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Quantitative structure activity relationship (QSAR) studies were performed on a set of polymethine compounds to develop new fluorescent probes for detecting amyloid fibrils. Two different approaches were evaluated for developing a predictive model: part least squares (PLS) regression and an artificial neural network (ANN). A set of 60 relevant molecular descriptors were selected by performing principal component analysis on more than 1600 calculated molecular descriptors. Through QSAR analysis, two predictive models were developed. The final versions produced an average prediction accuracy of 72.5 and 84.2% for the linear PLS and the non-linear ANN procedures, respectively. A test of the ANN model was performed by using it to predict the activity, i.e., staining or non-staining of amyloid fibrils, using 320 compounds. The five candidates whose greatest activities were selected by the ANN model underwent confirmation of their predicted properties by empirical testing. The results indicated that the ANN model potentially is useful for facilitating prediction of activity of untested compounds as dyes for detecting amyloid fibrils. [ABSTRACT FROM AUTHOR]

Fluorescence spectroscopy was used to study the ability of dye 7519 to follow the transition of monomeric insulin into fibrils and applicability of the dye to the insulin aggregation inhibition assay. The commercially available classic amyloid stain, thioflavin T, was used as the reference dye. For selecting potential inhibitors, the QSAR approach was applied. Dye 7519 appeared to be suitable for monitoring insulin aggregation into fibrils in vitro. The properties of the dye allowed us to test it as a potential probe in the screening assay of potential inhibitors of insulin fibrillization. One hundred forty-four flavonoids were tested as potential inhibitors of amyloid fibril formation using the quantitative structure activity relationship approach. Among them, 10 candidates with high indexes of inhibition were selected for tests in vitro using dye 7519 and the reference amyloid dye thioflavine T. Using dye 7519 fluorescence, we found that two compounds had inhibitory effects on insulin amyloid formation. These results agree with inhibition data using the thioflavine T assay. Our studies demonstrated that the fluorescent cyanine dye 7519 is a sensitive probe for quantitative detection of insulin amyloid formation and can be applied to screen agents capable of affecting aggregation of amyloid proteins. [ABSTRACT FROM AUTHOR]

A fluorescein-tagged iron(ii) cage complex was obtained in a moderate total yield using a two-step synthetic procedure starting from its propargylamine-containing clathrochelate precursor. An 11-fold decrease in fluorescence quantum yield is observed in passing from the given fluorescein-based dye to its clathrochelate derivative. An excitation energy transfer from the terminal fluorescent group of the macrobicyclic molecule to its quasiaromatic highly π-conjugated clathrochelate framework can explain this effect. The kinetics of the hydrolysis of the acetyl groups of acetylated fluorescein azide and its clathrochelate derivative in the presence of one equivalent of BSA evidenced no strong supramolecular host-guest interactions between BSA and the tested compounds. Study of a chemical stability of the deacetylated iron(ii) clathrochelate suggested the formation of a supramolecular 1 : 1 BSA-clathrochelate assembly. Moreover, an addition of BSA or HSA to its solution caused the appearance of strong clathrochelate-based ICD outputs. The fluorescence emission anisotropy studies also evidenced the supramolecular binding of the fluorescein-tagged iron(ii) clathrochelate to the BSA macromolecule, leading to a high increase in this type of anisotropy. Subcellular uptake of the fluorescein-tagged molecules was visualized using fluorescence microscopy and showed its distribution to be mainly in the cytosol without entering the nucleus or accumulating in any other organelle. An X-rayed crystal of the above propargylamide macrobicyclic precursor with a reactive terminal C[triple bond, length as m-dash]C bond contains the clathrochelate molecules of two types, A and B. The encapsulated iron(ii) ion in these molecules is situated in the center of its FeN 6 -coordination polyhedron, the geometry of which is intermediate between a trigonal prism (TP) and a trigonal antiprism (TAP). The Fe-N distances vary from 1.8754(6) to 1.9286(4) Å and the heights h of their distorted TP-TAP polyhedra are very similar (2.30 and 2.31 Å); their values of φ are equal to 25.3 and 26.6°. In this crystal, the molecules of types A and B participate in different types of hydrogen bonding, giving H-bonded clathrochelate tetramers through their carboxylic and amide groups, respectively; these tetramers are connected to H-bonded chains., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

