Evaluation of the Structure-Function Relationship of SGNH Lipase from Streptomyces rimosus by Site-Directed Mutagenesis and Computational Approach.

Streptomyces rimosus extracellular lipase (SrL) is a multifunctional hydrolase belonging to the SGNH family. Here site-directed mutagenesis (SDM) was used for the first time to investigate the functional significance of the conserved amino acid residues Ser10, Gly54, Asn82, Asn213, and His216 in the active site of SrL. The hydrolytic activity of SrL variants was determined using para-nitrophenyl (pNP) esters with C4, C8, and C16 fatty acid chains. Mutation of Ser10, Asn82, or His216, but not Gly54, to Ala abolished lipase activity for all substrates. In contrast, the Asn213Ala variant showed increased enzymatic activity for C8 and C16 pNP esters. Molecular dynamics (MD) simulations showed that the interactions between the long alkyl chain substrate (C16) and Ser10 and Asn82 were strongest in Asn213Ala SrL. In addition to Asn82, Gly54, and Ser10, several new constituents of the substrate binding site were recognized (Lys28, Ser53, Thr89, and Glu212), as well as strong electrostatic interactions between Lys28 and Glu212. In addition to the H bonds Ser10-His216 and His216-Ser214, Tyr11 interacted strongly with Ser10 and His216 in all complexes with an active enzyme form. A previously unknown strong H bond between the catalytically important Asn82 and Gly54 was uncovered, which stabilizes the substrate in an orientation suitable for the enzyme reaction.

Anion-Binding Properties of Short Linear Homopeptides.

A comprehensive thermodynamic and structural study of the complexation affinities of tetra (L1), penta (L2), and hexaphenylalanine (L3) linear peptides towards several inorganic anions in acetonitrile (MeCN) and N,N-dimethylformamide (DMF) was carried out. The influence of the chain length on the complexation thermodynamics and structural changes upon anion binding are particularly addressed here. The complexation processes were characterized by means of spectrofluorimetric, 1H NMR, microcalorimetric, and circular dichroism spectroscopy titrations. The results indicate that all three peptides formed complexes of 1:1 stoichiometry with chloride, bromide, hydrogen sulfate, dihydrogen phosphate (DHP), and nitrate anions in acetonitrile and DMF. In the case of hydrogen sulfate and DHP, anion complexes of higher stoichiometries were observed as well, namely those with 1:2 and 2:1 (peptide:anion) complexes. Anion-induced peptide backbone structural changes were studied by molecular dynamic simulations. The anions interacted with backbone amide protons and one of the N-terminal amine protons through hydrogen bonding. Due to the anion binding, the main chain of the studied peptides changed its conformation from elongated to quasi-cyclic in all 1:1 complexes. The accomplishment of such a conformation is especially important for cyclopeptide synthesis in the head-to-tail macrocyclization step, since it is most suitable for ring closure. In addition, the studied peptides can act as versatile ionophores, facilitating transmembrane anion transport.

Structural Dynamics of the Bacillus subtilis MntR Transcription Factor Is Locked by Mn2+ Binding.

Manganese (II) ions are essential for a variety of bacterial cellular processes. The transcription factor MntR is a metallosensor that regulates Mn2+ ion homeostasis in the bacterium Bacillus subtilis. Its DNA-binding affinity is increased by Mn2+ ion binding, allowing it to act as a transcriptional repressor of manganese import systems. Although experimentally well-researched, the molecular mechanism that regulates this process is still a puzzle. Computational simulations supported by circular dichroism (CD), differential scanning calorimetry (DSC) and native gel electrophoresis (native-PAGE) experiments were employed to study MntR structural and dynamical properties in the presence and absence of Mn2+ ions. The results of molecular dynamics (MD) simulations revealed that Mn2+ ion binding reduces the structural dynamics of the MntR protein and shifts the dynamic equilibrium towards the conformations adequate for DNA binding. Results of CD and DSC measurements support the computational results showing the change in helical content and stability of the MntR protein upon Mn2+ ion binding. Further, MD simulations show that Mn2+ binding induces polarization of the protein electrostatic potential, increasing the positive electrostatic potential of the DNA-binding helices in particular. In order to provide a deeper understanding of the changes in protein structure and dynamics due to Mn2+ binding, a mutant in which Mn2+ binding is mimicked by a cysteine bridge was constructed and also studied computationally and experimentally.

