Synthetic mRNA Control Set for Nanopore-Based Pseudouridine Modification Profiling in Human Transcriptomes

用于人类转录组中基于纳米孔的假尿苷修饰分析的合成 mRNA 对照集

• 批准号: 10582330
• 负责人: Sara Hakim Rouhanifard
• 金额: $ 84.83万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10582330
• 关键词: Address; Algorithms; Bar Codes; Base Pairing; Cell Line; Cells; Chemicals; Consensus; Data; Data Set; Derivation procedure; Detection; Development; Disease; Enzymes; Foundations; Gene Expression; Generations; Gold; Human; Human Cell Line; Individual; Isomerism; Label; Ligation; Location; Machine Learning; Mammalian Cell; Maps; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Methods; Modification; Molecular; Monitor; Outcome; Phenotype; Pseudouridine; RNA; RNA-Binding Proteins; Resistance; Ribonucleases; Role; Running; Sampling; Signal Transduction; Site; Structure; Thermodynamics; Time; Training; Transcript; Translations; Uridine; Work; cell type; computational pipelines; computerized tools; developmental disease; high standard; immunogenicity; in vivo; nanopore; next generation sequencing; novel; recruit; single molecule; success; tool; transcriptome; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Mammalian cells expend large amounts of energy into generating enzyme-mediated RNA chemical modifications that can change the base-pairing, RNA structure, or recruitment of RNA-binding proteins, among other elusive roles. Pseudouridine (ψ)-modified mRNAs are more thermodynamically stable, more resistant to RNAse-mediated degradation, and have the potential to modulate immunogenicity and enhance translation in vivo. However, ψ detection is extremely challenging: ψ modifications do not affect Watson-Crick base pairing and are indistinguishable from uridine when using hybridization-based methods. Further, since ψ is an isomer of uridine, detection using mass spectrometry requires non-quantitative chemical derivatization methods. While recent studies have shown that RNA modifications can be detected through direct RNA nanopore sequencing by monitoring basecalling errors, we have recently shown that the accuracy and fidelity of this approach is relatively low and sequence dependent. Our team has recently used a ligation approach to produce synthetic mRNA controls that contain single ψ sites within relevant transcripts mammalian cells. Using these synthetic controls we performed nanopore-based RNA sequencing and developed computational tools that increase the accuracy of ψ-calling to 90+%, depending on the specific sequence. We are basing our work on our recent finding that achieving ψ quantification requires sequence-specific training using unique signal parameters. The initial success of our team has laid the foundation to 1) generate an expanded set of barcoded synthetic RNA constructs that contain single ψ sites, 2) obtain a rigorous set of quadruplicate nanopore runs with ~50,000 single-molecule reads per construct, 3) develop computational tools to allow highly accurate sequence-specific ψ-calling. We will develop a gold-standard set of synthetic mRNA transcripts as a training molecular set for quantitative ψ profiling in direct RNA nanopore sequencing of human transcriptomes. The molecular set will allow quantitative profiling of hundreds of putative ψ sites across mammalian samples. This proposal will serve an unmet need by addressing a critical bottleneck: the lack of available modified RNA modification gold standards, i.e., RNA molecules that contain a site-specific and structure-specific modification. In this collaborative project we will develop a complete pipeline for synthesis of gold standard molecules; use these molecules to measure the nanopore signals that ψ modifications produce; develop a machine-learning tool to accurately quantify these modifications; profile site-specific ψ modifications in various cell lines to obtain ψ-maps that can be used to assess relationships of ψ modifications with phenotypes.

项目摘要/摘要 哺乳动物细胞探索大量能量以产生酶介导的RNA化学 可以改变RNA结合蛋白的基础配对，RNA结构或募集的修饰 其他难以捉摸的角色。伪脲（ψ）修饰的mRNA在热力学上更稳定，对 RNase介导的降解，并有可能调节免疫原性并增强翻译 体内。但是，ψ检测是极其挑战：ψ修改不会影响沃森 - 克里克底座配对 在使用基于杂交的方法时，与尿苷无法区分。此外，由于ψ是异构体 在尿苷中，使用质谱法检测需要非定量化学衍生化方法。尽管 最近的研究表明，可以通过直接RNA纳米测序检测RNA修饰 通过监视基本错误，我们最近表明，这种方法的准确性和保真度是 相关的低和序列依赖性。我们的团队最近使用了连接方法来生产合成 mRNA对照在相关转录本内包含单单ψ位点的哺乳动物细胞中。使用这些合成 控制我们执行了基于纳米孔的RNA测序并开发了计算工具，以增加 根据特定序列的不同，ψ调用至90％+％的精度。我们正在以最新作品为基础 发现实现ψ量化需要使用唯一信号参数的序列特异性训练。这 我们团队的最初成功已为1）产生一组扩展的条形码合成RNA 包含单单ψ位点的构造，2）获得一组严格的四倍体纳米孔，〜50,000 单分子读取每个结构，3）开发计算工具以允许高度准确的序列特异性 ψ呼叫。我们将开发一组金标准的合成mRNA转录本作为训练分子集 人类转录组的直接RNA纳米孔测序中的定量ψ分析。分子集将 允许对哺乳动物样品中数百个推定的ψ位点进行定量分析。 该建议将通过解决关键瓶颈来满足未满足的需求：缺乏可用的修改RNA 修改金标准品，即包含位点特异性和结构特异性修饰的RNA分子。 在这个协作项目中，我们将开发完整的金标准分子合成的管道；使用 这些分子测量产生ψ修饰的纳米孔信号；开发机器学习 准确量化这些修改的工具；在各种细胞系中进行的位点特异性ψ修改以获得 可用于评估修饰与表型的关系的ψ映射。