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.

项目总结/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结合蛋白的募集的修饰， 其他难以捉摸的角色。假尿苷（Pseudouridine，PdR）修饰的mRNAs更稳定，更耐药， RNA酶介导的降解，并具有调节免疫原性和增强翻译的潜力， vivo.然而，DNA的检测是极具挑战性的：DNA的修饰不影响沃森-克里克碱基配对 并且当使用基于杂交的方法时与尿苷不可区分。此外，由于α是异构体， 尿苷的质谱检测需要非定量化学衍生方法。而 最近的研究表明，RNA修饰可以通过直接RNA纳米孔测序来检测， 通过监测碱基判定错误，我们最近表明，这种方法的准确性和保真度是 相对较低且依赖于序列。我们的团队最近使用连接方法来产生合成的 在相关转录本哺乳动物细胞内含有单个转录位点的mRNA对照。使用这些合成 我们进行了基于纳米孔的RNA测序，并开发了计算工具， 根据特定的序列，识别的准确性为90+%。我们的工作是基于我们最近的 发现实现DNA定量需要使用独特信号参数的序列特异性训练。的 我们团队的初步成功奠定了基础，1）生成一组扩展的条形码合成RNA 包含单个双位点的构建体，2）获得严格的一组四重纳米孔运行，其中约50，000 每个构建体的单分子读段，3）开发计算工具以允许高度准确的序列特异性 打电话给你。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， 在人类转录组的直接RNA纳米孔测序中的定量RNA分析。分子集将 允许对哺乳动物样品中数百个推定的突变位点进行定量分析。 这项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 修改黄金标准，即，含有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管道合成金标准分子;使用 这些分子来测量纳米孔修饰产生的信号;开发机器学习 工具，以准确地量化这些修改;概况在各种细胞系中的位点特异性修饰，以获得 可用于评估基因修饰与表型关系的基因图谱。