An ability of the ribbed-functionalized iron(ii) clathrochelates to induce a CD output in interactions with a protein, covalent bonding or supramolecular interactions with a low-molecular-weight chiral inductor, was discovered. The interactions of CD inactive, carboxyl-terminated iron(ii) clathrochelates with serum albumin induced their molecular asymmetry, causing an appearance of strong CD signals in the range of 350-600 nm, whereas methyl ester and amide clathrochelate derivatives remained almost CD inactive. The CD spectra of carboxyl-terminated clathrochelates on supramolecular interactions or covalent bonding with (R)-(+)-1-phenylethylamine gave a substantially lower CD output than with albumin, affected by both the solvent polarity and the isomerism of clathrochelate's ribbed substituents. In supramolecular assemblies, the bands were most intensive for ortho-substituted carboxyl-terminated clathrochelates. The ortho- and meta-phenylethylamide cage complexes in tetrachloromethane inverted the signs of their CD bands compared with those in acetonitrile. It was suggested that the tris-dioximate metal clathrochelates possess a Russian doll-like molecular system. Because of the distorted TP-TAP geometry, their coordination polyhedron had no inversion centre and possessed an inherent chirality together with the equiprobability of its left(Λ)- and right(Δ)-handle twists. The selective fixation of one of these C 3 -distorted conformations resulted in the appearance of the CD signal in the range of their visible metal-to-ligand charge transfer bands. Calculations by DFT methods were used to illustrate the possible conformations of the macrobicyclic molecules, as well as the intramolecular interactions between the cage framework and optically active distal substituents responsible for the chirality induction of the metal-centred coordination polyhedra.

The effect of various N,N'-substituents in the molecule of benzothiazole trimethine cyanine dye on its ability to sense the amyloid aggregates of protein was studied. The dyes are low fluorescent when free and in the presence of monomeric proteins, but their emission intensity sharply increases in complexes with aggregated insulin and lysozyme, with the fluorescence quantum yield reaching up to 0.42. The dyes carrying butyl, hydroxyalkyl, and phenylalkyl groups as N,N'-substituents possess the increased fluorescent sensitivity to fibrillar lysozyme, whereas the ones carrying quaternary amino groups are preferably sensitive to fibrillar insulin. This fluorescent sensitivity preference provided by the N,N'-functional groups could be explained by the interaction between these groups and protein side chains. The strongest fluorescent response (up to 70times) and the same sensitivity to aggregates of both proteins were exhibited by the dye D-51 carrying N-sulfoalkyl group. The studied cyanines allow the detection of fibrillar aggregates in the wide range up to 0.8 to 300μg/ml and permit monitoring the protein aggregation kinetics with high reproducibility. The modification of trimethine cyanine dyes by functional substituents in N,N'-positions is suggested as a tool for the design of fluorescent molecules with the enhanced fluorescent sensitivity to the fibrillar aggregates of proteins., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The macrocyclic compounds mono- and bis-iron(II) clathrochelates were firstly studied as potential anti-fibrillogenic agents using fluorescent inhibitory assay, atomic force microscopy and flow cytometry. It is shown that presence of the clathrochelates leads to the change in kinetics of insulin fibrillization reaction and reduces the amount of formed fibrils (up to 70%). The nature of ribbed substituent could determine the activity of clathrochelates-the higher inhibitory effect is observed for compounds containing carboxybenzenesulfide groups, while the inhibitory properties only slightly depend on the size of complex species. The mono- and bis-clathrochelate derivatives of meta-mercaptobenzoic acid have close values of IC₅₀ namely 16 ± 2 and 24 ± 5 μM, respectively. The presence of clathrochelates decreases the fibril diameter from 5-12 nm for free insulin fibrils to 3-8 nm for these formed in the clathrochelate presence, it also prevents the lateral aggregation of mature fibrils and formation of superfibrillar clusters. However the addition of clathrochelate results in more heterogeneous (both by size and structure) insulin aggregates population as compared to the free insulin. This way, cage complexes-iron(II) clathrochelates are proposed as efficient agents able to suppress the protein aggregation processes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Interaction of the iron(II) mono- and bis-clathrochelates with bovine serum albumin (BSA), β-lactoglobulin, lysozyme and insulin was studied by the steady-state and time-resolved fluorescent spectroscopies. These cage complexes do not make significant impact on fluorescent properties of β-lactoglobulin, lysozyme and insulin. At the same time, the monoclathrochelates strongly quench a fluorescence intensity of BSA and substantially decrease its excited state lifetime due to their binding to this protein. This occurs due to the excitation energy transfer from a tryptophan residue to a cage molecule or/and to the change of the tryptophan nearest environment caused by either clathrochelate binding or an alteration of the BSA conformation. The effect of the iron(II) bis-clathrochelate on BSA fluorescence is much weaker as compared to its monomacrobicyclic analogs as a result of an increase in its size.

