A tetrafluorophenyl activated ester self-assembled monolayer for the immobilization of amine-modified oligonucleotides

A tetrafluorophenyl (TFP) ester-terminated self-assembled monolayer (SAM) for the fabrication of DNA arrays on gold surfaces is described. Activated ester SAMs are desirable for biomolecule array fabrication because they readily react with amine-containing molecules to form a stable amide linkage. N-Hydroxysuccinimide (NHS) ester SAMs are commonly used for this purpose but are subject to a competing hydrolysis side reaction, limiting their effectiveness under basic conditions. TFP was evaluated here as an alternative activated ester leaving group with a potentially greater stability under basic conditions. It is shown that TFP SAMs are much more stable to basic pH than their NHS analogs and are also more hydrophobic, which is an advantage in the fabrication of high-density spotted arrays. DNA arrays prepared on TFP SAMs at pH 10 have a 5-fold greater surface density of DNA molecules, reduced fluorescence background, and smaller spot radii than those prepared on NHS SAM analogs.     

Synthesis of chloro-substituted analogs of Thiazole orange - Fluorophores for flow cytometric analyses

Synthesis, absorption and fluorescence properties of a series of asymmetrical monomethine cyanine dyes, chloro-containing analogs of Thiazole orange, are reported. Their staining ability was studied by flow cytometry. The saturating concentrations of each dye that gives a stable staining intensity have been determined. The ability of dyes B9, B11, B13 to stain live macrophages and apoptotic splenocytes was investigated. Positive signal in nucleus of adherent macrophages detected by fluorescent microscopy showed good specificity of B9, B11 and B13 dyes for DNA. In apoptotic assay cells positive for Annexin V were stained more brightly with the dyes B9, B11 and B13 than with propidium iodide. Despite that B13 showed high DNA selectivity it induces apoptosis of splenocytes and it is not suitable for detection of dead cells. The other synthesized chloro-containing analogs of Thiazole orange B9 and B11 can be successfully used for flow cytometric analyses of DNA content in live cells and for analyses of cell apoptosis.     

DRAMATIC IMPROVEMENTS IN VIRAL INACTIVATION WITH BROMINATED PSORALENS, NAPHTHALENES AND ANTHRACENES

Amino or polyamino derivatives of naphthalene (N-H), anthracene (A-H) and 8-alkoxypsoralen (PSR-H) were prepared along with their monobrominatcd analogs (N-Br, A-Br and PSR-Br). The ammonium salts of these compounds are all water soluble and bind strongly to calf thymus DNA and to λ phage, a double-helical DNA, protein-coated virus. Binding of the sensitizer to DNA occurs, presumably by a mixture of hydrophobic, intercalative and electrostatic interactions. Relative binding constants to calf thymus DNA and to λ phage were measured by the cthidium bromide fluorescence quenching assay. In general the brominated analogs bind more tightly to calf thymus DNA and to the virus than to the nonhalogenated analogs. It is demonstrated that the brominated aromatics are much more effective at inactivating λ phage upon photoactivation (λ 310 or 350 nm) than are their nonbrominated analogs. At identical sensitizer concentrations (by weight) and light flux N-Br, A-Br, and PSR-Br produce 5–6 more logs of viral inactivation than their nonbrominated counterparts (N-H, A-H and PSR-H, respectively). The bromine effect may originate from light-induced electron transfer and subsequent cleavage of the C-Br bond of the sensitizer radical anion bonds to form aryl radicals. Singlet oxygen cannot be responsible for the viral inactivation because the brominated sensitizers are equally effective in the presence and absence of oxygen. Dithiothreitol does not protect λ phage from light-induced inactivation by the brominated sensitizer thereby demonstrating that the photogenerated reactive intermediates responsible for the effect are complcxed to the virus and are not generated free in solution.     

