Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells

Cell. 2019 Jul 11;178(2):491-506.e28. doi: 10.1016/j.cell.2019.05.029. Epub 2019 May 30.


Transforming the vast knowledge from genetics, biochemistry, and structural biology into detailed molecular descriptions of biological processes inside cells remains a major challenge-one in sore need of better imaging technologies. For example, transcription involves the complex interplay between RNA polymerase II (Pol II), regulatory factors (RFs), and chromatin, but visualizing these dynamic molecular transactions in their native intracellular milieu remains elusive. Here, we zoom into single tagged genes using nanoscopy techniques, including an active target-locking, ultra-sensitive system that enables single-molecule detection in addressable sub-diffraction volumes, within crowded intracellular environments. We image, track, and quantify Pol II with single-molecule resolution, unveiling its dynamics during the transcription cycle. Further probing multiple functionally linked events-RF-chromatin interactions, Pol II dynamics, and nascent transcription kinetics-reveals detailed operational parameters of gene-regulatory mechanisms hitherto-unseen in vivo. Our approach sets the stage for single-molecule studies of complex molecular processes in live cells.

Publication types

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

MeSH terms

  • Cell Line, Tumor
  • Chromatin / metabolism
  • Genes, Reporter
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism
  • Humans
  • Kinetics
  • Mutagenesis
  • Nanotechnology*
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism*
  • Single Molecule Imaging / methods*
  • Single-Domain Antibodies / chemistry
  • Single-Domain Antibodies / immunology
  • Time-Lapse Imaging
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transcription, Genetic*


  • Chromatin
  • Single-Domain Antibodies
  • Transcription Factors
  • RNA Polymerase II