The pathogenesis of Parkinson's disease that is the second most common neurodegenerative disease is associated with formation of different aggregates of α-synuclein (ASN), namely oligomers and amyloid fibrils. Current research is aimed on the design of fluorescent dyes for the detection of oligomeric aggregates, which are considered to be toxic and morbific spices. Fluorescent properties of series of benzothiazole trimethine and pentamethine cyanines were characterized in free state and in presence of monomeric, oligomeric and fibrilar ASN. The dyes with wide aromatic systems and bulky phenyl and alkyl substituents that are potentially able to interact with hydrophobic regions of oligomeric aggregates were selected for the studies. For majority of studied dyes noticeable changes in fluorescence characteristics were shown in the presence of fibrillar or oligomeric ASN, while the dyes slightly responded on the presence of monomeric protein. For pentamethine cyanine SL-631 and trimethine cyanine SH-299 certain specificity to oligomeric aggregates over fibrils was observed. Using these dyes at 10(-6) M concentration permits the detection of oligomeric ASN in the concentrations range of at least 0.2-2 microM. Pentamethine cyanine SL-631 is proposed as dye for fluorescent detection of oligomeric aggregates of ASN, while trimethine cyanine SH-299 is shown to be a sensitive probe both on oligomeric and fibrillar ASN. It is proposed that wide aromatic system of SL-631 pentamethine dye molecule could better fix on the less dense and structured oligomeric formation, while less bulky and more "crescent-shape" molecule of trimethine dye SH-299 could easier enter into the groove of beta-pleated structure.

In present paper series of trimethine cyanines modified in 5,5'- or 6,6'- position with hydroxy- or methoxy- substituents is studied for their ability to interact selectively with fibrillar formations. Processes of dye aggregation that accompany this interaction were also investigated. Meso-methyl trimethynecyanines with 5,5'- methoxy (7519) and hydroxy (7515) substituents strongly (up to 40 times) increase fluorescence intensity in the presence of fibrillar insulin, and also give noticeable fluorescent response on the presence of various aggregated proteins (lysozyme, β-lactoglobulin, α-synuclein A53T). 7519 and 7515 dyes can be used for fluorometric detection of fibrillar insulin at concentrations of approximately 1.5-120 microg/ml. For meso-ethyl substituted dye 7514 the ability to form H- and J-aggregates upon interaction with insulin fibrils was suggested. The model of the H- and J-aggregate packing in the protein fibrillar structure has been proposed., (© Springer Science+Business Media, LLC 2010)