The Nature of the (Oligo/Hetero)Arene Linker Connecting Two Triarylborane Cations Controls Fluorimetric and Circular Dichroism Sensing of Various ds-DNAs and ds-RNAs.

A series of tetracationic bis-triarylborane dyes, differing in the aromatic linker connecting two dicationic triarylborane moieties, showed very high submicromolar affinities toward ds-DNA and ds-RNA. The linker strongly influenced the emissive properties of triarylborane cations and controlled the fluorimetric response of dyes. The fluorene-analog shows the most selective fluorescence response between AT-DNA, GC-DNA, and AU-RNA, the pyrene-analog's emission is non-selectively enhanced by all DNA/RNA, and the dithienyl-diketopyrrolopyrrole analog's emission is strongly quenched upon DNA/RNA binding. The emission properties of the biphenyl-analog were not applicable, but the compound showed specific induced circular dichroism (ICD) signals only for AT-sequence-containing ds-DNAs, whereas the pyrene-analog ICD signals were specific for AT-DNA with respect to GC-DNA, and also recognized AU-RNA by giving a different ICD pattern from that observed upon interaction with AT-DNA. The fluorene- and dithienyl-diketopyrrolopyrrole analogs were ICD-signal silent. Thus, fine-tuning of the aromatic linker properties connecting two triarylborane dications can be used for the dual sensing (fluorimetric and CD) of various ds-DNA/RNA secondary structures, depending on the steric properties of the DNA/RNA grooves.

Inhibitory Effect of DMSO on Halohydrin Dehalogenase: Experimental and Computational Insights into the Influence of an Organic Co-solvent on the Structural and Catalytic Properties of a Biocatalyst.

Although the application of organic solvents in biocatalysis is well explored, in-depth understanding of the interactions of solvent with proteins, in particular oligomeric ones, is still scant. Understanding these interactions is essential in tailoring enzymes for industrially relevant catalysis in nonaqueous media. In our study, the homotetrameric enzyme halohydrin dehalogenase (HHDH) from Agrobacterium radiobacter AD1 (HheC) was investigated, as a model system, in DMSO/water solvent mixtures. DMSO, the most commonly used co-solvent for biocatalytic transformations, was found to act as a mixed-type inhibitor with a prevalent competitive contribution. Even 5 % (v/v) DMSO inhibits the activity of HheC by half. Molecular dynamics (MD) simulations showed that DMSO keeps close to Ser-Tyr catalytic residues forming alternate H-bonds with them. Stability measurements paired with differential scanning calorimetry, dynamic light scattering methods and MD studies revealed that HheC maintains its structural integrity with as much as 30 % (v/v) DMSO.

Inhibitory Effect of DMSO on Halohydrin Dehalogenase: Experimental and Computational Insights into the Influence of an Organic Co-solvent on the Structural and Catalytic Properties of a Biocatalyst.

Invited for the cover of this issue are Zlatko Brkljaca, Maja Majeric Elenkov and co-workers at the Ruder Boskovic Institute and University of Zagreb. The image depicts the enzyme halohydrin dehalogenase HheC, which is made up of four identical subunits, with marked catalytic residues and volumetric maps of water and DMSO in the active site. Read the full text of the article at 10.1002/chem.202201923.

Styryl dyes with N-Methylpiperazine and N-Phenylpiperazine Functionality: AT-DNA and G-quadruplex binding ligands and theranostic agents.

