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Single-molecule and ensemble methods to probe RNP nucleation and condensate properties
- Source :
- Methods
- Publication Year :
- 2022
- Publisher :
- Elsevier BV, 2022.
-
Abstract
- Biomolecular condensates often consist of intrinsically disordered protein and RNA molecules, which together promote the formation of membraneless organelles in cells. The nucleation, condensation, and maturation of condensates is a critical yet poorly understood process. Here, we present single-molecule and accompanying ensemble methods to quantify these processes more comprehensively. In particular, we focus on how to properly design and execute a single-molecule nucleation assay, in which we detect signals arising from individual units of fluorescently labeled RNA-binding proteins associating with an RNA substrate. The analysis of this data allows one to determine the kinetics involved with each step of nucleation. Complemented with meso-scale techniques that measure the biophysical properties of ribonucleoprotein condensates, the methods described herein are powerful tools that can be adopted for studying any protein-RNA interactions that promote phase separation.
- Subjects :
- 0303 health sciences
Chemistry
030302 biochemistry & molecular biology
Condensation
Kinetics
Nucleation
RNA-Binding Proteins
RNA
Ensemble learning
Article
General Biochemistry, Genetics and Molecular Biology
Intrinsically Disordered Proteins
03 medical and health sciences
Ribonucleoproteins
Chemical physics
Molecule
Molecular Biology
030304 developmental biology
Ribonucleoprotein
Subjects
Details
- ISSN :
- 10462023
- Volume :
- 197
- Database :
- OpenAIRE
- Journal :
- Methods
- Accession number :
- edsair.doi.dedup.....a3102ca179d71899d9bf2d89ad6cf491