Development and Characterization of 50 nanometer diameter Genetically Encoded Multimeric Nanoparticles

The mechanisms that regulate the physical properties of the cell interior remain poorly understood, especially at the mesoscale. Many crucial macromolecules and molecular assemblies such as ribosomes, RNA polymerase, and biomolecular condensates span the mesoscale size range, and changes in mesoscale physical properties have been suggested to be crucial for both normal physiology and disease. Therefore, we need better tools to study the cellular environment at this scale. Physical properties of the cell can be inferred through analysis of the motion of tracer nanoparticles, an approach called nanorheology. This requires the introduction of nanoparticles to cells, which can be labor intensive and slow. Genetically Encoded Multimeric nanoparticles (GEMs) were recently developed to address this limitation. GEMs consist of scaffold proteins fused to fluorescent tags that self-assemble into bright and stable nanoparticles of defined geometry. However, extremely sensitive microscopes were required to track previous (40nm-GEMs). Here, we describe the development and characterization of 50 nm diameter GEMs (50nm-GEMs) that are brighter and probe a slightly longer length scale. 50nm-GEMs will make high-throughput nanorheology accessible to a broader range of researchers and reveal new insights into the biophysical properties of cells.