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.
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 (Figure 1).
Figure 1. 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. In collaboration with BioRad Inc. we developed novel squaraine dyes for using as unspecific fluorescent detection of proteins in separation systems.1. 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 US2006207881, 21.09.2006.
2. 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.
3. 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 WO 2008/027821 A1, 06.03.2008.
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. To date a limited number of fluorescent dyes are known, which interact selectively with such aggregated proteins, moreover, most of these dyes are applicable only in certain test systems. Our studies present cyanine dyes as a new class of amyloid-specific sensitive fluorescent probes.
Recently mono- (T-284) and trimethine (SH-516) cyanines were firstly proposed as highly efficient dyes for detection of amyloid formations, particularly alpha-synuclein aggregates (ASN), hallmark of Parkinson diseases [1]. These dyes allow quantitative detection of as little as ~1 nmol/ml of fibrillar ASN, a comparable detection limit with that of commercially available dyes.
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. 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 2. Amyloid sensitive monomethine and trimethinecyanine dyes.
The good sensitivity and data reproducibility offered by these dyes enables the development of reliable fluorometric assays for monitoring amyloid fibril formation. Such an assay may be adapted for high throughput screening of potential inhibitors of ASN aggregation.
Now in collaboration with the groups of Prof. V. Subramaniam (University of Twente, the Netherlands) and Prof. O.I. Tolmachev (Institute of Organic Chemistry, Kyiv, Ukraine) the research project Development of fluorescent dyes for specific detection of amyloid protein nanostructures causing neurodegenerative diseases is carried out in frames of Science and Technology Center in Ukraine (STCU) and National Academy of Sciences of Ukraine (NASU) grant.
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.
The two-photon excitation (TPE) of the fluorescent probes is the modern approach in biological and medical studies that is used more and more widely for the detection of biological molecules and fluorescent imaging of biological objects. The using of TPE of fluorescent probes in procedures of biological objects detection permits deeper penetration of exciting beam into the tissue; excitation of visual fluorescence in near infrared spectral region, where the biological objects are transparent; decreased photodamage of the studied object; and obtaining of three-dimensional image of biological object.
Series of homodimer (Figure 3, a) and monomer benzothiazole styryl dyes containing spermine-like linkage/tail group were proposed as efficient TPE probes for DNA detection. Such dyes bind to DNA with up to three orders of magnitude emission intensity enhancement and have moderate values of the two-photon absorption cross-section (TPACS) 4.7-7.4x10-50 cm4s.
Besides, novel styryl dyes based on the tetrahydropyrrolo[1,2-a]-thieno[2,3-d]pyrimidinium heterocycle (Figure 3, b) were developed. Dyes of this class demonstrate bright emission in RNA presence, while their emission intensity in DNA presence is considerably (up to 8 times) lower. Fluorescence spectra after two-photon absorption of the 1064 nm radiation of YAG:Nd+3 20 ns pulsed laser were obtained for these dyes in the presence of RNA. Thus we consider oxo-thieno[2,3-d]pyrimidinium styrylcyanines to be promising probes for RNA detection upon TPE.
Figure 3. Homodimer styrylcyanine dyes promising for the TPE fluorescent detection of DNA.
Recently it was shown that the homodimer styryl dye is able to penetrate into the cell, and could be successfully used for the TPE fluorescent imaging of the living cell (Fig. 4).
Figure 4. TPE fluorescent image of the HeLa cells stained with the dye DBos-21. Fluorescent emission excited with the femtosecond pulsed Ti:Sapphire laser, excitation wavelength 880 nm.
For the benzothiazole styrylcyanine dyes the mode of their interaction with dsDNA was studied. Basing on the obtained results, the intercalation mechanism of binding to dsDNA is proposed for the monomer dye containing spermine-like N-alkyl tail group, as well as for the homodimer dye containing positively charged N-alkyl linkage group.
