Table 1. Dye fluorescence in the presence or absence of peptides or DNA. (Click to enlarge)

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To further examine the utility of the IG dyes for amyloid structure/function studies, we performed experiments to determine whether the association of dyes with amyloid allows for fluorescence resonance energy transfer (FRET) as was observed between ThT and chemically similar dyes (17). Figure 2A shows the fluorescence spectra of Aβ_1–42 peptide incubated with ThT in the presence or absence of BOBO-3 or dyes alone. The emission spectrum of ThT overlaps to some degree with the BOBO-3 excitation maxima and when both dyes are incubated together with the Aβ_1–42 peptide, a decrease in ThT fluorescence is observed with a concomitant increase in BOBO-3 emission (∼600 nm), indicating that energy transfer is occurring between these dyes. Figure 2B shows that FRET can also be observed when YOYO-1, which has similar emission spectra as ThT, is incubated with the Aβ_1–42 peptide and BOBO-3.These data suggest that the ThT and BOBO-3 are bound in close proximity in the Aβ_1–42 structure.

Figure 2. Resonance energy transfer between ThT and IG dyes. (Click to enlarge)

The different fluorescent properties of the IG dyes also allows for direct competition studies. Figure 3A shows the fluorescence of Aβ_1–42 peptide with BOBO-1, YOYO-3, and POPO-3 in the presence of increasing concentrations of Congo red, which is not fluorescent and does not interfere with IG dye light adsorption in these assays (data not shown). Congo red shows a competitive inhibition of the IG dyes with an apparent IC50 of ∼0.2 μM, indicating that their binding site(s) overlap to some degree. Compounds that are based on Congo red (i.e., chrysamine G and K114) (38, 39) also competed for IG dyes binding to Aβ_1–42 and hamster prion (data not shown). Although the IG dyes are not chemically similar to Congo red, chrysamine G, or K114, they do share a dimeric structure. To examine the effects dimeric structure has on the binding of IG dyes to Aβ_1–42, we measured changes in TOTO-3 fluorescence in the presence of increasing concentrations of chrysamine G and half-chrysamine G. Figure 3B shows that like Congo red, chrysamine G shows a typical competitive binding to the Aβ_1–42 with an IC50 of ∼1 μM. Half-chrysamine G, which is, as the name implies, a monomeric form of chrysamine G, was a much poorer competitor, which agrees with previous studies showing lower affinity of the monomeric chrysamine G for Abeta peptides (40). Interestingly, the IC50 values for both Congo red and chrysamine G were close to the concentrations of the IG dyes used in these experiments (0.25 and 1 μM, respectively), which suggests that the IG dyes, Congo red, and chrysamine G have similar affinities and are binding to overlapping sites. Congo red and chrysamine G have been shown to compete for amyloid binding, which suggests that their binding sites overlap (41).

Figure 3. Competition studies using IG dyes. (Click to enlarge)

The converse was also observed using TO-PRO-3, which is a monomeric form of the TOTO-3 dye with similar excitation/emission maxima (642/661), in competition studies with increasing concentrations of Congo red and chrysamine G (data not shown).

Taken together, our data suggest IG dyes are binding to amyloids in a similar manner as the well-established ThT and Congo red dyes. In this study, we only show data for six dyes, but the manufacturer has 16 dimeric/monomeric dyes of this class and ones that extend fluorescence emission into the near infrared.

Based on the above results and their availability, we think IG dyes could become important tools for biochemical studies of amyloid structure, as they complement existing probes and could benefit efforts to find effective therapeutics. However, these dyes would probably not be suitable for in vivo imaging, due to their DNA binding.

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