The oxazole yellow dye, YOYO-1 (a symmetric homodimer), is a commonly used molecule for staining DNA. We applied the brightness analysis to study the intercalation of YOYO-1 into the DNA. We distinguished two binding modes of the dye to dsDNA: mono-intercalation and bis-intercalation. Bis-intercalation consists of two consecutive mono-intercalation steps, characterised by two distinct equilibrium constants (with the average number of base pair per binding site equals 3.5): K1=3.36±0.43×107M-1 and K2=1.90±0.61×105M-1, respectively. Mono-intercalation dominates at high concentrations of YOYO-1. Bis-intercalation occurs at low concentrations.

Optical mapping, a single DNA molecule genome analysis platform that can determine methylation profiles, uses fluorescently labeled DNA molecules that are elongated on the surface and digested with a restriction enzyme to produce a barcode of that molecule. Understanding how the cyanine fluorochromes affect enzyme activity can lead to other fluorochromes used in the optical mapping system. The effects of restriction digestion on fluorochrome labeled DNA (Ethidium Bromide, DAPI, H33258, EthD-1, TOTO-1) have been analyzed previously. However, TOTO-1 is a part of a family of cyanine fluorochromes (YOYO-1, TOTO-1, BOBO-1, POPO-1, YOYO-3, TOTO-3, BOBO-3, and POPO-3) and the rest of the fluorochromes have not been examined in terms of their effects on restriction digestion. In order to determine if the other dyes in the TOTO-1 family inhibit restriction enzymes in the same way as TOTO-1, lambda DNA was stained with a dye from the TOTO family and digested. The restriction enzyme activity in regards to each dye, as well as each restriction enzyme, was compared to determine the extent of digestion. YOYO-1, TOTO-1, and POPO-1 fluorochromes inhibited ScaI-HF, PmlI, and EcoRI restriction enzymes. Additionally, the mobility of labeled DNA fragments in an agarose gel changed depending on which dye was intercalated.

Intercalating fluorescent probes are widely used to visualize DNA in studies on DNA-protein interactions. Some require the presence of adenosine triphosphate (ATP). We have investigated the mechanical properties of DNA stained with the fluorescent intercalating dyes YOYO-1 and YOYO-3 as a function of ATP concentrations (up to 2 mM) by stretching single molecules in nanofluidic channels with a channel cross-section as small as roughly 100×100 nm2. The presence of ATP reduces the length of the DNA by up to 11 %. On the other hand, negligible effects are found if DNA is visualized with the minor groove-binding probe 4\',6-diamidino-2-phenylindole. The apparent drop in extension under nanoconfinement is attributed to an interaction of the dye and ATP, and the resulting expulsion of YOYO-1 from the double helix.

Identification of the chemical and biological properties of amyloid fibrils is important for understanding their roles in human diseases and to clarify the mechanisms that govern their formation. In pursuit of these goals, small molecule fluorescent dyes have received increasing attention as probes of amyloid conformations. In this study, we report on the ability of YOYO-1, a homodimeric derivative of oxazole yellow, to detect fibrils formed by the Alzheimer\'s disease related Aβ(1-42) peptide. We find that YOYO-1 binds to Aβ(1-42) fibrils with the long axes of its oxazole yellow moieties parallel to the fibril axis, resulting in a 200x emission enhancement; a result that shows that YOYO-1 is a sensitive amyloid probe. Further, YOYO-1 exhibits characteristic absorption shifts upon binding to the Aβ(1-42) fibrils that we attribute to a self-stacking to non-stacking transition in its homodimer configuration; herein we show how this phenomenon can be exploited to estimate the degree of dye binding. Furthermore, we show that YOYO-1 can be used to monitor the kinetics of amyloid formation reactions. Taken together, our results show that YOYO-1 is a sensitive amyloid probe that can operate with both absorption and fluorescence read-outs, and this suggests that this commercially available dye could become a useful complement to thioflavin-T for in vitro amyloid-sensing applications.

The oxazole homodimer YOYO-1 has served as a valuable tool for the detection and quantification of nucleic acids. While the base specificity and selectivity of binding of YOYO-1 has been researched to some extent, the effect of unorthodox nucleic acid conformations on dye binding has received relatively less attention. In this work, we attempt to correlate the quadruplex-forming ability of G-rich sequences with binding of YOYO-1. Oligonucleotides differing in the number of tandem G repeats, total length, and length of loop sequence were evaluated for their ability to form quadruplexes in presence of sodium (Na(+)) or potassium (K(+)) ions. The fluorescence behavior of YOYO-1 upon binding such G-rich sequences was also ascertained. A distinct correlation was observed between the strength and propensity of quadruplex formation, and the affinity of YOYO-1 to bind such sequences. Specifically, as exemplified by the oligonucleotides 5\'-G4T2G4-3\' and 5\'-G3TG3TG3-3\', sequences possessing longer G-rich regions and shorter loop sequences formed stronger quadruplexes in presence of K(+) which translated to weaker binding of YOYO-1. The dependence of binding of YOYO-1 on sequence and structural features of G-rich DNA has not been explored previously and such studies are expected to aid in more effective interpretation of applications involving the fluorophore.