A FLUORESCENCE STUDY OF THE BINDING OF HOECHST 33258 and DAPI TO HALOGENATED DNAs

We have studied the time-resolved and the steady-state fluorescence of the DNA groove binders 4',6-diamidino-2-phenylindole (DAPI) and Hoechst 33258 with the double stranded DNAs poly(dA-dU) and poly(dI-dC) and their halogenated analogs, poly(dA-I5dU) and poly(dI-Br5dC). These studies were prompted by earlier observations that steady-state fluorescence of Hoechst 33258 is quenched on binding to halogenated DNAs (presumably due to an intermolecular heavy atom effect involving the halogen atom in the major groove), and recent studies which clearly point to a binding-site in the minor groove of DNA. Measurements of the time resolved fluorescence decay demonstrate that the fluorescence of Hoechst 33258 is quenched on binding to the halogenated DNAs, in agreement with previous observations. However, quenching studies carried out using the free halogenated bases IdUrd and BrdCyd in solution yielded bimolecular rate constants more than one order of magnitude larger than those expected for an intermolecular heavy atom effect. Moreover, the quenching of the Hoechst 33258 fluorescence was accompanied by an accelerated photochemical destruction of Hoechst 33258. We therefore conclude that the fluorescence quenching observed with halogenated DNAs is probably due to a photochemical reaction involving Hoechst 33258, rather than direct contact of Hoechst 33258 with the halogen substituents in the major groove of the DNA. The fluorescence decay measurements however, do provide clear evidence for at least two different modes of binding. Taking into account the alternating sequences used in this study and the possibility of two different conformations for bound dye, at least four different modes of binding are plausible. Our present data do not allow us to distinguish between these alternatives. The time-resolved fluorescence decays and fluorescence quantum yields of DAPI are not affected by the presence of the heavy atom substituents in the DNA major groove. Based on this observation and earlier reports that DAPI binds in one of the DNA grooves, we conclude that the high affinity sites for DAPI on DNA are located in the minor groove.     

Template-Dependent Incorporation of Spin-Labeled Thymidine Analogs into Viral DNA

The synthesis of novel pppU_d analogs substituted at position C(5) with tethered nitroxide radicals is reported. It is shown that these analogs can serve as good substrates for E. coli DNA polymerase (Pol I) and T-4 DNA polymerase using lambda-phage DNA as template. The template-dependent incorporation of the substrates was established by radio-labeling and ESR experiments.     

Synthesis of Distamycin Analogs and Their Interactions with Calf Thymus DNA

Two distamycin analogs (PyPyPy‐γ‐Dp and PyPyPyPy‐γ‐Dp) were synthesized by a haloform reaction and the DCC/HOBT coupling reaction in a ample and fast way without amino protection. By using calf thymus DNA, the interaction between the analogs and DNA duplex was studied by CD, and ITC.     

Highly efficient modification of DNA polymerase β under conditions of direct and sensitized activation of photoreactive DNAs. Modification of cell extract proteins

dUTP and dCTP derivatives containing a 4-azido-2,3,5,6-tetrafluorobenzylideneaminooxy group were incorporated into the 3′-end of the DNA primer within complexes with the DNA-matrix as analogs of natural dTTP by virtue of catalytic activity of DNA polymerase β or endogenous DNA polymerases of the cell extract. The photoreactive DNAs synthesized in situ were used for affinity modification of DNA polymerase β and DNA-binding proteins of the cell extract. For the photoreactive DNA based on these analogs, the efficiency of formation of covalent adducts with DNA polymerase β under the highest degree of DNA complexation with the enzyme was determined. The yield of covalent DNA adducts with the enzyme was 28–47%, depending on the type of the analog. The effect of the sequence of the DNA template near the localization of the photoreactive group on the redistribution of covalent cross-links between the possible targets was demonstrated. A possibility of increasing the efficiency of DNA polymerase β modification in the presence of a substantial excess of photoreactive DNA using a sensitizer, a dUTP derivative containing a pyrene residue, was studied. When photoreactive DNA containing a 2,3,5,6-tetrafluoro-4-azidobenzoyl (FAB) group was used, about 60% of DNA polymerase β was covalently attached to DNA. Photoreactive dNTP analogs ensuring a high level of protein modification in the cell extract were found. __________ Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1273–1283, May, 2005.     

Photophysics and Photochemical Studies of the Vitamin B6 Group and Related Derivatives

The photophysics and photochemical properties of vitamin B6 constituents and analogs were studied as function of pH and solvent. The p K of the phenolic oxygen and the pyridine ring nitrogen depends on the electron donor-acceptor ability of the 4-substituent, and agrees with the calculated proton affinity. For all studied compounds, the fluorescence properties showed that the phenolic oxygen is 8 units more acidic in the lowest singlet excited state than in the ground state. The pyridine N-atom is slightly more basic in the excited state. At pH of biological significance, pH 6–8, pyridoxamine and 4-pyridoxic acid are the more efficient chromophores with higher fluorescence yield and longer lifetime. Spectroscopic studies showed that the tautomeric equilibrium depends on the nature of the 4-substituent. The quenching of the singlet excited state of pyridoxamine and 4-pyridoxic acid by amino acids, free or in a peptide, and DNA bases at pH 7 was studied by time-resolved fluorescence techniques. The quenching rate constants are well correlated with the redox properties of the pyridoxinic compound and amino acids, and are related to the free energy change in the electron transfer process. Guanosine and pyrimidine bases also are efficient quenchers, involving an electron transfer reaction.     

