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Updated: Nov 16, 2025
Author Spotlight: Evaluation of Protein-Condensate Dynamics in Live Human Cells
Published on: January 5, 2024
Single-molecule and ensemble methods to probe RNP nucleation and condensate properties
Kevin Rhine1, Sophie Skanchy2, Sua Myong3
1Program in Cell, Molecular, Developmental Biology, and Biophysics, Johns Hopkins University, 3400 N Charles St, Baltimore, MD 21218, United States; Department of Biology, Johns Hopkins University, 3400 N Charles St, Baltimore, MD 21218, United States.
We developed new single-molecule and ensemble methods to quantify the nucleation and condensation of biomolecular condensates. These techniques provide a comprehensive understanding of protein-RNA interactions driving phase separation in cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Biomolecular condensates, crucial for cellular function, are formed by intrinsically disordered proteins and RNA.
- The processes of nucleation, condensation, and maturation of these condensates are not well understood.
- Understanding these dynamics is key to deciphering cellular organization and function.
Purpose of the Study:
- To present novel single-molecule and ensemble methods for quantifying condensate formation.
- To detail the design and execution of a single-molecule nucleation assay for protein-RNA interactions.
- To provide tools for comprehensive analysis of phase separation dynamics.
Main Methods:
- Development of a single-molecule nucleation assay using fluorescently labeled RNA-binding proteins.
- Detection of individual protein-RNA associations with an RNA substrate.
- Complementary use of meso-scale techniques to measure condensate biophysical properties.
Main Results:
- Quantification of nucleation kinetics for individual protein-RNA association events.
- Comprehensive characterization of condensate formation and maturation processes.
- Demonstration of the utility of the methods for studying diverse protein-RNA interactions.
Conclusions:
- The presented single-molecule and ensemble methods offer a powerful approach to study biomolecular condensate formation.
- These techniques enable detailed kinetic analysis of nucleation and condensation.
- The methods are broadly applicable to various protein-RNA interactions driving cellular phase separation.
