Abstract:The excited-state dynamics of the DNA bisintercalator YOYO-1 and of two derivatives has been investigated using ultrafast fluorescence up-conversion and time-correlated single photon counting. The free dyes in water exist in two forms: nonaggregated dyes and intramolecular H-type aggregates, the latter form being only very weakly fluorescent because of excitonic interaction. The excited-state dynamics of the nonaggregated dyes is dominated by a nonradiative decay with a time constant of the order of 5 ps associated with large amplitude motion around the monomethine bridge of the cyanine chromophores. The strong fluorescence enhancement observed upon binding of the dyes to DNA is due to both the inhibition of this nonradiative deactivation of the nonaggregated dyes and the dissociation of the aggregates and thus to the disruption of the excitonic interaction. However, the interaction between the two chromophoric moieties in DNA is sufficient to enable ultrafast hopping of the excitation energy as revealed by the decay of the fluorescence anisotropy. Finally, these dyes act as solvation probes since a dynamic fluorescence Stokes shift was observed both in bulk water and in DNA. Very similar time scales were found in bulk water and in DNA.

The fluorescence enhancement mechanisms of a series of DNA stains of the oxazole yellow (YO) family have been investigated in detail using steady‐state and ultrafast time‐resolved fluorescence spectroscopy. The strong increase in the fluorescence quantum yield of these dyes upon DNA binding is shown to originate from the inhibition of two distinct processes: 1) isomerisation through large‐amplitude motion that non‐radiatively deactivates the excited state within a few picoseconds and 2) formation of weakly emitting H‐dimers. As the H‐dimers are not totally non‐fluorescent, their formation is less efficient than isomerisation as a fluorescent contrast mechanism. The propensity of the dyes to form H‐dimers and thus to reduce their fluorescence contrast upon DNA binding is shown to depend on several of their structural parameters, such as their monomeric (YO) or homodimeric (YOYO) nature, their substitution and their electric charge. Moreover, these parameters also have a substantial influence on the affinity of the dyes for DNA and on the ensuing sensitivity for DNA detection. The results give new insight into the development and optimisation of fluorescent DNA probes with the highest contrast.

The thiazole orange dye TOTO binds to double-stranded DNA (dsDNA) by a sequence selective bis-intercalation. Each chromophore is sandwiched between two base pairs in a (5'-CpT-3'):(5'-ApG-3') site, and the linker spans two base pairs in the minor groove. We have used one- and two-dimensional NMR spectroscopy to examine the dsDNA binding of an analogue of TOTO in which the linker has been modified to contain a bipyridyl group (viologen) that has minor groove binding properties. We have investigated the binding of this analogue, called TOTOBIPY, to three different dsDNA sequences containing a 5'-CTAG-3', a 5'-CTTAG-3', and a 5'-CTATAG-3' sites, respectively, demonstrating that TOTOBIPY prefers to span three base pairs. The many intermolecular NOE connectivities between TOTOBIPY and the d(CGCTTAGCG):d(CGCTAAGCG) oligonucleotide in the complex shows that the bipyridyl-containing linker is positioned in the minor groove and spans three base pairs. Consequently, we have succeeded in designing and synthesizing a ligand that recognizes an extended recognition sequence of dsDNA as the result of a concerted intercalation and minor groove binding mode.

By condensation of quaternary benzothiazolium, quinolinium and acridinium salts having an active methyl group with 1-(3-bromopropyl)-4-chloroquinolinium or 1-(3-bromo-2-hydroxypropyl)-4-chloroquinolinium salts in the presence of a basic agent such as triethylamine, 8 asymmetric and symmetric monomethine cyanine dyes bearing 3-bromopropyl substituent with one or two positive charges, were synthesized. Additionally, two of the dyes were quaternized with pyridine, and monomethine cyanines with two and three positive charges are prepared. Most of the dyes showed high molar absorptivity (70 000±100 000 l mol À1 cm À1 ). The acridinium dyes showed broad peaks with lower intensity of 30 000 l mol À1 cm À1 . In the presence of nucleic acid in aqueous solutions, a strong enhancement of thē uorescence of these new dyes was observed. #