Two of earlier reported dsDNA sensitive cyanine dyes-monomethine Cyan 40 and meso-substituted trimethine Cyan 2 were studied for their ability to interact with non-canonical DNA conformations. These dyes were characterized by spectral-luminescent methods in the presence of G-quadruplex, triplex and dsDNA motifs. We have demonstrated that Cyan 2 binds strongly and preferentially to triple- and quadruple-stranded DNA forms that results in a strong enhancement of the dye fluorescence, as compared to dsDNA, while Cyan 40 form fluorescent complexes preferentially only with the triplex form. Highly fluorescent complexes of Cyan 2 with DNA triplexes and G-quadruplexes and Cyan 40 with DNA triplexes are very stable and do not dissociate during gel electrophoresis, leading to preferential staining of the above DNA forms in gels. The data presented point to the intercalation mechanism of the Cyan 2 binding to G4-DNA, while the complexes of Cyan 40 and Cyan 2 with triplex DNA are believed to be formed via groove binding mode. The Cyan dyes can provide a highly sensitive method for detection and quantification of non-canonical structures in genome.

A key feature of Parkinson's disease is the formation and accumulation of amyloid fibrils of the natively unfolded protein α-synuclein (ASN) inside neurons. Recently we have proposed novel sensitive monomethinecyanine dye T-284 as fluorescent probe for quantitative detection of ASN amyloid fibrils. In this study the T-284 dye complex with ASN fibril was characterized by means of fluorescence anisotropy, atomic force microscopy and time-resolved fluorescence techniques to give further insights into the mode of dye interaction with amyloid fibrils. The fluorescence anisotropy of T-284 was shown to noticeably increase upon addition of aggregated proteins indicating on stable dye/amyloid fibril complex formation. AFM imaging of fibrillar wild-type ASN revealed differences in heights between ASN fibrils alone and in presence of the T-284 dye (6.37 ± 1.0 nm and 8.0 ± 1.1 nm respectively), that is believed to be caused by embedding of T-284 dye molecules in the "binding channel" running along the fibril. Fluorescence decay analysis of the T-284 in complexes with fibrillar ASN variants revealed the fluorescence lifetime values for T-284/fibril complexes to be an order of magnitude higher as compared to the free dye. Also, the fluorescence decay of free T-284 was bi-exponential, while dye bound to protein yields tri-exponential decay. We suppose that in complexes with fibrillar ASN variants T-284 dye might exist in different "populations" due to interaction with fibrils in different conformers and ways. The exact binding mode of T-284 with ASN fibrils needs further studies. Studied parameters of dye/amyloid fibril complexes are important for the characterization and screening of newly-developed amyloid-sensitive dyes.

Spectral-fluorescent properties of benzothiazole styryl monomer (Bos-3) and homodimer (DBos-21) dyes in presence of DNA were studied. The dyes enhance their fluorescence intensity in 2-3 orders of magnitude upon interaction with DNA. Studied styrylcyanines in DNA presence demonstrate rather high values of two-photon absorption (TPA) cross-section, which are comparable with the values of TPA cross section of the rhodamine dyes. An applicability of the styrylcyanines as probes for the fluorescence microscopy of living cells was studied. It was shown that both dyes are cell-permeable but homodimer dye DBos-21 produces noticeably brighter staining of HeLa cells comparing with monomer dye Bos-3. Molecules of DBos-21 initially bind to the nucleic acids-containing cell organelles (presumable mitochondria) and are able to penetrate into the cell nucleus. Thus, homodimer styryl DBos-21 dye is viewed as efficient stain for single-photon and two-photon excitation fluorescence imaging of living cells.

