0006-3495/09/08/0835/9 $2.00 doi: 10.1016/j.bpj.2009.05.024Interaction of Oxazole Yellow Dyes wi

imaging in which we study the interaction of individualcule fluorescence imaging of DNA (8–10). The dyes are virtually nonfluorescent in solution, but undergo a strong increase in fluorescence quantum yield of a few orders of magnitude when bound to DNA (5,6). YO is a mono-interca-lator, which consists of only a single aromatic moiety. In the case of YOYO, which consists of two aromatic moieties con-nected with a linker, both moieties intercalate between bp leaving one position in between unoccupied (bis-intercala-tion) (10). The estimated binding constant for YOYO to DNA is 1010–1012 M1. YO has a lower affinity (11). Absorption spectroscopy, linear dichroism, and 1H NMR spectroscopy have been used to characterize the structure, binding mode, optical, and photophysical properties of the DNA molecules with the oxazole yellow dyes YO and YOYO. The mechanical properties of individual DNA mole-cules at various dye concentrations have beenmeasured. From these force extension data we calculated fractional elongation and fitted this with a McGhee-von Hippel binding isotherm (18). This fit resulted in the binding constants and the binding site sizes as a function of force for both dye molecules. For a 100 nM concentration of YO and YOYO we have simultaneously measured the force and the total fluorescence intensity and directly related the number of fluorescent mole-cules bound to the DNA to the force applied. These data showed clearly that the number of fluorophores increases as the force increases. They also showed that the kinetics of YOYO binding was relatively slow. Force relaxation experi-ments were carried out to get more detailed insight into these kinetics of dye molecules binding to DNA. The binding and unbinding rate of YOYO was two orders of magnitude lowe