Two newly synthesized symmetrical heptamethine cyanine dyes, AK7-5 and AK7-6, absorbing in the region of low autofluorescence of biological samples, have been tested for their ability to detect proteins aggregated into amyloid fibrils. In aqueous solution these probes possess three absorption bands corresponding to the monomer, dimer and H-aggregate species. The association of the dye with fibrillar lysozyme was followed by the enhancement of the monomer band and the reduction of the H-band. The absorption spectra measured at various fibril concentrations were analyzed in terms of the model allowing for the shift of equilibria between various dye species due to the binding of monomers and dimers of AK7-5 and AK7-6 to amyloid fibrils. The association constants and stoichiometries of the dye-fibril complexation have been evaluated. In contrast to fibrillar lysozyme, the native protein brought about strong J-aggregate formation accompanied by a marked drop in the absorbance of the dye monomer species. Quantum chemical calculations and simple docking studies showed that AK7-5 and AK7-6 monomers can bind to the grooves, running parallel to the fibril axis. Due to their ability to distinguish between the native and fibrillar protein states, the novel cyanines are recommended as complementary to existing amyloid markers.

The applicability of the three-step Förster resonance energy transfer (FRET) to detection of insulin amyloid fibrils was evaluated, using the chromophore system, containing Thioflavin T (ThT), 4-dimethylaminochalcone (DMC), and two squaraine dyes, referred to here as SQ1 and SQ4. The mediator chromophore DMC was found to enhance the fluorescence intensity of the terminal acceptor, SQ1, excited at 440 nm (at the absorption maximum of the principal donor, ThT), in fibrillar insulin compared to the system without DMC, providing the evidence for the cascade energy transfer in the chain ThT→DMC→SQ4→SQ1. Furthermore, the resulting Stokes shift in the four-chromophore system was 240 nm, as compared to 45 nm for the fibril-bound ThT, suggesting that higher signal-to-noise ratio is the advantage of amyloid fibril detection by multistep FRET. The maximum efficiencies of energy transfer in the insulin fibrils estimated from the quenching of the donor fluorescence in the presence of acceptor for the donor-acceptor pairs ThT-DMC, DMC-SQ4 and SQ4-SQ1 were 40%, 60% and 30% respectively, while negligible FRET occurred in the non-fibrillized protein. The most pronounced differences between fibrillar and non-fibrillized insulin were observed in the 3D fluorescence spectra. Specifically, two intensive spots centered at the emission wavelengths ~ 650 nm (SQ4) and ~ 685 nm (SQ1) were revealed at the excitation wavelength ~ 440 nm in the 3D patterns of insulin amyloid aggregates. In contrast, in the case of the non-fibrillized protein, the barely noticeable spots centered at the same wavelengths, as well as higher fluorescence intensities at the excitation above 550 nm were observed, suggesting the predominant impact of the direct excitation of SQ1 and SQ4 on their fluorescence responses. The inter-chromophore distances calculated from the experimental values of the energy transfer efficiency assuming the isotropic rotation of the dyes, were found to be 2.4, 4.5 and 4.3 nm for the ThT-DMC, DMC-SQ4 and SQ4-SQ1 pairs, respectively, revealing the different fibril binding sites for the examined dyes. The quantum-chemical calculations and simple docking studies provided evidence for the SQ1, SQ4 and ThT, DMC binding to the wet and dry interface of the insulin amyloid protofilament, respectively. The dye-protein complexes are likely to be stabilized by the hydrophobic, van der Waals, aromatic and electrostatic interactions. In summary, the above technique based on the multistep FRET can be employed for the identification and characterization of amyloid fibrils in vitro along with the classical ThT assay, allowing the increase of the amyloid detection sensitivity and lowering the probability of the pseudo-positive result. The applicability of the multistep FRET for amyloid visualization in vivo can be also tested by the involvement of the near-infrared fluorescent dyes to the cascade.

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