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.

项目概要/摘要 哺乳动物细胞消耗大量能量来产生酶介导的 RNA 化学物质 可以改变碱基配对、RNA 结构或 RNA 结合蛋白招募的修饰，其中 假尿苷 (ψ) 修饰的 mRNA 具有更热力学稳定性，更耐受其他难以捉摸的作用。 RNAse 介导的降解，并具有调节免疫原性和增强翻译的潜力 然而，ψ 检测极具挑战性：ψ 修饰不会影响 Watson-Crick 碱基配对。 当使用基于杂交的方法时， 和 与尿苷无法区分。 此外，由于 ψ 是异构体。 对于尿苷，使用质谱检测需要非定量化学衍生方法。 最近的研究表明，可以通过直接 RNA 纳米孔测序来检测 RNA 修饰 通过监控碱基识别错误，我们最近表明这种方法的准确性和保真度是 我们的团队最近使用连接方法来生产合成的。 使用这些合成的哺乳动物细胞内含有单个 ψ 位点的 mRNA 对照。 我们进行了基于纳米孔的 RNA 测序并开发了计算工具来提高 ψ 调用的准确度达到 90+%，具体取决于具体序列，我们的工作基于我们最近的工作。 发现实现 ψ 量化需要使用独特的信号参数进行特定序列的训练。 我们团队的初步成功为 1) 生成一组扩展的带条形码的合成 RNA 奠定了基础 包含单个 ψ 位点的构建体，2) 获得一组严格的四重纳米孔运行，约 50,000 每个构建体的单分子读取，3) 开发计算工具以实现高度准确的序列特异性 我们将开发一套黄金标准的合成 mRNA 转录本作为训练分子集。 ψ 人类转录组直接 RNA 纳米孔测序中的分析。 允许对哺乳动物样本中数百个假定的 ψ 位点进行定量分析。 该提案将通过解决一个关键瓶颈来满足未满足的需求：缺乏可用的修饰 RNA 修饰金标准，即包含位点特异性和结构特异性修饰的 RNA 分子。 在这个合作项目中，我们将开发一个完整的金标准分子合成管道； 这些分子用于测量 ψ 修饰产生的纳米孔信号，开发机器学习； 准确量化这些修饰的工具；分析各种细胞系中的位点特异性 ψ 修饰，以获得 ψ-图谱可用于评估 ψ 修饰与表型的关系。