Single-molecule fluorescence spectroscopy of TOTO on poly-AT and poly-GC DNA.
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.
Bowen
Benjamin P
BP
Department of Chemical and Materials Engineering, Arizona State University, Tempe, AZ 85287, USA. bpb@asu.edu
Enderlein
Jörg
J
Woodbury
Neal W
NW
D001483:Base Sequence; D052160:Benzothiazoles; D004247:DNA / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006207:Half-Life; D007364:Intercalating Agents / Q000737:chemistry; D011067:Poly dA-dT / Q000737:chemistry*; D011089:Polydeoxyribonucleotides / Q000737:chemistry*; D011804:Quinolines; D011806:Quinolinium Compounds / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013844:Thiazoles / Q000737:chemistry*
D052160:Benzothiazoles; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D011089:Polydeoxyribonucleotides; D011804:Quinolines; D011806:Quinolinium Compounds; D013844:Thiazoles; C051000:thiazole orange; C075295:1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-thiazole)-2-methylidene)quinolinium; D011067:Poly dA-dT; C019530:poly(dC-dG); D004247:DNA