Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances

Biophys J. 2007 Jan 1;92(1):303-12. doi: 10.1529/biophysj.106.093211. Epub 2006 Oct 13.


We introduce three-color alternating-laser excitation (3c-ALEX), a fluorescence resonance energy transfer (FRET) method that measures up to three intramolecular distances and complex interaction stoichiometries of single molecules in solution. This tool extends substantially the capabilities of two-color ALEX, which employs two alternating lasers to study molecular interactions (through probe stoichiometry S) and intramolecular distances (through FRET efficiency E), and sorts fluorescent molecules in multi-dimensional probe-stoichiometry and FRET-efficiency histograms. Probe-stoichiometry histograms allowed analytical sorting, identification, and selection of diffusing species; selected molecules were subsequently represented in FRET-efficiency histograms, generating up to three intramolecular distances. Using triply labeled DNAs, we established that 3c-ALEX enables 1), FRET-independent analysis of three-component interactions; 2), observation and sorting of singly, doubly, and triply labeled molecules simultaneously present in solution; 3), measurements of three intramolecular distances within single molecules from a single measurement; and 4), dissection of conformational heterogeneity with improved resolution compared to conventional single-molecule FRET. We also used 3c-ALEX to study large biomolecules such as RNA polymerase-DNA transcription complexes, and monitor the downstream translocation of RNA polymerase on DNA from two perspectives within the complex. This study paves the way for advanced single-molecule analysis of complex mixtures and biomolecular machinery.

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

  • Base Sequence
  • Biophysics / instrumentation
  • Biophysics / methods*
  • DNA / chemistry
  • DNA Fragmentation
  • DNA-Directed RNA Polymerases / chemistry
  • Diffusion
  • Fluorescence Resonance Energy Transfer / instrumentation*
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / pharmacology
  • Lasers
  • Molecular Conformation
  • Molecular Sequence Data
  • Oligodeoxyribonucleotides / chemistry
  • Protein Transport
  • Spectrometry, Fluorescence / methods*
  • Transcription, Genetic


  • Fluorescent Dyes
  • Oligodeoxyribonucleotides
  • DNA
  • DNA-Directed RNA Polymerases