We examined the practical aspects of using fluorescent mono (T-284) and trimethinecyanine (SH-516) dyes for detecting and quantifying fibrillar alpha-synuclein (ASN). We studied the interaction of cyanine dyes with fibrillar proteins using fluorescence spectroscopy and atomic force microscopy. The commercially available classic amyloid stain thioflavin T (Thio T) was used as the reference dye. T-284 and SH-516 dyes can be used for fluorometric quantification of fibrillar wild-type ASN at concentrations of approximately 1.5-20 microg/ml. Both dyes appeared suitable for step-wise monitoring of ASN variants (wild-type and mutants A30P and A53T) aggregation into fibrils in vitro, demonstrating good reproducibility, exceeding that for the commonly used Thio T. Our assay may be used for screening in vitro of agents capable of affecting the aggregation of ASN. In addition, T-284 and SH-516 cyanine dyes were shown to recognize amyloid proteins of various amino acid compositions selectively. T-284 demonstrated particular sensitivity to wild-type and A53T ASN, while for SH 516, the fluorescence response to fibrillar proteins was nearly the same except for lysozymes. T-284 and SH-516 cyanine dyes are sensitive and specific fluorescent probes for monitoring ASN fibril formation process in vitro, quantification of fibrillar ASN in solution, and fluorescent detection of various fibrillar protein species.

Series of squaraine benzothiazole and benzoselenazole dyes were studied as possible fluorescent probes for the detection of proteins, particularly albumins. It was shown that majority of the studied squaraines give significant fluorescent response on the human serum albumin (HSA) and bovine serum albumin presence. For squaraine dyes with N-hexyl pendent groups (P-1, P-2, P-3, P-5) about 100-540-fold fluorescence intensity increase upon albumins addition was observed. At the same time in presence of other proteins, namely insulin, avidin from hen egg white, immunoglobulin G (IgG), carbonic anhydrase fluorescence enhancement values were considerably lower -up to 43 times in IgG presence. It was noted that generally, squaraines with long N-hexyl pendent groups demonstrate higher emission increase values upon proteins addition comparing with their analogues with short N-ethyl tails. It was shown that fluorescence intensity enhancement for benzothiazole squaraine dye P-3, relates linearly to the HSA concentration over the wide range-from 0.2 to 500 microg/ml. Together with noticeable selectivity of this dye to albumins, existence of wide dynamic range gives possibility to propose P-3 dye as probe for HSA quantification.

With the aim of searching of novel amyloid-specific fluorescent probes the ability of series of mono- and trimethine cyanines based on benzothiazole, pyridine and quinoline heterocycle end groups to recognize fibrillar formations of alpha-synuclein (ASN) was studied. For the first time it was revealed that monomethine cyanines can specifically increase their fluorescence in aggregated ASN presence. Dialkylamino-substituted monomethine cyanine T-284 and meso-ethyl-substituted trimethine cyanine SH-516 demonstrated the higher emission intensity and selectivity to aggregated ASN than classic amyloid stain Thioflavin T, and could be proposed as novel efficient fluorescent probes for fibrillar ASN detection. Studies of structure-function dependences have shown that incorporation of amino- or diethylamino- substituents into the 6-position of the benzothiazole heterocycle yields in a appearance of a selective fluorescent response to fibrillar alpha-synuclein presence. Performed calculations of molecular dimensions of studied cyanine dyes gave us the possibility to presume, that dyes bind with their long axes parallel to the fibril axis via insertion into the neat rows (so called 'channels') running along fibril.

In the presented work studies of the interaction mode of monomer and two homodimer benzothiazole styryl dyes containing spermine-like linkage/tail group with the double stranded (ds) DNA are reported. For these dyes, equilibrium constant of dye binding to DNA (K(b)), as well as the number of dsDNA base pairs occupied by one bound dye molecule (n) were determined. The data obtained show that the presence of spermine-like group containing quaternary nitrogen (Bos-5) results in increase of K(b) value as compared to this of unsubstituted analogue (Sbt). Besides, for the dimer dyes containing benzothiazole styryl chromophores, the K(b) value is either five times higher (DBos-13) or almost the same (DBsu-10) as compared to this of corresponding monomer Sbt, depending on the position in the benzothiazole ring where the linker is attached. Moreover, the n values for both dimers are significantly different as well, pointing to the bis-intercalative binding mechanism for DBos-13 and for the groove-binding one for DBsu-10. The conclusion about the dimer dyes-dsDNA binding mechanisms is also supported by the study of the fluorescent response of these dyes on the presence of AT- and GC-containing polynucleotides.

