Amyloids are fibrillar protein aggregates associated with a number of neurodegenerative pathologies including Alzheimer and Creutzfeldt–Jakob disease. The study of amyloids is usually based on fluorescence with the dye thioflavin-T. Although a number of amyloid binding compounds have been synthesized, many are nonfluorescent or not readily available for research use. Here we report on a class of commercial benzothiazole/benzoxazole containing fluorescent DNA intercalators from Invitrogen that possess the ability to bind amyloid Aβ_1-42 peptide and hamster prion. These dyes fluoresce from 500–750 nm and are available as dimers or monomers. We demonstrate that these dyes can be used as acceptors for thioflavin-T fluorescence resonance energy transfer as well as reporter groups for binding studies with Congo red and chrysamine G. As more potential therapeutic compounds for these diseases are generated, there is a need for simple and inexpensive methods to monitor their interactions with amyloids. The fluorescent dyes reported here are readily available and can be used as tools for biochemical studies of amyloid structures and in vitro screening of potential therapeutics.

Amyloidosis includes neuropathologies such as Alzheimer, Parkinson's, Huntington's, and Creutzfeldt–Jakob disease (1, 2). Amyloidosis is also found in other apparently disparate diseases such as type 2 diabetes (3). A characteristic pathology of these diseases are deposits of proteinaceous aggregates that are resistant to normal proteolysis. These proteins are often cross-β sheet polymers, and although they do not share significant amino acid sequence similarities, the fibrils appear to share similar morphology and mechanisms of toxicity (4). X-ray diffraction, electron microscopy and solid-state nuclear magnetic resonance have shown that amyloid fibrils are long and unbranched with an organized core structure composed of either parallel or anti-parallel β-sheets (5-7).

To date, one of the most common methods to study amyloid fibrils is through the use of dyes, including Congo red and thioflavin-T (ThT). Congo red is nonfluorescent, but shows apple-green birefringence under polarized light when bound to amyloids (8). ThT is a small benzothiazole compound that is weakly fluorescent in solution, but becomes highly fluorescent when bound to amyloid, with absorption/emission maxima of 440 nm/480 nm (9). The mechanism of how these dyes bind amyloid is not fully understood, but studies suggest that ThT and Congo red orient with their long axis parallel to the long axis of the fibrils (10-12).

ThT and Congo red are not uniquely specific amyloid stains. ThT can fluorescence by binding non-β-sheet cavities, such as those in acetylcholinesterase and γ-cyclodextrin, and ThT does not bind β-sheet rich structures, such as transthyretin (11). Similarly, Congo red binds many enzymes, such as dehydrogenases, and other proteins that have a nucleotide binding site (13). However, the potential diagnostic value of identifying a specific amyloid stain prompted synthesis and study of Congo red and ThT analogs. Additionally, the search for unrelated chemicals that specifically bind amyloids has yielded many diverse compounds (14).

Despite the expanding number of amyloid binding compounds being discovered, options for studying their biochemical interactions with amyloids are still somewhat limited. This is in part due to the optical properties of many amyloid binding compounds. Most ThT/Congo red analogs and related compounds absorb light at wavelengths that overlap with endogenous protein absorption and fluorescence (15, 16). Since many of these compounds have nondistinct optical properties, in vivo and in vitro biochemical studies rely heavily on radiolabeled compounds (17-20). Although many amyloid binding dyes that fluoresce at longer wavelengths have been synthesized (21-24), most have not found their way into common usage. The reasons for this could be that many of these dyes are either not available commercially or require expertise in synthetic organic chemistry to produce. Additionally, many amyloid binding compounds have been patented.

The benzothiazole moiety of ThT is critical for binding amyloid (9). Benzothiazoles are a versatile group found in many compounds that bind both proteins and DNA and include Thiazole Orange, Stains-All, PicoGreen, and luciferin. We noticed that a well-characterized class of DNA intercalating dyes from Invitrogen is chemically similar to ThT, in that they have a benzothiazole component or the chemically related benzoxazole (Figure 1). This prompted us to examine whether this class of dyes is also able to bind amyloid peptides. Our results demonstrate that these dyes fluoresce in the presence of purified Alzheimer Aβ_1–42 peptide and recombinant hamster prions. Moreover, they proved to be versatile with respect to resonance energy transfer and in competition studies with Congo red and chrysamine G. It is hoped that because of their ready availability, these dyes can be used for screening potential therapeutic drugs and increase the arsenal available to researchers studying these debilitating diseases.

Figure 1. Structures of ThT (A) and the IG dyes BOBO-1 (B), POPO-3 (C), YOYO-3 (D), and TOTO-3 (E) along with their fluorescence emission spectra in the presence of Alzheimer Aβ_1–42 peptide (10–30 μg/mL). (Click to enlarge)

Materials and methods

ThT and Congo red were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chrysamine G and half-chrysamine G were purchased from Anaspec (Fremont, CA, USA). Structures of Congo red, chrysamine G, and half chrysamine G can be found at http://aatbio.com/. Recombinant hamster prion protein in fibrillar β-sheet conformation expressed and purified according to (25, 26) was purchased from UMBI Medical Biotechnology Center (Baltimore, MD, USA). Synthetic Aβ_1–42 peptide was purchased from rPeptide (Bogart, GA, USA). The Aβ_1–42 peptide from this manufacturer has been shown by electron microscopy to form fibrils in solution (27). Glass capillary PCR tubes were purchased from Roche (Indianapolis, IN, USA). Nucleic Acid Stains Dimer Sampler kit (containing 10 μL of each dye: BOBO-1, BOBO-3, POPO-1, POPO-3, TOTO-1, TOTO-3, YOYO-1, and YOYO-3), TO-PRO-3, ethidium bromide (EthBr), and λ DNA standard were purchased from Invitrogen (Grand Island, NY, USA). All chemicals were used as received.

