Polysome Shadowing

多核糖体阴影

基本信息

批准号：
10574132
负责人：
Joshua Arribere
金额：
$ 19.25万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA CRUZ
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10574132
关键词：
Affect Algorithms Behavior Binding Biological Models Biology Cells Chemicals Complex Computational algorithm Data Detection Development Devices Diagnostic Disease Equipment Free Ribosome Frequencies Goals Health Human In Vitro Individual Investigation Laboratories Length Life Measurement Medical Messenger RNA Methods Modeling Modification Monitor Nucleic acid sequencing Organism Performance Phenotype Polyribosomes Population Process Protein Analysis Protein Biosynthesis Protein Isoforms Protocols documentation RNA Reagent Research Personnel Ribosomal Proteins Ribosomes Sampling Scientist Site System Techniques Technology Time Transcript Translations Visualization Work base clinical application improved in vivo interest mRNA Translation mRNA sequencing markov model model organism nanopore novel ribosome profiling sequencing platform single molecule tool transcriptome transcriptome sequencing translation assay

Affect Algorithms Behavior Binding Biological Models Biology Cells Chemicals Complex Computational algorithm Data Detection Development Devices Diagnostic Disease Equipment Free Ribosome Frequencies Goals Health Human In Vitro Individual Investigation Laboratories Length Life Measurement Medical Messenger RNA Methods Modeling Modification Monitor Nucleic acid sequencing Organism Performance Phenotype Polyribosomes Population Process Protein Analysis Protein Biosynthesis Protein Isoforms Protocols documentation RNA Reagent Research Personnel Ribosomal Proteins Ribosomes Sampling Scientist Site System Techniques Technology Time Transcript Translations Visualization Work base clinical application improved in vivo interest mRNA Translation mRNA sequencing markov model model organism nanopore novel ribosome profiling sequencing platform single molecule tool transcriptome transcriptome sequencing translation assay

展开

项目摘要

Project Summary/Abstract The synthesis of proteins takes place by ribosomal translation of mRNA molecules. Despite its central importance in biology, there are only a handful of techniques to analyze ribosome::mRNA complexes in vivo, and none of these are able to identify sites of ribosome binding on intact and individual mRNA molecules. A facile technique to simultaneously sequence mRNA molecules and visualize sites of ribosome::mRNA interactions would greatly expand the ability to study protein synthesis, with impacts from model organism biology through human clinical applications. This application’s broad, long-term objective is to develop a technology to simultaneously identify all sites of ribosome binding on a single mRNA molecule, sequence the mRNA molecule, and do so across all mRNA molecules in a sample. To achieve the objective, we will combine the relatively old technology of chemical footprinting to mark sites of ribosome::mRNA interaction and the nascent technology of single molecule mRNA sequencing on the Oxford nanopore device to identify modification sites. Specifically, we will: (1) Identify appropriate chemical footprinting reagents and evaluate nanopore performance in modification detection and sequence identification on RNAs treated with the reagents; (2) Chemically footprint ribosome::mRNA complexes, and compare information obtained to the existing state of the art (ribosome footprint profiling); (3) Develop computational algorithms to identify footprints via statistical inference, using both simulated and real-world data. Results from these aims will provide critical data on the feasibility of ribosome footprinting with whole transcript single molecule sequencing. The resulting protocol will equip researchers and clinicians to visualize and quantify facets of protein synthesis in their experimental system of choice for diagnostic purposes, high-throughput phenotyping, and/or mechanistic analysis of protein synthesis.

项目摘要/摘要蛋白质的合成是通过mRNA分子的核糖体翻译进行的。尽管有它在生物学中的核心重要性，只有少数技术可以分析核糖体:: mRNA复合体在体内，并且这些都无法识别核糖体位点对完整和单个mRNA分子的结合。同时轻松的技术序列mRNA分子和可视化核糖体位点:: mRNA相互作用将极大扩大研究蛋白质合成的能力，并通过模型有机体生物学的影响人类临床应用。该应用程序的广泛长期目标是开发在单个mRNA分子上同时识别所有核糖体结合的技术，对mRNA分子进行序列，并在样品中的所有mRNA分子中这样做。实现目的，我们将将化学足迹的相对旧技术结合到标记地点核糖体:: mRNA相互作用和单分子mRNA的新生技术在牛津纳米孔设备上进行测序以识别修饰位点。具体来说，我们将：（1）确定适当的化学足迹试剂并评估纳米孔的性能用试剂处理的RNA上的修改检测和序列鉴定；（2）化学上足迹核糖体:: mRNA复合物，并比较获得的信息现有的艺术状态（核糖体足迹分析）；（3）开发计算算法使用模拟和现实世界数据通过统计推断识别足迹。结果这些目标将提供有关核糖体足迹的可行性的关键数据转录本单分子测序。最终的协议将为研究人员和临床医生在其实验系统中可视化和量化蛋白质合成的面出于诊断目的，高通量表型和/或机械分析的选择蛋白质合成。