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

Analyst. 2024 Nov 4;149(22):5476-5481. doi: 10.1039/d4an00758a.

Abstract

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.

MeSH terms

  • Alkynes* / chemistry
  • Amino Acids* / chemistry
  • Cycloaddition Reaction*
  • HeLa Cells
  • Histones / chemistry
  • Histones / metabolism
  • Humans
  • Huntingtin Protein / chemistry
  • Huntingtin Protein / genetics
  • Huntingtin Protein / metabolism
  • Spectrum Analysis, Raman* / methods
  • Vimentin / chemistry
  • Vimentin / metabolism

Substances

  • Amino Acids
  • Alkynes
  • Histones
  • Huntingtin Protein
  • Vimentin
  • HTT protein, human