New monomethine, unsymmetrical styryl dyes consisting of benzothiazole and N-methylpiperazine or N-phenylpiperazine scaffolds were synthesized, and their binding affinities for different ds-polynucleotides and G-quadruplex were studied. Substitution of piperazine unit with methyl or phenyl group strongly influenced their binding modes, binding affinities, spectroscopic responses and antiproliferative activities. Compounds with N-methylpiperazine substituents showed a significant preference for AT-DNA polynucleotides and demonstrated AT-minor groove binding, which manifested in strong fluorescence increase, significant double helix stabilization, and positive induced circular dichroism spectra. These compounds formed complexes with G-quadruplex by π-π stacking interactions of dye with the top or bottom G-tetrad. Bulkier compounds with N-phenylpiperazine function are probably bound to ds-polynucleotide by partial intercalation between base pairs. On the other hand, they showed stronger stabilization of G-quadruplex compared to methyl-substituted compounds. Fluorimetric titrations pointed to possible mixed stoichiometry's: 1:1 complex with π-π stacking interactions of dye on the top or bottom G-tetrad and 1:2 complex with dye positioned between two G-quadruplex molecules. Bulkier dyes with N-phenylpiperazine fragments demonstrated micromolar and submicromolar antiproliferative activity that was especially pronounced for leukaemia and lymphoma. Flow cytometric assay shows dose- and time-dependent increase in SubG0/G1 phase. Furthermore, the compounds enter the cells readily and accumulate in the mitochondrial space, co-localize with the standard mitochondrial markers.

Anion-Sensing Properties of Cyclopentaphenylalanine.

Cyclic pentaphenylalanine was studied as an efficient anion sensor for halides, thiocyanate and oxoanions in acetonitrile and methanol. Stability constants of the corresponding complexes were determined by means of fluorimetric, spectrophotometric, 1H NMR, and microcalorimetric titrations. A detailed structural overview of receptor-anion complexes was obtained by classical molecular dynamics (MD) simulations. The results of 1H NMR and MD studies indicated that the bound anions were coordinated by the amide groups of cyclopeptide, as expected. Circular dichroism (CD) titrations were also carried out in acetonitrile. To the best of our knowledge, this is the first example of the detection of anion binding by cyclopeptide using CD spectroscopy. The CD spectra were calculated from the structures obtained by MD simulations and were qualitatively in agreement with the experimental data. The stoichiometry of almost all complexes was 1:1 (receptor:anion), except for dihydrogen phosphate where the binding of dihydrogen phosphate dimer was observed in acetonitrile. The affinity of the cyclopeptide receptor was correlated with the structure of anion coordination sphere, as well as with the solvation properties of the examined solvents.

Bithiophene-Cored, mono-, bis-, and tris-(Trimethylammonium)-Substituted, bis-Triarylborane Chromophores: Effect of the Number and Position of Charges on Cell Imaging and DNA/RNA Sensing.

The synthesis, photophysical, and electrochemical properties of selectively mono-, bis- and tris-dimethylamino- and trimethylammonium-substituted bis-triarylborane bithiophene chromophores are presented along with the water solubility and singlet oxygen sensitizing efficiency of the cationic compounds Cat1+ , Cat2+ , Cat(i)2+ , and Cat3+ . Comparison with the mono-triarylboranes reveals the large influence of the bridging unit on the properties of the bis-triarylboranes, especially those of the cationic compounds. Based on these preliminary investigations, the interactions of Cat1+ , Cat2+ , Cat(i)2+ , and Cat3+ with DNA, RNA, and DNApore were investigated in buffered solutions. The same compounds were investigated for their ability to enter and localize within organelles of human lung carcinoma (A549) and normal lung (WI38) cells showing that not only the number of charges but also their distribution over the chromophore influences interactions and staining properties.

Tetracationic Bis-Triarylborane 1,3-Butadiyne as a Combined Fluorimetric and Raman Probe for Simultaneous and Selective Sensing of Various DNA, RNA, and Proteins.