The dimeric cyanine dyes, YOYO-1 and TOTO-1, are widely used as DNA probes because of their excellent fluorescent properties. They have a higher fluorescence quantum yield than ethidium homodimer, DAPI and Hoechst dyes and bind to double-stranded DNA with high affinity. However, these dyes are limited by heterogeneous staining at high dye loading, photocleavage of DNA under extended illumination, nicking of DNA, and inhibition of the activity of DNA binding enzymes. To overcome these limitations, seven novel cyanine dyes (Cyan-2, DC-21, DM, DM-1, DMB-2OH, SH-0367, SH1015-OH) were synthesized and tested for fluorescence emission, resistance to displacement by Mg(2+), and the ability to function as reporters for DNA unwinding. Results show that Cyan-2, DM-1, SH-0367 and SH1015-OH formed highly fluorescent complexes with dsDNA. Of these, only Cyan-2 and DM-1 exhibited a large fluorescence enhancement in buffers, and were resistant to displacement by Mg(2+). The potential of these two dyes to function as reporter molecules was evaluated using continuous fluorescence, DNA helicase assays. The rate of DNA unwinding was not significantly affected by either of these two dyes. Therefore, Cyan-2 and DM-1 form the basis for the synthesis of novel cyanine dyes with the potential to overcome the limitations of YOYO-1 and TOTO-1.

We ascertained the ability to detect fibrillar beta-lactoglobulin (BLG) of a series of mono-, tri-, penta-, and heptamethinecyanines based on benzothiazole and benzimidazole heterocycles, and of benzothiazole squaraine. Fluorescence properties of these cyanine dyes were measured in the unbound state and in the presence of monomeric and fibrillar BLG and compared with those for the commercially available benzothiazole dye Thioflavin T. The correlation between the chemical nature of the dye molecules and the ability of dyes to bind aggregated proteins was established. We found that meso-substituted cyanines with amino substituents in heterocycle in contrast to the corresponding unsubstituted dyes have a binding preference to fibrillar BLG and a noticeable fluorescence response in the presence of the aggregated protein. For the squaraines and benzimidazole penthamethinecyanines studied, fluorescence emission increased both in the presence of native and fibrillar protein. The trimethinecyanines T-49 and SH-516 exhibit specifically increased fluorescence in the presence of fibrillar BLG. These dyes demonstrated the same or higher emission intensity and selectivity to aggregated BLG as Thioflavin T, and are proposed for application in selective fluorescent detection of aggregated proteins.

The series of novel monomer and homodimer styryl dyes based on (p-dimethylaminostyryl) benzothiazolium residues were synthesized and studied as possible fluorescent probes for nucleic acids detection. Spectral-luminescent and spectral-photometric properties of obtained dyes in the unbound state and in DNA presence were studied. Fluorescence emission induced by two-photon excitation of dye-DNA complexes in aqueous buffer solution was registered. Two-photon absorption cross section values of the studied dyes in DNA presence were evaluated.

The series of recently synthesized monomeric and homodimeric cyanine dyes based on monomethine cyanine chromophore with oxazolo[4,5-b]pyridinium and quinoline end groups [Vassilev A, Deligeorgiev T, Gadjev N, Drexhage K-H. Synthesis of novel monomeric and homodimeric cyanine dyes based on oxazolo[4,5-b]pyridinium and quinolinium end groups for nucleic acid detection, Dyes Pigm 2005;66:135-142] were studied as possible fluorescent probes for nucleic acids detection. Significant fluorescence enhancement and intensity level (quantum yield up to 0.75) was observed for all the dyes in the presence of DNA. The oxazolo[4,5-b]pyridinium cyanines demonstrated high sensitivity as fluorescent stains for post-electrophoretic visualization of nucleic acids in agarose gels upon both VIS and UV transillumination, and the visualized band contained 0.8 ng of dsDNA.

