Single-molecule Fluorescence Spectroscopy of TOTO on Poly-AT and Poly-GC DNA

• Published in: Photochemistry and photobiology Vol. 78; no. 6; pp. 576 - 581
• Main Authors: Bowen, B P, Enderlein, J, Woodbury, N W
• Format: Journal Article
• Language: English
• Published: 01.12.2003
• Subjects: TOTO
• ISSN: 0031-8655
• DOI: 10.1043/0031-8655(2003)078(0576:SFSOTO)2.0.CO;2

Excited state lifetime and amplitude measurements were made on thiazole orange dimer (TOTO), a dimeric DNA-intercalating fluorophore, at single-molecule concentrations. As expected from previous study, the excited state lifetime of TOTO intercalated in DNA is dependent on the sequence of the double-stranded DNA, having values of 2.2 ns in poly-GC DNA and 1.8 ns in poly-AT DNA. The distribution of excited state lifetimes of single molecules of TOTO intercalated into oligonucleotides having varying proportions of poly-GC sequences relative to poly-AT sequences were analyzed as a function of the fraction of poly-GC. By using excited state lifetime distributions from the purely GC and purely AT oligonucleotides as a basis set, it was possible to estimate the GC content of oligonucleotides with intermediate GC composition to within a few percent error. This serves as a model for the analysis of equilibrium binding distributions in DNA using single-molecule methods.