A bis-triarylborane tetracation (4-Ar2 B-3,5-Me2 C6 H2 )-C≡C-C≡C-(3,5-Me2 C6 H2 -4-BAr2 [Ar=(2,6-Me2 -4-NMe3 -C6 H2 )+ ] (24+ ) shows distinctly different behaviour in its fluorimetric response than that of our recently published bis-triarylborane 5-(4-Ar2 B-3,5-Me2 C6 H2 )-2,2'-(C4 H2 S)2 -5'-(3,5-Me2 C6 H2 -4-BAr2 ) (34+ ). Single-crystal X-ray diffraction data on the neutral bis-triarylborane precursor 2 N confirm its rod-like dumbbell structure, which is shown to be important for DNA/RNA targeting and also for BSA protein binding. Fluorimetric titrations with DNA/RNA/BSA revealed the very strong affinity of 24+ and indicated the importance of the properties of the linker connecting the two triarylboranes. Using the butadiyne rather than a bithiophene linker resulted in an opposite emission effect (quenching vs. enhancement), and 24+ bound to BSA 100 times stronger than 34+ . Moreover, 24+ interacted strongly with ss-RNA, and circular dichroism (CD) results suggest ss-RNA chain-wrapping around the rod-like bis-triarylborane dumbbell structure like a thread around a spindle, a very unusual mode of binding of ss-RNA with small molecules. Furthermore, 24+ yielded strong Raman/SERS signals, allowing DNA or protein detection at ca. 10 nm concentrations. The above observations, combined with low cytotoxicity, efficient human cell uptake and organelle-selective accumulation make such compounds intriguing novel lead structures for bio-oriented, dual fluorescence/Raman-based applications.

Synthesis, DNA/RNA-interaction and biological activity of benzo[k,l]xanthene lignans.

Interactions of two newly synthesized and six previously reported benzoxanthene lignans (BXLs), analogues of rare natural products, with DNA/RNA, G-quadruplex and HSA were evaluated by a set of spectrophotometric methods. Presence/absence of methoxy and hydroxy groups on the benzoxanthene core and minor modifications at C-1/C-2 side pendants - presence/absence of phenyl ring and presence/absence of methoxy and hydroxy groups on phenyl ring - influenced the fluorescence changes and the binding strength to double-stranded (ds-) and G-quadruplex structures. In general, compounds without phenyl ring showed stronger fluorescence changes upon binding than phenyl-substituted BXLs. On the other hand, BXLs with an unsubstituted phenyl ring showed the best stabilization effects of G-quadruplex. Circular dichroism spectroscopy results suggest mixed binding mode, groove binding and partial intercalation, to ds-DNA/RNA and end-stacking to top or bottom G-tetrads as the main binding modes of BXLs to those targets. All compounds exhibited micromolar binding affinities toward HSA and an increased protein thermal stability. Moderate to strong antiradical scavenging activity was observed for all BXLs with hydroxy groups at C-6, C-9 and C-10 positions of the benzoxanthene core, except for derivative bearing methoxy groups at these positions. BXLs with unsubstituted or low-substituted phenyl ring and one derivative without phenyl ring showed strong growth inhibition of Gram-positive Staphylococcus aureus. All compounds showed moderate to strong tumor cell growth-inhibitory activity and cytotoxicity.

Fine structural tuning of styryl-based dyes for fluorescence and CD-based sensing of various ds-DNA/RNA sequences.

