Imaging specific proteins in living cells with small unnatural amino acid attached Raman reporters

Fluorescence labeling via fluorescent proteins (FPs) or immunofluorescence has been routinely applied for microscopic imaging of specific proteins. However, due to these over-weight and oversized labels (e.g. GFP, 238 aa, 27 kDa, ∼4 nm in size), the potential physiological malfunctions of the target proteins are largely underestimated in living cells. Herein, for living cells, we report a small and minimally-invasive Raman reporter (about 2 aa and <1 kDa), which can be site-specifically introduced into proteins by genetic codon expansion. After a single unnatural amino acid (UAA) is precisely incorporated into the target protein, the strained alkyne can rapidly undergo copper-free Diels–Alder cycloaddition reactions with the tetrazine-functionalized Raman reporter, which features a fine vibrational spectrum in contrast to fluorescence. In our experimental results, the UAA-based Raman tag was successfully incorporated into vimentin, histone 3.3 and huntingtin (Htt74Q) proteins in living HeLa cells and further utilized for stimulated Raman imaging. The site-specific bioorthogonal fusion of small Raman tags with intracellular proteins will pave the way for minimally-invasive protein labeling and multi-color imaging in living cells.