High-resolution fluorescence imaging, combined with the latest labeling techniques available today – have yet been able to provide the necessary spatiotemporal resolution to record all cellular processes in live cells.
Fluorescent proteins (Fl-proteins) such as GFP had a vast impact on biology following their discovery, as they were the first to enable the study of proteins in live cells. However, Fl-proteins have many drawbacks in the super resolution microscopy era. Fluorescent organic dyes (Fl-dyes), which offer improved photophysical capabilities, are a good substitute to Fl-proteins. However today, all available methods for attaching Fl-dyes to proteins in live cells still relies on the addition of a relatively large protein tag, which in turn reinstate one of the major drawbacks of Fl-proteins (their size).
Based on recent advancement in genetic code expansion (AMBER suppression) and bioorthogonal chemistry, we are developing an innovative system to specifically label cellular proteins with Fl-dyes in live cells. This is done by directly, and non-invasively, attaching the small Fl-dye to a selected amino acid site on a given protein.
Reducing the size of the tag to a minimum and utilizing the superb photophysical parameters of Fl-dyes will offer a unique tool to study cellular proteins in live cells at close-to-physiological conditions.
We will present data showing the development and feasibility of the approach, and will demonstrate its applicability and efficiency using our benchmark protein α-tubulin.