Evaluation of binding of perylene diimide and benzannulated perylene diimide ligands to dna by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) and spectroscopic studies in solution were used to evaluate the self-association, G-quadruplex DNA binding, and selectivity of a series of perylene diimides (PDIs) (PIPER, Tel01, Tel11, Tel12, and Tel18) or benzannulated perylene diimide ligands (Tel34 and Tel32). Fluorescence and resonance light scattering spectra of Tel01, Tel12, Tel32, and Tel34 reveal that these analogs undergo self-association in solution. UV-Vis and fluorescence titrations with G-quadruplex, duplex, or single-stranded DNA demonstrate that all the analogs, with the exception of Tel32, bind to G-quadruplex DNA, with those PDIs that are self-associated in solution showing the highest degree of selectivity for binding G-quadruplex DNA. Parallel ESI-MS analysis of the stoichiometries demonstrates the ability of the ligands, with the exception of Tel32, to bind to G-quadruplex DNA. While most ligands show major 1:1 and 2:1 binding stoichiometries as expected in the case of end-stacking, interestingly, three of the most quadruplex-selective ligands show a different behavior. Tel01 forms 3:1 complexes, while Tel12 and Tel32 only form 1:1 complexes. Collisional activation dissociation patterns are compatible with ligand binding to G-quadruplex DNA via stacking on the ends of the terminal G-tetrads. Experiments with duplex and single strand DNA were performed to assess the binding selectivities of the ligands. PIPER, Tel11, and Tel18 demonstrated extensive complexation with duplex DNA, while Tel11 and Tel18 bound to single strand DNA, confirming the lack of selectivity of these two ligands. Our results indicate that Tel01, Tel12, and Tel34 are the most selective for G-quadruplex DNA.     

Synthesis,DNA-binding,and cytotoxic studies on three copper(II) complexes of unsymmetrical synthetic analogues of curcumin

Copper(II) chelates of three unsymmetrical synthetic analogs of curcumin, namely (2E)-2-(4-hydroxy-3-methoxybenzylidene)-5-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)cyclopentanone(1), (2E)-2-(3,4-dihydroxybenzylidene)-5-((E)-3-(3,4-dihydroxyphenyl)acryloyl)cyclopentanone(2), and (2E)-2-(3,4-dimethoxybenzylidene)-5-((E)-3-(3,4-dimethoxyphenyl)acryloyl)cyclopeantanone(3) have been synthesized and characterized by physicochemical and spectroscopic methods. The ligands were in their enolic form and metal complexes have 1 : 2 metal:ligand stoichiometry. The DNA-binding properties of the ligands and their metal complexes were studied by absorption titrations, fluorescence quenching experiments, and viscosity measurements with calf-thymus DNA. The interactions of copper(II) complexes were higher than that of free ligands. The observed intrinsic binding constants reveal moderate interaction of copper(II) complexes with calf-thymus DNA. The binding involves intercalative mode through non-covalent interactions and produced conformational changes in the structure of DNA. The compounds were investigated for their possible cytotoxic and antitumor activities. All the compounds were cytotoxic towards Dalton’s lymphoma ascites cells. It was found that copper chelates are remarkably active compared to free curcumin analogs. Concentrations needed for 50% cell death were 10–22 μg mL^?1 for copper complexes and 27–52 μg mL^?1 for curcumin analogs. Copper complex of 2 with two hydroxyl groups in the phenyl ring was most active towards Dalton’s lymphoma ascites cells (increase in life span 77.91%). Copper(II) complex of 3, which possesses methoxy groups in the phenyl ring system, showed the lowest activity towards increase in lifespan of tumor-bearing mice (increase in lifespan 60.14%). Copper chelates of all curcuminoid analogs showed a significant reduction in solid tumor volume in mice.     

Biologically active fluorescent farnesol analogs

We describe ten polyene analogs of farnesol, typified by 3,7,11-trimethyl-2,4,6,8,10-dodecapentenaldehyde oxime, which preserve the length, cross-section, and approximate hydrophobicity of farnesol. Four of the ten display strong quorum-sensing activity in the human pathogen Candida albicans, with IC(50) values for inhibition of germ-tube formation as low as 10 microM. The polyenes display absorption maxima between 320 and 380 nm, with the extinction coefficients for the oximes approaching 100,000. All but two of the analogs are fluorescent, with excitation maxima varying over the range of 320-370 nm. Oxime anti-4, which can undergo fluorescence excitation at wavelengths beyond 400 nm, is demonstrated to be useful for confocal fluorescence microscopic imaging of fungal cells. The farnesol analogs are also expected to be useful for detection of farnesol binding proteins and in determination of farnesol pharmacokinetics.     