1. V.P. Tokar, M.Yu. Losytskyy, V.B. Kovalska, D.V. Kryvorotenko, A.O. Balanda, V. M. Prokopets, M.P. Galak, I.M. Dmytruk, V.M. Yashchuk and S.M. Yarmoluk. Fluorescence of Styryl Dyes-DNA Complexes Induced by Single- and Two-Photon Excitation // Journal of Fluorescence. 2006. Vol. 16, Nr 6. P. 783-791.
2. V.B. Kovalska, D.V. Kryvorotenko, A.O. Balanda, M.Yu. Losytskyy, V.P. Tokar, S.M. Yarmoluk. Fluorescent homodimer styrylcyanines: synthesis and spectral-luminiscent stidies in nucleic acids and protein complex // Dyes and Pigments. 2005. Vol. 67, Ή 1. P. 47-54.
3. V.M. Yashchuk, V.Yu. Kudrya, M.Yu. Losytskyy, V.P. Tokar, S.M. Yarmoluk, I.M. Dmytruk, V.M. Prokopets, V.B. Kovalska, A.O. Balanda, D.V. Kryvorotenko, T.Yu. Ogul'chansky. The optical biomedical sensors for DNA detection and imaging based on two-photon excited luminescent styryl dyes: phototoxic influence on the DNA // Proceedings of SPIE. 2007. Vol. 6796. 67960M. 14 pp.
4. N. Akbay, M.Yu. Losytskyy, V.B. Kovalska, A.O. Balanda and S.M. Yarmoluk. The Mechanism of Benzothiazole Styrylcyanine Dyes Binding with dsDNA: Studies by Spectral-Luminescent Methods // Journal of Fluorescence. 2008. V.18, Iss.1. P.139-147.
Fluorescence detection of proteins at long-wavelength excitation is widely used for biomedical application due to such benefits of near-infrared-based methods as possibility to use non-expensive diode lasers as excitation sources, and decreased autofluorescence from biomolecules beyond 600 nm. Due to their physical-chemical properties such as light absorption in the visible and near-infrared (NIR) regions, and sharp and intensive fluorescence squaraine dyes are suitable for these purposes.
A wide series of squaraine dyes based on indolenine, benzoxazole, benzothiazole and benzoselenazole heterocycles were tested for their sensitivity to various proteins. 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. 5) also gives fluorescent response to the presence of HSA and ovalbumine.
The thousands times emission increase in the presence of BSA/SDS micelles was demonstrated for symmetric benzothiazole and benzoselenazole dyes with N-hexyl tails (Fig. 5). Majority of the 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.
Figure 5. Structures of studied squaraine dyes.
Studies of applicability of various squaraine dyes for specific and non-specific detection of protein are now carried out.
1. 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. 285292.
2. Volkova KD, Kovalska VB, Losytskyy MY, Bento A, Reis LV, Santos PF, Almeida P, Yarmoluk SM. Studies of Benzothiazole and Benzoselenazole Squaraines as Fluorescent Probes for Albumins Detection.// J Fluorescence. - in press.
Development of fluorescent probes that selectively interact with double stranded (ds) DNA regions is actual for several modern diagnostics and research methods such as synthesizing cDNA for library production and purifying DNA fragments for subcloning, quantification DNA amplification products and detecting DNA molecules in drug preparations.
Development of dyes which interact with DNA via groove-binding is one of the ways to obtain probes that are highly specific to dsDNA. For the cyanine dyes, groove binding becomes more prevalent for the cyanines with more than one methine group in polymethine chain. Recently we proposed trimethine cyanine dyes with crescent shape of molecules (Fig. 6a) as efficient groove binders, interacting with dsDNA with up to hundred times emission increasing and demonstrating high selectivity to dsDNA as compared with RNA. Besides we have shown that modification of the bridged pentamethine dye in a cyclopentene or cyclohexene groups (Fig. 6b) could be used for obtaining of the dyes highly specific to dsDNA.