A set of styryl- and bis-styryl dyes, varying in length, aromatic surface, net positive charge and steric positioning or bulkiness of substituents, was tested for interactions with various ds-DNA or ds-RNA. Most of the compounds showed strong affinity toward ds-DNA/RNA, directly correlated to the synergistic contribution of the aromatic-conjugated surface and net positive charge. The volume or positioning of terminal aromatic substituents directly controlled the binding mode of the core structure, shifting between DNA/RNA groove binding or DNA/RNA intercalation. Consequently, upon binding to DNA/RNA the fluorimetric and induced CD (ICD) response varied for different compounds, for instance one derivative showed specific fluorescence increase with AT-DNA, while another derivative showed specific ICD response with AU-RNA. Preliminary screening on human tumour cell lines revealed an efficient cellular uptake for all dyes. Only mono-styryl-quinoline derivatives showed a strong antiproliferative activity combined with efficient fluorescent localisation, thus showing promising theragnostic potential, while other compounds were negligibly cytotoxic but still efficient fluorescent markers of cytoplasmic organelles.

Unique Aggregation of Sterigmatocystin in Water Yields Strong and Specific Circular Dichroism Response Allowing Highly Sensitive and Selective Monitoring of Bio-Relevant Interactions.

We demonstrated the hitherto unknown property of the mycotoxin sterigmatocystin (STC) to provide homogeneous solutions in aqueous medium by forming a unique aggregate type (not formed by analogous aflatoxins), characterized by exceptionally strong circular dichroism (CD) bands in the 300-400 nm range. Results showed that these CD bands do not originate from intrinsic STC chirality but are a specific property of a peculiar aggregation process similar to psi-DNA CD response. Transmission electron microscopy (TEM) experiments revealed a fine fiber network resembling a supramolecular gel structure with helical fibers. Thermodynamic studies of aggregates by differential scanning calorimetry (DSC) revealed high reversibility of the dominant aggregation process. We demonstrated that the novel STC psi-CD band at 345 nm could be applied at biorelevant conditions (100 nanomolar concentration) and even in marine-salt content conditions for specific and quantitative monitoring of STC. Also, we showed that STC strongly non-covalently interacts with ds-DNA with likely toxic effects, thus contrary to the previous belief requiring prior enzyme epoxidation.

Phase-Dependent Adsorption of Myelin Basic Protein to Phosphatidylcholine Lipid Bilayers.

The dense packing of opposite cytoplasmic surfaces of the lipid-enriched myelin membrane, responsible for the proper saltatory conduction of nerve impulses through axons, is ensured by the adhesive properties of myelin basic protein (MBP). Although preferentially interacting with negatively charged phosphatidylserine (PS) lipids, as an intrinsically disordered protein, it can easily adapt its shape to its immediate environment and thus adsorb to domains made of zwitterionic phosphatidylcholine (PC) lipids. As the molecular-level interaction pattern between MBP and PC lipid membranes suffers from scarce characterization, an experimental and computational study of multilamellar liposomes (MLVs) composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in the presence of bovine MBP is presented here. Calorimetric and temperature-dependent UV-Vis measurements identified DPPC pretransition temperature (Tp) and calorimetric enthalpy (ΔHcal) as the physicochemical parameters most responsive to the presence of MBP. Besides suggesting an increase in ß-sheet fractions of structured MBP segments as DPPC lipids undergo from the gel (20 °C) to the fluid (50 °C) phase, FTIR spectra unraveled the significant contribution of lysine (Lys) residues in the adsorption pattern, especially when DPPC is in the fluid (50 °C) phase. In addition to highlighting the importance of Lys residues in the MBP adsorption on DPPC lipid bilayer, employing salt bridges (SBs) and hydrogen bonds (HBs), MD data suggest the crucial importance of the orientation of MBP with respect to the surface of the DPPC lipid bilayer.

Impact of C-terminal domains of paralogous single-stranded DNA binding proteins from Streptomyces coelicolor on their biophysical properties and biological functions.

