Methodologies for studying the spliceosome's RNA dynamics with single-molecule FRET

The spliceosome is an extraordinarily dynamic molecular machine in which significant changes in composition as well as protein and RNA conformation are required for carrying out pre-mRNA splicing. Single-molecule fluorescence resonance energy transfer (smFRET) can be used to elucidate these dynamics both in well-characterized model systems and in entire spliceosomes. These types of single-molecule data provide novel information about spliceosome components and can be used to identify sub-populations of molecules with unique behaviors. When smFRET is combined with single-molecule fluorescence colocalization, conformational dynamics can be further linked to the presence or absence of a given spliceosome component. Here, we provide a description of experimental considerations, approaches, and workflows for smFRET with an emphasis on applications for the splicing machinery.