Evaluation of complexes of DNA duplexes and novel benzoxazoles or benzimidazoles by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used to compare the metal ion binding and metal-mediated DNA binding of benzoxazole (1, 2, 3, 4) and benzimidazole (5) compounds and to elucidate the putative binding modes and stoichiometries. The observed metal versus non-metal-mediated DNA binding, as well as the specificity of DNA binding, is correlated with the biological activities of the analogs. The ESI-MS spectra for the antibacterial benzoxazole and benzimidazole analogs 4 and 5 demonstrated non-specific and non-metal-mediated binding to DNA, with the appearance of DNA complexes containing multiple ligands. The anticancer analog 2 demonstrates a clear preference for metal-mediated DNA interactions, with an apparent selectivity for Ni2+ -mediated binding over the more physiologically relevant Mg2+ or Zn2+ cations. Complexation between DNA and the biologically inactive analog 1 was not observed, either in the absence or presence of metal cations.     

SELECTIVE DNA THYMINE DIMERIZATION DURING UVA IRRADIATION IN THE PRESENCE OF A SATURATED PYRIDOPSORALEN

Abstract— It has been recently shown that UVA (320–400 nm) irradiation of DNA in the presence of pyridopsoralens induces the formation of thymine cyclobutane dimers in addition to monoadducts. In this work, we measured the potency of a saturated pyridopsoralen to photosensitize DNA, despite its inability to covalently attach to DNA. First, from spectroscopic fluorescence measurements, we have shown that both analogs, saturated and unsaturated pyridopsoralens, namely 4',5'-dihydro-7-methyl-pyrido[3,4-clpsoralen (DH-MePyPs) and 7-methylpyrido[3,4-c]psoralen, exhibit a similar global affinity for DNA. Secondly, we demonstrated, by footprinting experiments, that exposure of a DNA sequence to 365 nm UV radiation in the presence of DH-MePyPs results in selective cyclobutane thymine dimerization. Thymines located in the immediate proximity of the 5'-TA-3' step are exclusively affected and the frequency of this photoprocess depends on flanking sequences. We thus probe a selective thymine dimer photosensitizer. Results are discussed in terms of drug affinity and physical properties of the helix at the binding site.     

The extent of DNA deformation in DNA photolyase-substrate complexes: a solution state fluorescence study.

Cyclobutylpyrimidine dimers (CPDs) are the major UV photoproduct formed in DNA containing adjacent pyrimidines. These lesions can be repaired by DNA photolyase, a flavoprotein that utilizes blue light in a direct reversal of the cyclobutane ring. Previous studies have shown that the CPD is base flipped into the protein, with concomitant disruption of the substrate around the CPD. In this study, we use a fluorescent cytidine analog, pyrrolo-dC (PC), to probe how far base flipping propagates along the duplex. From these measurements, the degree of base destacking in the two bases flanking the adenines opposing the CPD appears to be minimal, which was consistent with the protein:substrate crystal structure. Fluorescence-detected melting temperatures for duplexes with and without a CPD were obtained, suggesting that a 5'-pyrimidine-PC-purine-3' motif is more stable than the 5'-purine-PC-pyrimidine-3' motif. This stability trend was reflected in the fluorescence intensities of ss-PC oligos but not for duplexes. The melting point depression due to the PC probe was found to be comparable to other popular fluorescent base analogs.     

Computational design and characterization of new thieno-expanded tricyclic purine analogs

Unnatural nucleobases are under intense research due to their widespread applications in nucleic acids research. In this work, four new thieno-expanded purine analogs comprising ^ttzA, ^tthA, ^ttzG, and ^tthG were computationally designed based on the isomorphic tz- and th-bases. These base analogs can also be seen as modified derivatives of the previously reported tricyclic purine analogs (^ttA and ^ttG). The structural, electronic, and photophysical properties are studied by means of DFT and TDDFT calculations. We find out that these new bases can form stable Watson-Crick base pairs with natural counterparts, thus potentially mimicking natural nucleobases in DNA/RNA duplexes. Calculations reveal that these bases have smaller AIPs and HOMO-LUMO gaps than natural ones, suggesting that they are candidates for applications in nanowire technology. Particularly, the photophysical properties were explored, and the results are compared with those for tz-, th-, and tt-bases. The nature of the low-lying excited states is discussed, and analyses reveal that the thiophene-homologation would not change excitation maxima of ^thA and ^tzA, while it will result in large red-shifts of those of ^thG and ^tzG. Meanwhile, thiophene insertion has relatively larger influences on the emissions ^thA and ^tzG, for which the fluorescence was 37 nm blue-shifted and 19 nm red-shifted, respectively. Taking these new bases as derivatives of ^ttA and ^ttG, it was found that the modifications would result in large red-shifts of both the excitation maxima and the fluorescence. The effects of water solution and base paring on the phtotophysical properties were also considered.     

