Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances.
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.
Lee
Nam Ki
NK
School of Chemistry, Seoul National University, Seoul, Korea.
Kapanidis
Achillefs N
AN
Koh
Hye Ran
HR
Korlann
You
Y
Ho
Sam On
SO
Kim
Younggyu
Y
Gassman
Natalie
N
Kim
Seong Keun
SK
Weiss
Shimon
S
D001483:Base Sequence; D001703:Biophysics / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000737:chemistry; D053938:DNA Fragmentation; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D007834:Lasers; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D021381:Protein Transport; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014158:Transcription, Genetic
D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D004247:DNA; D012321:DNA-Directed RNA Polymerases