Figure 6. Trimethine (a) and pentamethine (b) cyanine dyes that are considered to bind with the DNA groove.
The fluorescent aggregates that are specifically formed in DNA presence could be also used for the selective detection of dsDNA. The trimethinecyanine dye Cyan βiPr (Fig. 7) was revealed to form fluorescent J-aggregates on dsDNA that was observed neither for the free dye solution, nor in the presence of RNA or protein. The high preference of Cyan βiPr to the J-aggregates formation on AT-containing polynucleotides as compared to GC-containing ones points at the formation of the aggregates in the dsDNA minor groove (Fig. 8). These fluorescent aggregates were successfully used for the visualization of AT-rich DNA regions using fluorescence microscopy (Fig. 9).
Figure 7. Chemical structure of trimethine cyanine dye Cyan βiPr that forms fluorescent J-aggregates on AT-sequences of dsDNA.
Fig. 8. Model of J-aggregate of Cyan βiPr that is specifically formed in the minor groove of poly(dA)-poly(dT) polynucleotide. The model was bulit using Hyperchem 5.0 basing on the data of spectral-luminescent studies.
Fig. 9. Fluorescence microscopy image of Cyan βiPr J-aggregates formed on poly(dA)-poly(dT) polynucleotide molecules.
1. V.B. Kovalska, K.D. Volkova, M.Yu. Losytskyy, O.I. Tolmachev, A.O. Balanda and S.M. Yarmoluk. 6-Disubstituted benzothiazole trimethine cyanines new fluorescent dyes for DNA detection // Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2006. Vol. 65, No. 2. P. 271-277.
2. M.Yu. Losytskyy, K.D. Volkova, V.B. Kovalska, I.E. Makovenko, Yu.L. Slominskii, O.I. Tolmachev, S.M. Yarmoluk. Fluorescent Properties of Pentamethine Cyanine Dyes with Cyclopentene and Cyclohexene Group in Presence of Biological Molecules // Journal of Fluorescence. 2005. Vol.15, No. 6. P. 849-857.
3. V.B. Kovalska, M.Yu. Losytskyy and S.M. Yarmoluk. Luminescence spectroscopic studies of trimethinecyanines substituted in polymethine chain with nucleic acids and proteins // Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2004. Vol. 60, No. 1-2. P. 129-136.
4. M.Yu. Losytskyy, V.M. Yashchuk, S.S. Lukashov and S.M. Yarmoluk. Davydov Splitting in Spectra of Cyanine Dye J-Aggregates, Formed on the Polynucleotides // Journal of Fluorescence. 2002. Vol. 12, No. 1. P. 109-112.
Method for covalent protein labeling with fluorescent hemicyanine dyes was developed as a simple and convenient procedure that does not require preparation of any active intermediates. In this method low-fluorescent dye (ABTN) undergoes transformation into moderate-fluorescent dye (HBTN) upon conjugation to biomolecules via aliphatic amino groups (Scheme 1). Significant spectral-luminescent changes accompany the transformation of predecessor ABTN into derivative HBTN dye that allows monitoring a conjugation reaction. Contrary to the weakly fluorescent ABTN dye, HBTN forms highly fluorescent complexes with DNA or BSA. This makes HBTN an attractive dye for using in techniques that require labeling of biomolecules. The described conjugation method was successfully used for the fluorescent labeling of bovine serum albumin and its visualization on polyacrylamide gel.
Scheme 1. The reaction of ABTN with amines converts it into fluorescent dye HBTN.O.M. Kostenko, V.B. Kovalska, K.D. Volkova, P. Shaytanov, I.O. Kocheshev, Yu.L. Slominskiy, I.V. Pisareva, S.M. Yarmoluk. New method for covalent fluorescent biomolecules labelling with hemicyanine dye // Journal of Fluorescence. 2006. Vol. 16. P. 589-593.
©
YARMOLUK RESEARCH LAB