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Single‐molecule Fluorescence Spectroscopy of TOTO on Poly‐AT and Poly‐GC DNA ¶
Author(s) -
Bowen Benjamin P.,
Enderlein Jörg,
Woodbury Neal W.
Publication year - 2003
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1562/0031-8655(2003)0780576sfsoto2.0.co2
Subject(s) - oligonucleotide , excited state , dna , molecule , dimer , chemistry , intercalation (chemistry) , fluorophore , fluorescence , analytical chemistry (journal) , crystallography , physics , atomic physics , chromatography , biochemistry , organic chemistry , quantum mechanics
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.