Single-stranded DNA-binding proteins (SSB) are crucial in DNA metabolism. While Escherichia coli SSB is extensively studied, the significance of its C-terminal domain has only recently emerged. This study explored the significance of C-domains of two paralogous Ssb proteins in S. coelicolor. Mutational analyses of C-domains uncovered a novel role of SsbA during sporulation-specific cell division and demonstrated that the C-tip is non-essential for survival. In vitro methods revealed altered biophysical and biochemical properties of Ssb proteins with modified C-domains. Determined hydrodynamic properties suggested that the C-domains of SsbA and SsbB occupy a globular position proposed to mediate cooperative binding. Only SsbA was found to form biomolecular condensates independent of the C-tip. Interestingly, the truncated C-domain of SsbA increased the molar enthalpy of unfolding. Additionally, calorimetric titrations revealed that C-domain mutations affected ssDNA binding. Moreover, this analysis showed that the SsbA C-tip aids binding most likely by regulating the position of the flexible C-domain. It also highlighted ssDNA-induced conformational mobility restrictions of all Ssb variants. Finally, the gel mobility shift assay confirmed that the intrinsically disordered linker is essential for cooperative binding of SsbA. These findings highlight the important role of the C-domain in the functioning of SsbA and SsbB proteins.

Selenium-Substituted Monomethine Cyanine Dyes as Selective G-Quadruplex Spectroscopic Probes with Theranostic Potential.

The binding interactions of six ligands, neutral and monocationic asymmetric monomethine cyanine dyes comprising benzoselenazolyl moiety with duplex DNA and RNA and G-quadruplex structures were evaluated using fluorescence, UV/Vis (thermal melting) and circular dichroism (CD) spectroscopy. The main objective was to assess the impact of different substituents (methyl vs. sulfopropyl vs. thiopropyl/thioethyl) on the nitrogen atom of the benzothiazolyl chromophore on various nucleic acid structures. The monomethine cyanine dyes with methyl substituents showed a 100-fold selectivity for G-quadruplex versus duplex DNA. Study results indicate that cyanines bind with G-quadruplex via end π-π stacking interactions and possible additional interactions with nucleobases/phosphate backbone of grooves or loop bases. Cyanine with thioethyl substituent distinguishes duplex DNA and RNA and G-quadruplex structures by distinctly varying ICD signals. Furthermore, cell viability assay reveals the submicromolar activity of cyanines with methyl substituents against all tested human cancer cell lines. Confocal microscopy analysis shows preferential accumulation of cyanines with sulfopropyl and thioethyl substituents in mitochondria and indicates localization of cyanines with methyl in nucleus, particularly nucleolus. This confirms the potential of examined cyanines as theranostic agents, possessing both fluorescent properties and cell viability inhibitory effect.

Highly selective preparation of N-terminus Horseradish peroxidase-DNA conjugate with fully retained enzymatic activity: HRP-DNA structure - activity relation.

Within the last decade, the field of bio-nanoengineering has achieved significant advances allowing us to generate, e.g., nanoscaled molecular machineries with arbitrary shapes. To unleash the full potential of novel methods such as DNA origami technology, it is important to functionalise complex molecules and nanostructures precisely. Thus, considerable attention has been given to site-selective modifications of proteins allowing further incorporation of various functionalities. Here, we describe a method for the covalent attachment of oligonucleotides to the glycosylated horseradish peroxidase protein (HRP) with high N-terminus selectivity and significant yield while conserving the enzymatic activity. This two-step process includes a pH-controlled metal-free diazotransfer reaction using imidazole-1-sulfonyl azide hydrogen sulfate, which at pH 8.5 results in an N-terminal azide-functionalized protein, followed by the Cu-free click SPAAC reaction to dibenzocyclooctyne- (DBCO) modified oligonucleotides. The reaction conditions were optimised to achieve maximum yield and the best performance. The resulting protein-oligonucleotide conjugates (HRP-DNA) were characterised by electrophoresis and mass spectrometry (MS). Native-PAGE experiments demonstrated different migration patterns for HRP-DNA and the azido-modified protein allowing zymogram experiments. Structure-activity relationships of novel HRP-DNA conjugates were assessed using molecular dynamics simulations, characterising the molecular interactions that define the structural and dynamical properties of the obtained protein-oligonucleotide conjugates (POC).

