Fluorescent dyes for study of beta-pleated protein formations

One of the directions of our researches is the development of dyes for fluorescent detection of beta-pleated protein formations, which are considered to be the hallmarks of a number of neurodegenerative disorders. Recently we proposed cyanine dyes as a new class of amyloid-specific sensitive fluorescent probes [1, 2]. Thus mono- (T-284) and trimethine (SH-516) cyanines ( Fig. 1) allow quantitative detection of as little as ~1 nmol/ml of fibrillar alpha-synuclein (ASN), a comparable detection limit with that of commercially available dyes [3].
These dyes appeared to have ability to follow the step-by-step transition of monomeric ASN proteins into fibrils, demonstrating good results reproducibility, much better than it was observed for commonly used dye Thioflavin T [3]. The data of fluorescent, fluorescence polarization, and life-time spectroscopy, as well as atomic force microscopy carried out together with Prof. V. Subramaniam (University of Twente, the Netherlands) allowed us to get insight into mechanism of cyanine dye/amyloid fibril complex formation.

Figure 1. Amyloid sensitive cyanine dyes (T-284, SH-516 and 7519).

The fluorescent assay for screening of potential inhibitors of amyloid fibril formation was developed on the base of proposed cyanine dyes. Thus benzothiazole trimethinecyanine dye 7519 (Fig. 1) was successfully applied in study of series of flavonoids as inhibitors of insulin aggregation in vitro [4]. Besides, using the mentioned assay we firstly demonstrated the high activity of phthalocyanines with out-planed ligands as inhibitors of fibrillogenesis (Fig. 2).

Figure 2. Zirconium phthalocyanine containing lysine fragments as out-planed substituents.

1. K.D. Volkova, V.B. Kovalska, A.O. Balanda, R.J. Vermeij, V. Subramaniam, Yu.L. Slominskii and S.M. Yarmoluk. Cyanine dye-protein interactions: looking for fluorescent probes for amyloid structures // Journal of Biochemical and Biophysical Methods. – 2007. – Vol. 70, Iss. 5. – P. 727-733.
2. K.D. Volkova, V.B. Kovalska, A.O. Balanda, M.Yu Losytskyy, A.G. Golub, R.J. Vermeij, V. Subramaniam, O.I. Tolmachev and S.M. Yarmoluk. Specific fluorescent detection of fibrillar α-synuclein using mono- and trimethine cyanine dyes // Bioorganic and Medicinal Chemistry. – 2008. – Vol. 16, Iss. 3. – P. 1452-1459.
3. K.D. Volkova, V.B. Kovalska, G.M. Segers-Nolten, G. Veldhuis, V. Subramaniam, S.M. Yarmoluk. Explorations of the application of cyanine dyes for quantitative α-synuclein detection // Biotechnic & Histochemistry. – 2009. – 84. – P. 55-61.
4. K.D. Volkova, V.B. Kovalska, M.Yu. Losytskyy, G. Veldhuis, G. M. J. Segers-Nolten, O.I. Tolmachev, V. Subramaniam, S.M. Yarmoluk. Studies of interaction between cyanine dye T-284 and fibrillar alpha-synuclein // Journal of Fluorescence. – 2010. – Vol. 20, N 6. – P. 1267-1274.
5. K.D. Volkova, V.B. Kovalska, D. Inshin, Y.L. Slominskii, O.I. Tolmachev, S.M. Yarmoluk . Novel fluorescent trimethine cyanine dye 7519 for amyloid fibril inhibition assay // Biotechnic & Histochemistry. – Early Online, 1-4.

Dyes for fluorescent detection of non-canonical DNA structures (triplexes and quadruplexes)

In living organisms mostly genomic DNAs exist in a double-stranded (ds) helical form, but occurrence of some other conformations also seems possible. The guanine-rich nucleic acid sequences are known to fold into four-stranded (G-quadruplexes, G4-DNA) or triple-stranded (triplex) structures in which the nucleotide bases are connected by means of Hoogsteen base-pairing bonding (Fig. 3). The problem of the possible role of triplex and quadruplex DNA motifs in gene regulation and genomic stability still remains unsolved. Therefore, development of a specific probe for distinguishing quadruplex or triplex conformations from a canonical ds one may have a significant scientific and practical importance.

Figure 3. Structures of G-G-C triad in poly(dG-dG)-poly(dC) (A) and G-tetrad in G4-DNA (B).

Interaction of earlier reported DNA sensitive cyanine dyes, monomethine Cyan 40 and trimethine Cyan 2 (Fig. 4), with dsDNA and non-canonical, triplex and G-quadruplex DNA motifs was studied by spectral-luminescent methods. Cyan 40 demonstrated strong preference for triplex over duplexes and quadruplex DNA forms; whereas Cyan 2 was less specific with respect to non-cannonical DNA structures and binds with high affinity to either triplex or quadruplex DNA forms. We suggested that the complexes of Cyan 40 and Cyan 2 with triplex DNA are formed via groove binding mode, while Cyan 2 intercalates into G4-DNA.

Figure 4. Chemical structures of Cyan 40 and Cyan 2.

We consider the studied cyanine dyes can provide a highly sensitive method for detection and quantification of non-canonical DNA structures in genome and could be used for the search and development of agents that specifically bind with such DNA motifs and inhibit their functioning.

V.B. Kovalska, M.Yu. Losytskyy, S.M. Yarmoluk, I. Lubitz, A.B. Kotlyar. Mono and Trimethine Cyanines Cyan 40 and Cyan 2 as Probes for Highly Selective Fluorescent Detection of Non-canonical DNA Structures // Journal of Fluorescence. – Early Online. – DOI 10.1007/s10895-010-0709-y.

