CLAP-seq: An Aptamer-Based Platform for Transcriptome-Wide Mapping of RNA Modifications

CLAP-seq：基于适配体的 RNA 修饰全转录组图谱平台

• 批准号: 9812571
• 负责人: Jonathan Thomas Sczepanski
• 金额: $ 17.76万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-02 至 2021-08-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9812571
• 关键词: Affinity; Antibodies; Base Pairing; Binding; Biology; Chemicals; DNA; Data; Data Set; Development; Developmental Process; Disease; Engineering; Ensure; Enzymes; Generations; Genetic Transcription; Goals; Health; Human; Human Development; Image; In Vitro; Individual; Knowledge; Lead; Link; Maps; Methods; Mission; Modification; National Institute of Child Health and Human Development; Nucleic Acids; Nucleosides; Nucleotides; Oligonucleotides; Physiological; Post-Transcriptional RNA Processing; Post-Translational Protein Processing; Property; Protocols documentation; Publishing; RNA; RNA Binding; Readiness; Reagent; Reporting; Research; Research Project Grants; Ribose; Role; Route; Site; Specificity; Techniques; Technology; Validation; aptamer; base; chemical binding; crosslink; enantiomer; epitranscriptome; epitranscriptomics; frontier; human disease; innovation; innovative technologies; loss of function; next generation sequencing; novel; stereochemistry; transcriptome

Project Summary/Abstract Beginning in the 1950s, more than 100 types of posttranscriptional modifications have been identified in cellular RNA. Today, the study of RNA post-transcriptional modifications – known as epitranscriptomics – is a rapidly developing field, which promises to greatly enhance our understanding of human health and disease. Despite the profound implications already assigned to many RNA modifications, their precise functions remain poorly understood. This can be attributed to the lack of sensitive and robust sequencing technologies to detect these epitranscriptomics marks in a transcriptome-wide manner. A key bottleneck is the lack of sensitive and specific enrichment techniques (affinity- or reactivity-based) for RNA molecules containing these modifications. The proposed research takes direct aim at this critical deficit using the aptamer approach, employing in vitro selection methods to identify nucleic acid molecules that bind chemically modified RNAs. These aptamers are unique in that they are comprised of L-(deoxy)ribose-based nucleic acids (L-DNA and L-RNA), which are mirror images (enantiomers) of natural D-nucleotides. L-Aptamers, which are completely orthogonal to natural biology, are extremely well suited for binding RNA targets. Therefore, in vitro selection will be used to isolate novel L- aptamers capable of binding chemically modified mononucleotides, which will enable selective capture of RNA molecules containing the same modified residue. These L-aptamers will then be used in Cross-Linking- Aptamer Pull-down and sequencing (CLAP-seq), the first transcriptome-wide profiling technology employing aptamer-based RNA enrichment prior to next-generation sequencing. CLAP-seq not only promises to open a general and robust route towards transcriptome-wide profiling of the growing list of RNA modifications, but also promises to reinforce our current view of the epitranscriptome. Accordingly, the development of CLAP-seq will have a profound impact on the field of epitranscriptomics, which is well aligned with the mission of the NICHD and the goal of this FOA: to promote research into the role of RNA chemical modifications in development and related disease.

项目总结/摘要 从20世纪50年代开始，已经鉴定了100多种类型的转录后修饰， 细胞RNA今天，RNA转录后修饰的研究-被称为表转录组学-是一项重要的研究。 这是一个快速发展的领域，有望大大提高我们对人类健康和疾病的理解。 尽管许多RNA修饰已经具有深刻的意义，但它们的精确功能仍然存在。 不太了解。这可归因于缺乏灵敏和稳健的测序技术来检测 这些表型转录组学标记以转录组范围的方式。一个关键的瓶颈是缺乏敏感和 特异性富集技术（基于亲和力或反应性）用于含有这些修饰的RNA分子。 拟议中的研究采用适体方法直接瞄准这一关键缺陷， 选择方法来鉴定结合化学修饰的RNA的核酸分子。这些适体是 其独特之处在于它们由基于L-（脱氧）核糖的核酸（L-DNA和L-RNA）组成，它们是镜像的。 天然D-核苷酸的图像（对映体）。L-适体，这是完全正交的自然生物学， 非常适合结合RNA靶点。因此，体外筛选将用于分离新的L- 能够结合化学修饰的单核苷酸的适体，其将能够选择性捕获RNA 含有相同修饰残基的分子。这些L-适体然后将用于交联-聚合。 适体下拉和测序（CLAP-seq），第一个转录组范围的分析技术， 在下一代测序之前进行基于适体的RNA富集。CLAP-seq不仅承诺打开一个 这是一种通用和强大的途径，可以对不断增长的RNA修饰列表进行全转录组分析，而且 有望加强我们目前对墓志铭的看法。因此，CLAP-seq的开发将 对表观转录组学领域产生了深远的影响，这与NICHD的使命是一致的 本FOA的目标是：促进研究RNA化学修饰在发育中的作用， 相关疾病。