Polysome Shadowing

项目摘要/摘要 蛋白质的合成是通过核糖体翻译信使核糖核酸分子进行的。尽管它的 在生物学中的核心重要性，只有少数几种技术可以分析 体内存在核糖体：：mRNA复合体，但这些复合体都不能识别核糖体的位置 结合完整和单个的信使核糖核酸分子。一种简便的技术，可以同时 对核糖体：：mRNA相互作用的mRNA分子进行测序和位置可视化将大大 扩展研究蛋白质合成的能力，通过 人类临床应用。这个应用程序的广泛、长期目标是开发一个 同时识别核糖体结合在单个mRNA分子上的所有位置的技术， 对信使核糖核酸分子进行测序，并对样本中的所有信使核糖核酸分子进行测序。要实现 目标是，我们将结合相对较老的化学足迹技术来标记地点 核糖体：：信使核糖核酸相互作用与单分子信使核糖核酸的新生技术 对牛津纳米孔装置进行测序，以确定修饰位置。具体来说，我们会：(1) 确定合适的化学足迹试剂并评估纳米孔性能 用该试剂处理的RNA的修饰检测和序列鉴定； 核糖体：：mRNA复合体的化学足迹，并将获得的信息与 现有技术水平(核糖体足迹图谱)；(3)开发计算算法以 使用模拟和真实世界的数据，通过统计推断来识别足迹。结果 从这些目标将提供核糖体足迹与全长的可行性的关键数据 转录单分子测序。由此产生的议定书将为研究人员和 临床医生在他们的实验系统中可视化和量化蛋白质合成的各方面 用于诊断、高通量表型鉴定和/或机理分析的选择 蛋白质合成。

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.