Design of fluorescent dyes for biomedical applications

Search and development of novel highly efficient fluorescent probes for biology and medicine are among the main activities of our group. The «lead dye» method, an original approach to design of fluorescent probes with required properties, was proposed and applied by us for the performing of contract research projects. The wide house-stock collection containing about 2,000 dyes of various classes (polymethine cyanines, styryles, coumarines, and metallocomplexes) is used as a basis for performing of such studies.
In collaboration with BioRad Inc. we developed squaraine dyes for using as unspecific fluorescent detection of proteins in separation systems.
Series of sensitive fluorescent dyes for nonspecific detection of proteins in gels (LUCY dyes) was developed by our group for Sigma-Aldrich Inc. Main advantages of the stains Lucy 506, Lucy 569 and Lucy 565 are their high sensitivity, simple and rapid staining protocol and low protein-to protein variability. These dyes show a wide linear concentration range and allow detection of about 5-10 ng of protein per band (Fig. 5).

Figure 5. Limits of proteins detection on SDS-polyacrylamide gel by LUCY-506 and LUCY-565 proteins gel stains developed by us for Sigma-Aldrich Inc., in comparison with SYPRO Ruby stain (Invitrogen Corp.).

Recently in collaboration with Sigma-Aldrich we successfully finished the research project aimed on the development of novel high-sensitive fluorescent probe for DNA visualization in gels. With the Nancy - 520 fluorescent stain developed in this project one can visualize as low as 0.5 ng/band of dsDNA in agarose gel. Besides, Nancy-520 can be used to determine dsDNA concentrations in solution, with a linear range between 0 and 2 µg/ml DNA.

1. A. Rueck, B. Schoenenberger, S. Yarmoluk, V. Kovalska, M. Losytskyy, Y. Slominskii. Nucleic acid fluorescent stains // US Patent Application Publication US2010041045, 18.02.2010.
2. T.R. Berkelman, S.M. Yarmoluk, V.B. Kovalska, M.Yu. Losytskyy, K.D. Volkova. Use of squaraine dyes to visualize protein during separations // Patent Application Publication WO 2008/027821 A1, 06.03.2008.
3. K.D. Volkova, V.B. Kovalska and S.M. Yarmoluk. Modern techniques for protein detection on polyacrylamide gels: problems arising from the use of dyes of undisclosed structures for scientific purposes // Biotech. Histochem. – 2007. – Vol. 82, Iss. 4&5. – P. 201-208.
4. V. Kovalska, D. Kryvorotenko, M. Losytskyy, P. Nording, A. Rueck, B. Schoenenberger, S. Yarmoluk, F. Wahl. Detection of polyamino acids using trimethincyanine dyes // US Patent Application Publication US2006207881, 21.09.2006.

Long-wavelength dyes for fluorescent detection of proteins

Albumins are the major plasma proteins circulating in the bloodstream. Since serum albumin is a reliable prognostic indicator for morbidity and mortality, liver and other diseases and act as carriers for nutritional factors and drugs, analytical methods of albumin study are in high demand. The advantages of fluorometric methods over other methods are their high sensitivity, selectivity and convenience. Squaraine dyes have been reported as efficient noncovalent fluorescent labels for albumins, exhibiting high quantum yields when bound to these proteins.
A wide series of squaraine dyes based on indolenine, benzoxazole, benzothiazole and benzoselenazole heterocycles were tested for their sensitivity to various albumins. Studied symmetric benzothiazole and benzoselenazole squaraines demonstrated bright fluorescence both in HSA and BSA presence. Dyes containing N-ethyl tail groups have shown significant sensitivity to HSA. Using of some of these benzothiazole dyes allows quantification of HSA in the range from 0.2 µg/ml to 500 µg/ml that is comparable with commercially used dyes such as CBB and Pyrogallol Red Protein.
It was shown that unsymmetric indolenine squaraine dyes increase their emission intensity considerably in the presence of BSA. The 3-oxo-substituted indolenine dye (Fig. 6) also gives fluorescent response to the presence of HSA and ovalbumine.

Figure 6. Structures of studied squaraine dyes.

For the dyes giving significant fluorescent response on HSA presence, the study of dye-HSA binding mechanism and site specificity is being carried out. We consider these researches to result in the development of squaraine - based fluorometric assay for the study of pharmakokinetics and pharmacodynamics of potential drugs and characterization of their binding to HSA.

1. K.D. Volkova, V.B. Kovalska, M.Yu. Losytskyy, L.V. Reis, P.F. Santos, P. Almeida, D.E. Lynch, S.M. Yarmoluk. Aza-substituted squaraines for the fluorescent detection of albumins // Dyes and Pigments, in press.
2. K.D. Volkova, V.B. Kovalska, M.Y. Losytskyy, A. Bento, L.V. Reis, P.F. Santos, P. Almeida, S.M. Yarmoluk. Studies of benzothiazole and benzoselenazole squaraines as fluorescent probes for albumins detection // J. Fluorescence. – 2008. – Vol. 18, № 5. – P. 877-882.
3. K.D. Volkova, V.B. Kovalska, A.L. Tatarets, L.D. Patsenker, D.V. Kryvorotenko, S.M. Yarmoluk. Spectroscopic study of squaraines as protein-sensitive fluorescent dyes // Dyes and Pigments. – 2007. – Vol. 72, No. 3 – P. 285-292.

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