The influence of methyl-, 2-hydroxyethyl-, dimethyl-, diethyl- and benzoyl-amino substituents in the 6,6'-positions of benzothiazole heterocycle of trimethine cyanines on their spectral-luminescent properties and behavior in presence of DNA, RNA and BSA was studied. It was shown that incorporation of 6,6'-substituents generally leads to the increase in dyes tendency to aggregation, resulting in the considerable decrease in the emission intensity of the disubstituted dyes as compared to the unsubstituted ones. Emission of the studied 6,6'-disubstited dyes in DNA presence is considerably more intensive than in presence of RNA, that points on the existing of DNA binding preference for the mentioned dyes. Insertion of benzoyl-amino groups into the 6,6'-positions permitted us to design the DNA-sensitive dyes on the basis of symmetric trimethine cyanines with unsubstituted polymethine chain, while typically such dyes slightly respond on the presence of biopolymers. 6,6'-Benzoyl-amino-disubstituted trimethine cyanines are proposed as efficient dyes for DNA detection.

Fluorescent chromophore, alkylamino-(tetra-hydronaphthalenylidene)- benzothiazolium derivatives (HBTN dyes), are proposed as covalent labels for proteins via aliphatic amino groups. Spectral-luminescent properties of 3-methyl-2-{(E)-[7-(methylamino)-4,4a,5,6-tetra-hydronaphthalen-2(3H)-ylidene]methyl}-1,3-benzothiazol-3-ium chloride (HBTN, R=Me) and its predecessor, 2-[(E)-(7-methoxy-4,4a,5,6-tetrahydronaphthalen-2(3H)-ylidene)methyl]-3-methyl-1,3-benzothiazol-3-ium chloride (ABTN), are studied for free dyes and in the presence of DNA and BSA. Considerable spectral-luminescent changes accompany the transformation of ABTN into HBTN that allows monitoring conjugation reaction. In presence of DNA and BSA the HBTN increases its emission in 15 and 4 times respectively and becomes strongly fluorescent. The conditions for labeling are developed and a model conjugate of HBTN dye with BSA is synthesized. It was shown that using of HBTN dye as a fluorescent label allows detection by eye of about 3 mug/band of BSA on polyacrylamide gel upon UV-irradiation.

A series of pentamethine cyanine dyes with cyclohexene or cyclopentene group in polymethyne chain, assumed as DNA groove-binders, were studied as fluorescent probes for nucleic acids as well as for native and denatured proteins. It was revealed that the presence of methyl or dimethyl substituent in 5 position of the cyclohexene group hinders the formation of dye-DNA fluorescent complex, while the methyl substituent in 2 position leads to the increasing of the dye-DNA complex fluorescence intensity. The dyes SL-251, SL-1041, and SL-1046 containing methyl group in the 2 position of the cyclic group, are reported as bright DNA-sensitive dyes. The study of the dyes DNA-binding specificity demonstrated significant AT-preference that points to the groove-binding interaction mode. At the same time, the dyes SL-251, SL-377, and SL-957 with the 2-methyl substituted cyclohexene group were shown to be sensitive fluorescent dyes both for nonspecific (in SDS presence) proteins detection and for native BSA.

Series of homodimer styryls containing on (p-dimethylaminostyryl) pyridinium residues that are connected with aliphatic linkage group was synthesized. Spectral luminescent properties of obtained dyes in free state and in nucleic acids presence were studied. It was shown that DNA binding affinity of the novel homodimers exceeds that of parent monomer (p-dimethylaminostyryl)pyridine iodide. For homodimers with the linkage 4-10 carbon atoms preference in binding to DNA than to RNA was observed. It could be concluded that parent monomer has different mechanisms of binding to nucleic acids than corresponding homodimer dye.

