利用共定位单分子光谱技术（CoSMoS）对前体mRNA剪接进行单分子分析。

Single-Molecule Analysis of Pre-mRNA Splicing with Colocalization Single-Molecule Spectroscopy (CoSMoS).

作者信息

Braun Joerg E, Serebrov Victor

机构信息

RNA Therapeutics Institute, Howard Hughes Medical Institute, and Department of Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, 01605, USA.

出版信息

Methods Mol Biol. 2017;1648:27-37. doi: 10.1007/978-1-4939-7204-3_3.

DOI:10.1007/978-1-4939-7204-3_3

PMID:28766287

Abstract

Recent development of single-molecule techniques to study pre-mRNA splicing has provided insights into the dynamic nature of the spliceosome. Colocalization single-molecule spectroscopy (CoSMoS) allows following spliceosome assembly in real time at single-molecule resolution in the full complexity of cellular extracts. A detailed protocol of CoSMoS has been published previously (Anderson and Hoskins, Methods Mol Biol 1126:217-241, 2014). Here, we provide an update on the technical advances since the first CoSMoS studies including slide surface treatment, data processing, and representation. We describe various labeling strategies to generate RNA reporters with multiple dyes (or other moieties) at specific locations.

摘要

近期用于研究前体信使核糖核酸（pre-mRNA）剪接的单分子技术进展，为剪接体的动态特性提供了深入见解。共定位单分子光谱法（CoSMoS）能够在细胞提取物的完整复杂性下，以单分子分辨率实时追踪剪接体组装过程。CoSMoS的详细方案先前已发表（安德森和霍斯金斯，《分子生物学方法》1126:217 - 241，2014年）。在此，我们提供自首次CoSMoS研究以来的技术进展更新，包括载玻片表面处理、数据处理和呈现方式。我们描述了各种标记策略，以在特定位置生成带有多种染料（或其他基团）的RNA报告分子。