Live cell PNA labelling enables erasable fluorescence imaging of membrane proteins

Nat Chem. 2021 Jan;13(1):15-23. doi: 10.1038/s41557-020-00584-z. Epub 2020 Dec 7.

Abstract

DNA nanotechnology is an emerging field that promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress is the ability to create a nucleic acid-protein junction in the context of living cells. Here we report a covalent labelling reaction that installs a biostable peptide nucleic acid (PNA) tag. The reaction proceeds within minutes and is specific for proteins carrying a 2 kDa coiled-coil peptide tag. Once installed, the PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization. We demonstrate the versatility of this approach by recruiting different fluorophores, assembling multiple fluorophores for increased brightness and achieving reversible labelling by way of toehold-mediated strand displacement. Additionally, we show that labelling can be carried out using two different coiled-coil systems, with epidermal growth factor receptor and endothelin receptor type B, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of epidermal growth factor receptor on CHO cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • ErbB Receptors / chemistry
  • ErbB Receptors / genetics
  • ErbB Receptors / metabolism*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Microscopy, Fluorescence*
  • Nucleic Acid Hybridization
  • Peptide Nucleic Acids / chemical synthesis
  • Peptide Nucleic Acids / chemistry*
  • Peptide Nucleic Acids / metabolism
  • Peptides / chemical synthesis
  • Peptides / chemistry
  • Peptides / metabolism
  • Protein Binding
  • Receptor, Endothelin B / chemistry
  • Receptor, Endothelin B / genetics
  • Receptor, Endothelin B / metabolism*
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification

Substances

  • Peptide Nucleic Acids
  • Peptides
  • Receptor, Endothelin B
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • ErbB Receptors

Associated data

  • figshare/10.6084/m9.figshare.c.5127728