Beyond genetic and epigenetic inheritance, we have applied expertise in single-molecule methodology to many other biological problems. Leveraging the power of single-molecule techniques in capturing transient, stochastic, and heterogeneous molecular events, these studies provide mechanistic insights into dynamic biological processes that are obscured in bulk assays due to ensemble averaging. The single-molecule biophysical experiments in these studies are corroborated by complementary approaches (structural, biochemical, genetic, etc.). These collaborative efforts demonstrate the versatility of the single-molecule toolkit, making it better appreciated by the wider scientific community.