The series of symmetrical beta-substituted and alpha,gamma-substituted trimethinecyanine dyes were studied for their absorption and fluorescent characteristics in unbound state and in the presence of nucleic acids and proteins. It was shown that beta-substituted and alpha,gamma-bridged trimethinecyanines containing extended heterocyclic systems or N-phenyl as well as N-cyclohexyl substituents demonstrate increased affinity to proteins. At the same time the presence of both N-phenyl and N-cyclohexyl substituents leads to the decrease of the dye fluorescence intensity in complexes with nucleic acids. For trimethinecyanines similarly to unsymmetrical monomethines the presence of N-omega-hydroxy alkyl substituents results in the increase of fluorescence intensity of dye-DNA complex and the emission decrease of dye-RNA complex.

Fifteen polymethine cyanine dyes were studied as fluorescent stains for DNA in electrophoretic gels. Among studied cyanines, two dyes CPent V and CCyan 2-O most effectively visualized covalently closed and linear double-stranded DNA molecules in gels under standard conditions using UV-illumination, green filter and black-and-white photo film. Ethidium bromide was 1.2-1.6 times more effective as compared to cyanine dyes in staining of DNA in the concentration range of 8-18 ng, while studied cyanines were more sensitive to DNA quantity above 50 ng.

Spectral properties of carbocyanine dye 3-methyl-2-[3-methyl-2-(3-methyl-2,3-dihydro-1,3-benzothiazole-2-iliden)-1- butenyl]-1,3-benzothiazole-3-il iodide (Cyan betaiPr) in water solution, as well as in the presence of different types of double stranded DNA have been studied. While in water solution of 'free' dye Cyan betaiPr stays mainly in monomeric form, in the presence of DNA the dye molecules form J-aggregates. The molecular structure of these J-aggregates causes the Davydov splitting of their absorption band, corresponding to the first electronic transition. A study of site-specificity showed that in the presence of poly (dA/dT) the majority of Cyan betaiPr molecules form J-aggregates, while in the presence of poly (dGC/dGC) dye molecules stay mainly in monomeric form and in presence of chicken erythrocytes DNA both J-aggregate and monomeric forms of dye are present. We suppose that Cyan betaiPr molecules aggregate in DNA groove, which serves as a template for J-aggregate forming. An increase of ionic strength of solution leads to the release of dye molecules from DNA grooves and prevents J-aggregates formation.

Novel monomethine pyridinium cyanine dyes of similar structure and containing 'affinity-modifying' groups of different chemical nature were studied by spectral-luminescent methods as possible fluorescent probes for the nucleic acids detection. It was shown that the nature of the functional groups in the dye linker influences the fluorescent properties of the dye-nucleic acids complexes. Incorporation of a hydroxyl group into the linker structure leads to a significant increase in the fluorescence intensity of the dye--double-stranded DNA complexes relative to the parent dye Cyan 40.

Absorption, fluorescence emission and excitation spectra of benzothiazole cyanine dyes--thiazole orange (TO) and 7-methyl-6-(3-methyl-2,3-dihydro-1,3-benzothiazol-2-ylidenmethyl) [1,3] dioxolo [4',5':4,5] benzo [d] [1,3] thiazolium methylmethosulfate (Cyan 13)--were investigated over a wide concentration range. The dyes form aggregates with a 'sandwich'-like structure in water solution. At low dye to DNA concentrations ratios, Cyan 13 and TO monomers appear to interact with the DNA. On increasing the dye to DNA concentrations ratio, free dye molecules aggregate with the DNA-bound ones. The spectra of the free dye aggregates and the aggregates formed on the DNA, are characterized by an anomalously large (more than 100 nm) Stokes shift. This suggests, that the pi-electron systems of the aggregates undergo substantial changes in excited state, compared to those of the monomers. The formation of aggregates consisting of the free and DNA-bound dye molecules can be explained using the half-intercalation model of the interaction of the cyanine dye monomers with the DNA.

Results of investigations of fluorescent properties of a beta-substituted carbocyanine and its complexes with nucleic acids in comparison with those for the unsubstituted dye are presented. Carbocyanine substituted in polymethine chain has shown promising properties for use as a fluorescent probe in homogeneous systems of nucleic acids detection.