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Three-Color Alternating-Laser Excitation of Single Molecules: Monitoring Multiple Interactions and Distances

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

^* School of Chemistry, Seoul National University, Seoul, Korea; Department of Chemistry and Biochemistry, and Department of Physiology, University of California, Los Angeles, California; and Clarendon Laboratory, Department of Physics, and IRC in Bionanotechnology, University of Oxford, Oxford, United Kingdom

Correspondence: Address reprint requests to Seong Keun Kim, School of Chemistry, Seoul National University, Seoul 151-747, Korea. Tel.: 82-2-880-6659; Fax: 82-2-889-5719; E-mail: seongkim{at}snu.ac.kr; or to Shimon Weiss, Dept. of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Dr., E. Los Angeles, CA 90095. Tel.: 310-794-0093; Fax: 310-267-4672; E-mail: sweiss{at}chem.ucla.edu.

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.

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