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

项目摘要/摘要 哺乳动物细胞耗费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，包括 其他难以捉摸的角色。假尿苷(ψ)修饰的mRNA在热力学上更稳定，对 核糖核酸酶介导的降解，并有可能调节免疫原性和增强翻译 活着。然而，ψ检测极具挑战性：ψ修饰不会影响沃森-克里克碱基配对 并且在使用基于杂交的方法时与尿苷难以区分。此外，由于ψ是一种异构体 对于尿苷，使用质谱仪进行检测需要非定量的化学衍生化方法。而当 最近的研究表明，通过直接的RNA纳米孔测序可以检测到RNA的修饰 通过监测基线误差，我们最近已经表明，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了一种连接方法来生产合成的 在相关转录本哺乳动物细胞中包含单个ψ位点的mR NA控制。使用这些合成的 我们进行了基于纳米孔的RNA测序，并开发了计算工具来增加 根据特定的顺序，ψ呼叫的准确率达到90+%。我们的工作是基于我们最近的 发现实现ψ量化需要使用唯一信号参数的特定序列训练。这个 我们团队的初步成功为1)生产扩展的条形码合成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，约为50,000 每个构建体的单分子读数，3)开发计算工具以实现高度精确的序列特异性 ψ-呼叫。我们将开发一套黄金标准的合成信使核糖核酸转录本作为训练分子集 人类转录本直接ψ纳米孔测序中的定量rna图谱分析。分子集将会 允许对哺乳动物样本中数以百计的假定ψ位点进行定量分析。 这项提议将通过解决一个关键的瓶颈来满足一个未得到满足的需求：缺乏可用的修饰RNA 修饰黄金标准，即包含特定位点修饰和特定结构修饰的RNA分子。 在这个合作项目中，我们将开发一条合成黄金标准分子的完整流水线；使用 这些分子测量ψ修饰产生的纳米孔信号；开发一种机器学习 准确量化这些修改的工具；分析各种细胞系中特定部位的ψ修改，以获得 ψ-可用于评估ψ修饰与表型的关系的图谱。

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.