A quick and simplified method (QuickRibo-mRNA) for isolation of ribosome protected mRNA fragments for translatome identification

一种快速简化的方法 (QuickRibo-mRNA)，用于分离核糖体保护的 mRNA 片段以进行翻译组鉴定

• 批准号: 10325082
• 负责人: Prashant K. Khade
• 金额: $ 30.18万
• 依托单位: RIBO-THERAPEUTICS LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2022-12-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10325082
• 关键词: 2019-nCoV; Address; Affect; Animals; Back; Benchmarking; Biology; Buffers; California; Categories; Cell Count; Cell physiology; Cells; Code; Codon Nucleotides; Complex; Consumption; Cycloheximide; Cytolysis; Data; Development; Diagnostic Reagent Kits; Disease; Elements; Filtration; Future; Gel; Gene Expression; Genes; Genetic Translation; Genome; Genomics; Goals; Impact evaluation; Intercistronic Region; Italy; Length; Libraries; Location; Manufacturer Name; Maps; Measurement; Messenger RNA; Methodology; Methods; MicroRNAs; Modeling; Molecular Weight; Monitor; Morphologic artifacts; Neurology; Nucleotides; Open Reading Frames; Pathogenesis; Peptides; Periodicity; Phase; Phenotype; Physiological; Plants; Positioning Attribute; Preparation; Principal Investigator; Production; Proteins; Proteomics; Puromycin; RNA; RNA library; RNA purification; Reading Frames; Reproducibility; Research Personnel; Resolution; Ribonucleases; Ribosomes; Sensitivity and Specificity; Small RNA; Techniques; Technology; Therapeutic; Time; Tissue Sample; Tissues; Transcript; Translating; Translations; Universities; Untranslated Regions; Virus; Work; base; cancer immunotherapy; cancer therapy; cell type; commercialization; comparative efficacy; coronavirus disease; cost; cost effective; data quality; deep sequencing; efficacy evaluation; gel electrophoresis; improved; in vivo; insight; instrument; instrumentation; neoantigens; novel; novel virus; nuclease; professor; repaired; ribosome profiling; therapeutic RNA; trait; transcriptome; transcriptome sequencing; translatome; tumor immunology; web site

The goal of this proposal is to establish and validate a novel method for the isolation of ribosome protected mRNA footprints for translatome identification. Translatome or Ribosome profiling (RP), a deep sequencing technique of ribosome-protected mRNA footprints, is a powerful method that gives a “snapshot” of actively translating mRNA. Revolutionizing the capacity to capture every step of mRNA translation at sub-codon resolution, it potentially allows one to monitor the rate and location of mRNA translation in the cell and annotate the new protein coding sequences of genomes. RP, therefore, affords a vast opportunity to explore and model complex cellular processes in real time. This methodology has been mined extensively in numerous diseases including cancer, immunology and neurology. RP identified several new micro-peptides and protein from COVID mRNA, further expanding its potential as a novel target-identification method. In this context, RP has emerged as a potent method to identify mRNA frameshifting and novel ORFs, and providing valuable insights for understanding functional elements of the genome, disease pathogenesis and evaluation of impact treatment in the case of mRNA therapeutics and cancer therapy in a gene and the tissues specific manner. However, RP applications are stunted and limited to specialized labs because it requires expertise, specialized instruments and time-consuming - taking up to ~5 days on an average. In turn, this affects the cost, quality of the final data and thereby obscuring the mRNA translational landscape. Given the power and potential for widespread use, there is an urgent and unmet need for alternative/improved RP technology similar to RNA purification methods which are less time consuming, free of artifacts and inexpensive. In Phase-I application, we proposed two specific aims to refine the QuickRibo-mRNA method, validate and compare it with current RP methods to establish its efficacy. In aim-1 we will establish and validate the QuickRibo-mRNA methodology in various conditions and cell types in obtaining uniform ribosome protected mRNA fragments with improved percent read mapping to coding sequences. In aim-2 we will compare the efficacy of QuickRibo-mRNA with known ribosome profiling technologies. We believe that phase-I proposal will provide a benchmark for phase II, which can streamline the QuickRibo-mRNA method for performing ribosome profiling with high reproducibility and efficiency. In the end RP will be done proficiently in the simplest way with less time, instrumentation and expertise to identify gene “expression with reading frames and translation efficiency” before making any conclusions entirely based on RNA-gene “expression profile”.

该提案的目的是建立和验证一种新的方法，以隔离核糖体受保护 转换识别的mRNA足迹。 核糖体受到保护的mRNA足迹的技术是一种有力的方法 翻译mRNA。 解决方案，它有可能允许一个人监视mRNA翻译在细胞中的速率和位置并注释 因此，新的蛋白质编码序列RP为探索和模型提供 实时的复杂蜂窝过程。 包括癌症，免疫学和神经学。 mRNA，进一步扩展了其作为这种内容的新目标 ASA有效的方法来识别mRNA帧和11月，并提供有价值的见解 了解基因组，疾病发病机理和评估信任的功能元素 然而，mRNA疗法和癌症治疗以特定于ANE组织的方式 申请被发育不良并限于专业实验室，因为它需要专业知识，专业的工具 时间 - 耗时 - 依次最多5天，这会影响最终数据的成本，质量 从而掩盖了mRNA翻译景观。 对于替代/改进的RP技术，类似于RNA纯化方法，有一个紧急且未列出的RP技术 在这里，耗时较少，没有工件和便宜。 旨在完善QuickRibo-MRNA方法，验证并将其与当前的RP方法进行比较以建立其 疗效。 获得均匀核糖体保护的mRNA片段的类型，并改善百分比读取映射到编码 序列。 技术。我们认为，I期建议将为II期提供一个基准 QuickRibo-MRNA方法最终以繁殖和效率进行核糖体分析 RP将以最简单的方式熟练地完成，以更少的时间，仪器和专业知识来识别基因 在基于任何结论之前，“具有阅读帧和翻译效率的表达” RNA-GENE“ Express Croper”。