A novel genetically encodable tagging system for visualizing translation dynamics in living cells

Many diseases, such as cancers and Alzheimer's disease, are caused by disrupted intracellular protein homeostasis. Understanding how proteins are translated is critical for the development of therapeutics that can help maintain protein homeostasis and therefore fight diseases. Over the past six years, the use of repeat epitope tags combined with complementary fluorescent intrabodies, such as the SunTag or the spaghetti monster imaging systems, has made it possible to visualize translation dynamics at the single mRNA level in real time. By applying these tagging systems, many mechanisms of translation have been uncovered. However, the lack of comparable and orthogonal tagging systems has made applications that require multicolor imaging challenging. To address this challenge, we developed a novel genetically encodable tagging system. For this, we grafted complementary determining regions or loops identified through deep mining of the Protein Data Bank to a stable single-chain variable fragment (scFv) scaffold that folds correctly in the reduced intracellular environment. After the loop grafting, the binding of the wildtype scFv was rescued in living cells. Our FRAP data suggests the recovery halftime of this tagging system is ∼5 min, significantly longer than our recently developed anti-HA and anti-FLAG frankenbodies, even though the epitope tag is about the same length. We believe these favorable properties will make this novel tagging system a useful new tool in the expanding live-cell imaging toolbox.