Triplet fraction buildup effect of the DNA–YOYO complex studied with fluorescence correlation spectroscopy
Section snippets
Materials
Dimeric cyanine dye YOYO-1 iodide was purchased from Molecular Probes, USA. Sequence-grade oligodeoxyribonucleotides were purchased from Date Concept, Japan. Unless otherwise mentioned, other chemicals were purchased from Wako Chemicals, Japan. All materials were of analytical grade and used without further purification.
Preparation of DNA fragments
Fifty-base pair DNA fragments were prepared by annealing an arbitrary sequence (5′-TCT ACT GGG ACG GAA CAG CTT TGA GGT GCG TGT TTG TGC CTG TCC TGG GA-3′) and its complementary
Fluctuation of photon count rate
The different lengths of dsDNA fragments were diluted to various concentrations, mixed with 5 nM YOYO, and subjected to FCS analysis with 488-nm Ar+ laser for excitation, for which the power was fixed at 150 μW. In these measurements, DNA–YOYO complexes behave as fluorescent molecules because YOYO molecules in the free form are almost nonfluorescent [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. Typical fluctuations of count rate are shown in Fig. 1.
Conclusion
DNA fragments of various lengths were prepared and mixed with the dimeric cyanine dye YOYO. FCS analysis was carried out on these samples. By investigating photon count rate per DNA–YOYO complex, the interval between substantially emitting DNA binding YOYO molecules was estimated. We found that the buildup-triplet fraction varies depending on both the DNA length and the DNA/YOYO ratio. Furthermore, the mechanism underlying this phenomenon can be explained by considering the buildup-triplet
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