Fluorescence assays

Molar extinction coefficients (ε) were used to quantitate the following dyes: Chrysamine G (58,487 M-1cm-1 at 392 nm; Anaspec product info), half-chrysamine G (21,289 M-1cm-1 at 343 nm; Anaspec product info), Congo red (50,000 M-1cm-1 at 498 nm) (13), and ThT (24,420 M-1cm-1 at 420 nm) (28). Excitation/emission maxima for the Invitrogen dyes (IG dyes) provided by the manufacturer (in nm) are as follow: BOBO-1 (462/481), BOBO-3 (570/602), POPO-1 (434/456), POPO-3 (534/570), TOTO-1 (514/533), TOTO-3 (642/660), YOYO-1 (491/509), YOYO-3 (612/631), and TO-PRO-3 (642/661). Quantum yield of these fluorophores in buffer are usually less than 0.01 but increase up to 0.6 in the presence of DNA (http://probes.invitrogen.com/media/pis/mp03600.pdf).

Fluorescent assays were performed using the ultrasensitive Signalyte-II fluorometer (Creatv Microtech, Potomac, MD, USA), described in more detail in (29-31). The fluorometer used for these studies was equipped with four light-emitting diodes (LED) including red, amber, green, and blue LEDs with excitation maxima of 470, 530, 590, and 635 nm and long-pass emission filters with cut-on wavelength of 515, 570, 630, and 665 nm. TOTO-1 and POPO-1 were not used for these studies because the fluorometer used did not have the appropriate LEDs and filters. The fluorometer cuvettes are glass capillary PCR tubes. For the fluorescence measurements, dyes were mixed with protein or DNA samples at room temperature in a 96-well microtiter plate in duplicate for approximately 10 min before 40-μL aliquots were loaded into the glass tubes and measured. Concentrations of peptides used for the experiments are given in the figure legends. All fluorescent binding assays were either performed in PBS unless otherwise stated. Fluorescence was measured for up to 3 s per tube.

Results and discussion

Figure 1 shows the structures of ThT (Figure 1A) and the IG dyes BOBO-1 (Figure 1B), POPO-3 (Figure 1C), YOYO-3 (Figure 1D), and TOTO-3 (Figure 1E) along with their fluorescence emission spectra in the presence of Alzheimer Aβ_1–42 peptide. Hamster prion protein also showed analogous fluorescence spectra to the Aβ peptide (data not shown). In Figure 1, the ThT emission peak appears at 521 nm instead of the reported value of 480 nm. This apparent shift in peak value appears to be an artifact due to the 515 nm cut-off filter used in the Signalyte fluorometer, but there was negligible shift in the emission of Aβ_1–42, DNA, and prion with the different dyes (data not shown). Figure 1 shows the substantial structural similarities between the IG dyes and ThT. The IG dyes contain either positively charged N-methyl-benzothiazole or N-methyl-benzoxazole rings that are similar in dimension to the positively charged dimethyl benzothiazole ring of ThT. Although the IG dyes do not have an N′N-dimethylaniline moiety like ThT, they do have flat aromatic groups that can rotate independently of the benzothiazole/benzoxazole, a property that is important for the binding of ThT to amyloid (9).

The benzothiazole moiety of ThT is important for its binding to amyloids. The crystal structure of amyloid-like β2-microglobulin in complex with ThT shows that the benzothiazole moiety has a well-defined apolar binding pocket within the amyloid structure (9). Studies have also shown that chemical modification of the ThT benzothiazole moiety affects affinity for Aβ peptides (20, 32). Many other amyloid structures similarly possess a hydrophobic pocket that could accommodate benzothiazole compounds such as ThT (33). Benzothiazole compounds have also been shown to bind to huntingtin, which is associated with Huntington's disease, and inhibit its fibrillogenesis (34). Compounds containing the related benzoxazole moiety have also been shown to bind amyloids (35).

Since the IG dyes are DNA intercalators, it is possible that the observed fluorescence could be due to contaminating DNA in the recombinant peptide preparations. Therefore we compared fluorescence increase of the amyloid peptides and λ DNA standard against a panel of dyes.

Table 1 shows the increase in fluorescence observed when ThT, IG dyes, and EthBr (which is not structurally related to the aforementioned dyes) are incubated with a DNA standard, Aβ_1–42 peptide, or hamster prion. On the basis of weight, the amyloid peptides show a modest increase in fluorescence compared with DNA. Other benzothiazole containing dyes have also been reported to bind both DNA and the amyloid form of β-lactoglobulin (23, 36, 37). ThT showed an increase in fluorescence upon binding to both amyloid proteins, but much less so with DNA. Conversely, EthBr showed an increase in fluorescence upon binding to DNA, but almost no increase with the amyloid peptides. Although these results do not rule out contaminating DNA in the protein preparations, they suggest that any contamination would be low. This is supported by an HPLC trace provided by rPeptide, which shows no significant peaks, besides the product (data not shown).

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.

Competing interests

The authors declare no competing interests.

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