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

@article{citeulike:2613587, abstract = {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.}, address = {School of Chemistry, Seoul National University, Seoul, Korea.}, author = {Lee, N. K. and Kapanidis, A. N. and Koh, H. R. and Korlann, Y. and Ho, S. O. and Kim, Y. and Gassman, N. and Kim, S. K. and Weiss, S.}, citeulike-article-id = {2613587}, citeulike-linkout-0 = {http://dx.doi.org/10.1529/biophysj.106.093211}, citeulike-linkout-1 = {http://www.biophysj.org/cgi/content/abstract/92/1/303}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/17040983}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=17040983}, day = {1}, doi = {10.1529/biophysj.106.093211}, issn = {0006-3495}, journal = {Biophys J}, keywords = {fret, sms}, month = {January}, number = {1}, pages = {303--312}, posted-at = {2009-11-05 04:24:08}, priority = {2}, title = {Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances.}, url = {http://dx.doi.org/10.1529/biophysj.106.093211}, volume = {92}, year = {2007} }

TY - JOUR ID - citeulike:2613587 L3 - citeulike-article-id:2613587 N2 - 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. IS - 1 JF - Biophys J SN - 0006-3495 AD - School of Chemistry, Seoul National University, Seoul, Korea. EP - 312 TI - Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances. VL - 92 SP - 303 KW - fret KW - sms AU - Lee, NK AU - Kapanidis, AN AU - Koh, HR AU - Korlann, Y AU - Ho, SO AU - Kim, Y AU - Gassman, N AU - Kim, SK AU - Weiss, S PY - 2007/01/01/ UR - http://dx.doi.org/10.1529/biophysj.106.093211 ER -