Structural and dynamical changes of the Streptococcus gordonii metalloregulatory ScaR protein induced by Mn2+ ion binding.

Divalent metal ions are essential micronutrients for many intercellular reactions. Maintaining their homeostasis is necessary for the survival of bacteria. In Streptococcus gordonii, one of the primary colonizers of the tooth surface, the cellular concentration of manganese ions (Mn2+) is regulated by the manganese-sensing transcriptional factor ScaR which controls the expression of proteins involved in manganese homeostasis. To resolve the molecular mechanism through which the binding of Mn2+ ions increases the binding affinity of ScaR to DNA, a variety of computational (QM and MD) and experimental (ITC, DSC, EMSA, EPR, and CD) methods were applied. The computational results showed that Mn2+ binding induces a conformational change in ScaR that primarily affects the position of the DNA binding domains and, consequently, the DNA binding affinity of the protein. In addition, experimental results revealed a 1:4 binding stoichiometry between ScaR dimer and Mn2+ ions, while the computational results showed that the binding of Mn2+ ions in the primary binding sites is sufficient to induce the observed conformational change of ScaR.

Evans blue as a selective dye marker for white-light diagnosis of non-muscle-invasive bladder cancer: an in vitro study.

OBJECTIVE: To develop a diagnostic method relying on the preferential accumulation of a dye in non-muscle-invasive bladder cancer (NMIBC) that is visible in conjunction with white-light cystoscopy (WLC). MATERIALS AND METHODS: We investigated in detail the permeation of Evans blue in urothelial cell carcinoma (UCC) spheroids prepared from T24, J82 and RT-112 human cell lines and spheroids composed of normal human urothelial (NHU) cells. To gain more insight into the differential accumulation, all spheroids were investigated ultrastructurally using transmission electron microscopy (TEM). RESULTS: We found that, after exposure to Evans blue for 2 h, UCC spheroids accumulated dramatically more dye than spheroids composed of NHU cells. Using TEM it was found that the malignant spheroids contain similar ultrastructural characteristics, i.e. a wide intercellular space and a decreased number of desmosome-like cell attachments, to those from clinical samples of non-papillary carcinoma in situ of the bladder. CONCLUSION: We believe the present findings could be important for future developments in clinical diagnostics for early bladder cancer detection, staging and grading involving WLC.

Recognition of ATT Triplex and DNA:RNA Hybrid Structures by Benzothiazole Ligands.

Interactions of an array of nucleic acid structures with a small series of benzothiazole ligands (bis-benzothiazolyl-pyridines-group 1, 2-thienyl/2-benzothienyl-substituted 6-(2-imidazolinyl)benzothiazoles-group 2, and three 2-aryl/heteroaryl-substituted 6-(2-imidazolinyl)benzothiazoles-group 3) were screened by competition dialysis. Due to the involvement of DNA:RNA hybrids and triplex helices in many essential functions in cells, this study's main aim is to detect benzothiazole-based moieties with selective binding or spectroscopic response to these nucleic structures compared to regular (non-hybrid) DNA and RNA duplexes and single-stranded forms. Complexes of nucleic acids and benzothiazoles, selected by this method, were characterized by UV/Vis, fluorescence and circular dichroism (CD) spectroscopy, isothermal titration calorimetry, and molecular modeling. Two compounds (1 and 6) from groups 1 and 2 demonstrated the highest affinities against 13 nucleic acid structures, while another compound (5) from group 2, despite lower affinities, yielded higher selectivity among studied compounds. Compound 1 significantly inhibited RNase H. Compound 6 could differentiate between B- (binding of 6 dimers inside minor groove) and A-type (intercalation) helices by an induced CD signal, while both 5 and 6 selectively stabilized ATT triplex in regard to AT duplex. Compound 3 induced strong condensation-like changes in CD spectra of AT-rich DNA sequences.