URIL tags for intracellular RNA tracking and RNP proximity labeling

用于细胞内 RNA 追踪和 RNP 邻近标记的 URIL 标签

• 批准号: 10738661
• 负责人: Dennis Bong
• 金额: $ 42.47万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-27 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10738661
• 关键词: ALS pathology; Adolescent; Amyotrophic Lateral Sclerosis; Antibodies; Area; Attention; Benchmarking; Binding; Biology; Biotin; Biotinylation; C9ALS; C9ORF72; Cell Line; Cells; Chemicals; Complex; Cytoplasmic Granules; Data; Diagnostic; Disease; Dyes; Evaluation; Face; Fluorescence; Genetic Transcription; Goals; In Vitro; Investigation; Label; Link; Location; Methods; Modification; Neurodegenerative Disorders; Nucleic Acid Probes; Pathologic; Patients; Peptide Nucleic Acids; Plasmids; Property; Prosthesis; Proteins; Publishing; RNA; RNA Folding; RNA-Binding Proteins; Regulatory Pathway; Research; Ribonucleoproteins; Site; Streptavidin; Structure; Subcellular structure; System; Testing; Therapeutic; Transcript; Transfection; Validation; Variant; Western Blotting; cellular imaging; crosslink; early onset; insight; nucleic acid structure; protein TDP-43; tool; trafficking

PROJECT SUMMARY / ABSTRACT If a facile method to site-selectively install prosthetic groups at internal sites in genetically-encoded RNA were available, then it would be possible to modify RNAs and ribonucleoprotein complexes (RNPs) in native structural and intracellular context, thus elevating studies on RNA folding, trafficking, lifetime, interactomes and regulatory pathways. The objective of this application is to test the extent to which compact U-rich internal loop (URIL) sites can be used as a general targeting motif in structured RNAs. If it were possible to selectively target the URIL motif with chemical probes, then juxtaposition of the URIL site with protein binding RNA motifs would enable tracking and chemical modification of ribonucleoprotein complexes (RNPs). This would enable elucidation of motif-specific RNA location and interactome by fluorogenic and proximity (biotin) labeling of URIL RNPs; such unbiased motif-centered interactome readout is not possible with existing methods. We hypothesize that appropriately modified, URIL-targeting bifacial peptide nucleic acids (bPNAs) could enable intracellular fluorogenic URIL (FLURIL) RNA tracking and proximity labeling of URIL (PLURIL) RNPs, respectively. Our proposed plan begins with the synthesis of bPNA probes, followed by rigorous in vitro and intracellular evaluation, optimization and validation with existing tools and known interactome partners. FLURIL RNP tagging will be benchmarked against MS2-labeling, the gold standard in RNA tracking. PLURIL tagging will be tested by its efficacy in identification of known RNPs. Further, we will test the extent to which URIL tags can be used to probe disease-relevant RNP biology in the intracellular context of amyotrophic lateral sclerosis (ALS), using patient-derived cells. Investigation of ALS pathology is a highly active area, with attention focused on two major forms: C9-ALS and Fus-linked ALS. While C9-ALS represents a majority of ALS cases, Fus-linked ALS is most commonly found in juvenile, aggressive early-onset cases; notably, the pathological mechanisms of these two forms appear to be distinct. Dysregulated RNP biology centered on C9orf72 RNA (C9-ALS) and U1snRNA (Fus-linked ALS) identifies these transcripts as prime substrates for URIL tag probes. The rigor in the prior research lies in the substantive preliminary and published data supporting intracellular fluorogenic and proximity labeling of URIL-RNPs. These data form a strong scientific premise for the impactful and unique application of motif-specific URIL-tagging as a broadly enabling discovery tool in ALS pathology and other RNP-centered diseases.

项目概要/摘要 如果有一种简便的方法可以在基因编码 RNA 的内部位点选择性地安装假体基团 可用，那么就有可能修饰天然的 RNA 和核糖核蛋白复合物 (RNP) 结构和细胞内环境，从而提升对 RNA 折叠、运输、寿命、相互作用组和 监管途径。此应用的目的是测试紧凑的富 U 内部循环的程度 (URIL) 位点可用作结构化 RNA 中的通用靶向基序。如果可以选择性地 用化学探针靶向 URIL 基序，然后将 URIL 位点与蛋白质结合 RNA 基序并置 将使核糖核蛋白复合物（RNP）的跟踪和化学修饰成为可能。这将使 通过 URIL 的荧光和邻近（生物素）标记阐明基序特异性 RNA 位置和相互作用组 RNP；现有方法不可能实现这种以基序为中心的无偏见相互作用组读出。我们 假设经过适当修饰、靶向 URIL 的双面肽核酸 (bPNA) 可以实现 胞内荧光 URIL (FLURIL) RNA 追踪和 URIL (PLURIL) RNP 的邻近标记， 分别。我们提出的计划从 bPNA 探针的合成开始，然后是严格的体外和 使用现有工具和已知的相互作用组伙伴进行细胞内评估、优化和验证。氟里尔 RNP 标记将以 MS2 标记为基准，MS2 标记是 RNA 追踪的黄金标准。 PLURIL 标记 将通过其识别已知 RNP 的功效进行测试。此外，我们将测试 URIL 标记的程度 可用于探索肌萎缩侧索硬化症细胞内疾病相关的 RNP 生物学 （ALS），使用患者来源的细胞。 ALS 病理学研究是一个高度活跃的领域，备受关注 两种主要形式：C9-ALS 和 Fus 相关的 ALS。虽然 C9-ALS 代表了 ALS 病例的大多数， Fus相关的ALS最常见于青少年、侵袭性早发病例；值得注意的是，病理性 这两种形式的机制似乎不同。以 C9orf72 RNA 为中心的 RNP 生物学失调 (C9-ALS) 和 U1snRNA（Fus 连接的 ALS）将这些转录物识别为 URIL 标签探针的主要底物。 先前研究的严谨性在于支持细胞内的实质性初步和已发表的数据 URIL-RNP 的荧光和邻近标记。这些数据为有影响力的研究提供了强有力的科学前提 主题特异性 URIL 标记的独特应用作为 ALS 病理学中广泛的发现工具 和其他以 RNP 为中心的疾病。