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

Ki Lee Nam, Achillefs N. Kapanidis, Ran Koh Hye, You Korlann, On Ho Sam, Younggyu Kim, Natalie Gassman, Keun Kim Seong, Shimon Weiss

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158 Scopus citations


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.

Original languageEnglish
Pages (from-to)303-312
Number of pages10
JournalBiophysical Journal
Issue number1
StatePublished - Jan 2007
Externally publishedYes

Bibliographical note

Funding Information:
This work was funded by a National Research Laboratory grant and a Chemical Genomics grant of the Korean Science and Engineering Foundation and Ministry of Science and Technology to S.K.K., and National Institutes of Health grant GM65382 and GM069709-01A1 and Department of Energy grants FG03-02ER6339 and 04ER63938 to S.W.


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