Engineering Hydrogel Production in Mammalian Cells to Synthetically Mimic RNA Granules

Recent studies revealed the biological significance of dynamic multicomponent assemblies of biomolecules inside living cells. Protein and nucleic acid assemblies are biomolecular condensates or non-membrane-bound organelles that have attracted increasing attention. Synthetic tools that manipulate the dynamic assembly/disassembly process of the structures are useful in elucidating both biophysical mechanisms of their assembly/disassembly and physiological roles of the condensates. In this report, general protocols to form and observe synthetic polymer-based condensates in living cells are described using the tool iPOLYMER. Taking advantage of the modular design of the tool, both chemical and light stimuli can induce formation of synthetic condensates inside living cells, which are observed by laser-scanning confocal microscopy. The experimental design described herein should help those who conduct experiments on synthetic manipulation of biomolecular condensates using iPOLYMER and other tools for synthetic manipulation of condensates. Technical notes for using iPOLYMER, including basic protocols of chemical- or light-inducible dimerization techniques (CID/LID), choice of proper control experiments, and advantages/disadvantages are also presented.