Structures of terminal heterocyclic groups and fluorescence-spectral properties of polymethine dyes

The fluorescence and absorption bands of carbocyanines containing pyrylium or benzoxazolium groups, their nitrogen-, sulfur-, and selenium-containing analogs, their benzohomologs, or isomeric analogs of pyrylium as terminal groups were mathematically processed using the method of moments. The regularities in the displacement of the absorption and fluorescence bands following the replacement of the heteroatom and variations of the positions of substituents in the terminal groups were explained using perturbation theory. Based on quantum-chemical calculations, the changes in the bond orders in the ground and excited states of the dyes were studied. The correlations between the moments of the experimental absorption and fluorescence bands and the frequency and the form of the chromophore vibrations were analyzed. The transition from pyrylo-4-cyanines to isomeric pyrylo-2-cyanines leads to substantial broadening of the bands, an increase in the Stokes shifts, decreases in the coefficients of asymmetry, kurtosis, and fine structure of the bands, as well as a decrease in the quantum yield of fluorescence.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 2194–2203, September, 1996.     

Design and synthesis of cinanserin analogs as severe acute respiratory syndrome coronavirus 3CL protease inhibitors

The severe acute respiratory syndrome (SARS) coronavirus 3CL protease is an attractive target for the development of anti-SARS drugs. In this paper, cinanserin (1) analogs were synthesized and tested for the inhibitory activities against SARS-coronavirus (CoV) 3CL protease by fluorescence resonance energy transfer (FRET) assay. Four analogs show significant activities, especially compound 26 with an IC(50) of 1.06 microM.     

A novel spectrofluorimetric method for the determination of DNA

A new simple, selective and sensitive fluorescence quenching method was developed to determine nucleic acids (DNA) with the 9-anthracenecarboxylic acid (ACA)-cetyl trimethyl-ammonium bromide (CTAB) system. The fluorescence intensity of ACA was decreased by the addition (CTAB). However, the fluorescence intensity of the system increased dramatically when DNA was added to the solution. The fluorescence enhancement is probably based on the DNA interaction with CTAB. Under the optimum conditions, the changes of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of nucleic acids over the range 0.08-1.0 microg mL(-1) for CT (calf thymus) DNA or FS (fish sperm) DNA. Its detection limits are 0.02 microg mL(-1) for CT DNA and 0.019 microg mL(-1) for FS DNA. Based on this approach, a new quantitative method for DNA assay is presented in this paper.     

三聚氰胺对DNA潜在损伤作用的研究

在生理酸度条件下(pH 7.4),采用溴化乙锭(EB)为荧光探针的荧光光谱法、I-离子荧光猝灭效应、DNA熔点和粘度效应等手段,研究了三聚氰胺与DNA的相互作用。随着DNA的加入,三聚氰胺的荧光强度明显减小而且三聚氰胺能够猝灭DNA-EB复合物的荧光,说明三聚氰胺能够竞争置换EB而与DNA作用;三聚氰胺的加入使得DNA的粘度增大,DNA-EB的熔点降低;DNA的加入减小了I-对三聚氰胺荧光的猝灭程度。三聚氰胺以嵌插方式作用于DNA的亲核位点,意味着三聚氰胺进入生物体后有可能通过形成DNA加合物的形式造成DNA损伤,从而最终导致基因突变。     

LINKED-FUNCTION ANALYSIS OF FLUORESCENCE DECAY KINETICS: RESOLUTION OF SIDE-CHAIN ROTAMER POPULATIONS OF A SINGLE AROMATIC AMINO ACID IN SMALL POLYPEPTIDES

A linked-function approach to fluorescence decay data analysis is presented that permits complex systems to be resolved from a single decay curve. The method involves linking fluorescence decay parameters based on a relationship established by independent physical measurements. As an example, by correlating the fluorescence data with ¹H-NMR results, the complex fluorescence decay kinetics of tyrosine analogs and single tyrosyl residues in simple polypeptides can be explained by ground-state rotameric populations of the phenol ring about the Cα-Cβ bond.