A fluorogenic array for temporally unlimited single-molecule tracking

Nat Chem Biol. 2019 Apr;15(4):401-409. doi: 10.1038/s41589-019-0241-6. Epub 2019 Mar 11.

Abstract

We describe three optical tags, ArrayG, ArrayD and ArrayG/N, for intracellular tracking of single molecules over milliseconds to hours. ArrayG is a fluorogenic tag composed of a green fluorescent protein-nanobody array and monomeric wild-type green fluorescent protein binders that are initially dim but brighten ~26-fold on binding with the array. By balancing the rates of binder production, photobleaching and stochastic binder exchange, we achieve temporally unlimited tracking of single molecules. High-speed tracking of ArrayG-tagged kinesins and integrins for thousands of frames reveals novel dynamical features. Tracking of single histones at 0.5 Hz for >1 hour with the import competent ArrayG/N tag shows that chromosomal loci behave as Rouse polymers with visco-elastic memory and exhibit a non-Gaussian displacement distribution. ArrayD, based on a dihydrofolate reductase nanobody array and dihydrofolate reductase-fluorophore binder, enables dual-color imaging. The arrays combine brightness, fluorogenicity, fluorescence replenishment and extended fluorophore choice, opening new avenues for tracking single molecules in living cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cell Line
  • Color
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry*
  • Green Fluorescent Proteins
  • HeLa Cells
  • Humans
  • Microscopy, Fluorescence / methods*
  • Single Molecule Imaging / methods*
  • Single-Domain Antibodies

Substances

  • Fluorescent Dyes
  • Single-Domain Antibodies
  • Green Fluorescent Proteins