The growing demands of advanced fluorescence and super-resolution microscopy benefit from the development of small and highly photostable fluorescent dyes. However, direct attachment of organic dyes to the protein of interest remains challenging. This challenge can be addressed by labelling unnatural amino acids (UAAs) with click chemistry.
Techniques developed to expand the genetic code permit the residue-specific encoding of UAAs armed with “clickable” chemical handles into proteins. Such UAAs can be labeled with small organic dyes by bioorthogonal click chemistry in living cells. The minimal size of the UAA and the possibility to couple the fluorophores directly to the protein of interest with single-residue precision in living cells are the main advantages of this labelling approach. However, until recently, click chemistry-based protein labelling has only been used in conventional cell lines, and not in complex, non-dividing cells, such as primary neurons.
In this lecture, I will provide a brief introduction to the development of genetic code expansion technology. I will then discuss the application of click chemistry-based protein labeling in microscopy studies of mammalian cells, also highlighting our recent work on labeling various neuronal protein classes, including cytoskeletal elements and ion channels.
