus; Disease; Disease model; Escherichia coli; Frequencies; Genetic Transcription; Goals; Gold; Health; High-Throughput RNA Sequencing; Human; Hydrophobicity; Knowledge; Label; Length; Link; Location; Mass Spectrum Analysis; Metabolic Diseases; Methods; Methylation; Modification; Mus; Non-Insulin-Dependent Diabetes Mellitus; Nucleotides; Obesity; Organism; Peptides; Performance; Phenylalanine-Specific tRNA; Population; Positioning Attribute; Post-Transcriptional RNA Processing; Protein Isoforms; Proteomics; RNA; RNA Sequences; Reading; Regulation; Research; Research Personnel; Resolution; Ribosomal RNA; Running; Sampling; Site; Tail; Techniques; Technology; Testing; Time; Tissues; Transcript; Transfer RNA; Variant; Work; Yeasts; automated algorithm; base; cell type; comparative; epitranscriptomics; experimental study; genome-wide; human disease; improved; in vivo; instrument; malignant breast neoplasm; nervous system disorder; nucleobase; reading difficulties; reconstruction; reference genome; tool; transcriptome; transcriptome sequencing; two-dimensional

Project Summary Aberrant RNA base modifications have been correlated with the development of major diseases including breast cancer, type-2 diabetes, obesity, and neurological disorders, each affecting millions of Americans. However, these modifications are undetectable by current high-throughput RNA sequencing technologies, which do not directly sequence RNAs, but instead sequence cDNAs that only contain the four canonical deoxynucleotides. Other tools to sequence nucleobase modifications in RNA are usually tailored for a single specific modified nucleotide and cannot provide single-base-resolution spatial information for modifications. Thus, very few of the over 160 identified RNA modifications have been studied. To better understand RNA with its rich modifications, we have been developing a mass spectrometry (MS)-based 2-dimensional hydrophobic end-labeling sequencing strategy (2-D HELS MS Seq) as: 1) a de novo and accurate method to directly sequence RNA and 2) a general method to sequence all base modifications in any RNA type at single-base resolution. The method can currently sequence purified or mixed samples of short synthetic RNAs and simultaneously identify, locate, and quantify the frequency of a specific modification in a population. In this proposal, we focus on improving read-length, throughput, and sensitivity to sequence rare RNA modifications, quantify post-transcriptional base modifications, and detect active isoforms of mixed cellular RNA samples. We propose to (a) de novo MS sequence specific and total cellular tRNA (<100 nt) as proof-of-concept examples (Aim 1), (b) de novo sequence complex endogenous RNA samples (up to 100 strands, 950 nt per run) (Aim 2), and (c) quantify genome wide post- transcriptional RNA modifications in metabolic disease models (Aim 3). This project is highly significant as successful accomplishment of the proposed work will 1) bring the power of MS-based laddering technology to RNA, thus providing a method comparable to analysis of peptide modifications in proteomics, that can reveal the identity and position of various RNA modifications, 2) allow direct and de novo RNA sequencing without cDNA synthesis, and 3) allow accurate reading of multiple base modifications at single nucleotide resolution in one experiment without prior knowledge of sequences and modifications, helping to address a long-standing unmet need in the broad field of epitranscriptomics. Our tool will promote better understanding of functions of post- transcriptional modifications and isoforms including their correlations to human diseases; we will develop the method into a gold standard for verifying other techniques for sequencing and annotating genome-wide base modifications, thereby helping to build more accurate and inclusive reference epitranscriptomic databases.

项目摘要 异常的RNA碱基修饰与包括乳腺癌在内的主要疾病的发生有关。 癌症、2型糖尿病、肥胖症和神经系统疾病，每一种都影响着数百万美国人。然而，在这方面， 目前的高通量RNA测序技术无法检测到这些修饰， 直接对RNA进行测序，而是对仅含有四种典型脱氧核苷酸的cDNA进行测序。 对RNA中的核碱基修饰进行测序的其他工具通常是针对单个特定修饰的核碱基而定制的。 因此，它不能提供用于修饰的单碱基分辨率空间信息。因此， 已经研究了超过160种鉴定的RNA修饰。为了更好地了解RNA及其丰富的修饰， 我们一直在开发基于质谱（MS）的二维疏水末端标记测序 2-D HELS MS Seq策略：1）直接测序RNA的从头和准确方法，2）通用的 该方法以单碱基分辨率对任何RNA类型中的所有碱基修饰进行测序。该方法目前可以 对纯化的或混合的短合成RNA样品进行测序，并同时鉴定、定位和定量 一个群体中特定变异的频率。在这个提案中，我们专注于提高读长， 通量和对序列罕见RNA修饰的敏感性，定量转录后碱基修饰， 并检测混合细胞RNA样品的活性同种型。我们建议（a）从头MS序列特异性 和总细胞tRNA（<100 nt）作为概念验证实例（Aim 1），（B）从头序列复合物 内源性RNA样品（多达100条链，每次运行950 nt）（目的2），和（c）定量基因组范围的后- 在代谢疾病模型中的转录RNA修饰（Aim 3）。该项目意义重大， 所提出的工作的成功完成将1）将基于MS的梯形技术的力量， RNA，从而提供了一种与蛋白质组学中的肽修饰分析相当的方法，该方法可以揭示蛋白质组学中的肽修饰。 各种RNA修饰同一性和位置，2）允许直接和从头RNA测序而无需cDNA 合成，和3）允许在一个单核苷酸分辨率下精确阅读多个碱基修饰 在没有序列和修改的先验知识的情况下进行实验，有助于解决长期未得到满足的问题。 需要在广泛的领域epitranscriptomics。我们的工具将促进更好地了解后， 转录修饰和亚型，包括它们与人类疾病的相关性;我们将开发 方法转化为验证其他全基因组碱基测序和注释技术的金标准 这将有助于构建更准确和更具包容性的参考epitranscriptomic数据库。

项目成果

A General LC-MS-Based Method for Direct and De Novo Sequencing of RNA Mixtures Containing both Canonical and Modified Nucleotides.

一种基于 LC-MS 的通用方法，用于对含有规范和修饰核苷酸的 RNA 混合物进行直接和从头测序。

• DOI: 10.1007/978-1-0716-1374-0_17
• 发表时间: 2021
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Zhang,Ning;Shi,Shundi;Yuan,Xiaohong;Ni,Wenhao;Wang,Xuanting;Yoo,Barney;Jia,TonyZ;Li,Wenjia;Zhang,Shenglong
• 通讯作者: Zhang,Shenglong

Parent tRNA Modification Status Determines the Induction of Functional tRNA-Derived RNA by Respiratory Syncytial Virus Infection.

• DOI: 10.3390/v15010057
• 发表时间: 2022-12-24
• 期刊: Viruses
• 作者: Choi EJ;Wu W;Zhang K;Yuan X;Deng J;Ismail D;Buck DL;Thomason KS;Garofalo RP;Zhang S;Bao X
• 通讯作者: Bao X