SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Three-Color Alternating-Laser Excitation of Single Molecules: Monitoring Multiple Interactions and Distances
Nam Ki Lee 1, Achillefs N. Kapanidis 2, Hye Ran Koh 1, You Korlann 3, Sam On Ho 3, Younggyu Kim 3, Natalie Gassman 3, Seong Keun Kim 1* and Shimon Weiss 4
1 Seoul National University
2 University of Oxford
3 University of California, Los Angeles
4 University of California Los Angeles
* To whom correspondence should be addressed. E-mail: seongkim{at}snu.ac.kr.
Submitted on July 13, 2006
Revised on August 17, 2006
Accepted on 11 September 2006
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Abstract |
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We introduce 3-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. 3c-ALEX extends substantially the capabilities of 2-color ALEX, which employs two alternating lasers to study molecular interactions (through probe stoichiometry S) and intramolecular distances (through FRET efficiency E). 3c-ALEX 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 (i) FRET-independent analysis of three-component interactions; (ii) observation and sorting of singly, doubly, and triply labeled molecules simultaneously present in solution; (iii) measurements of three intramolecular distances within single molecules from a single measurement; and (iv) 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.
Key Words:
FRET, alternating-laser excitation, biomolecular interactions, molecular sorting, single-molecule fluorescence spectroscopy, transcription complexes
