We designed and synthesized nucleosides bearing aminophenyl- or aminonaphthyl-3-methoxychromone fluorophores attached at position 5 of cytosine or thymine and converted them to nucleoside triphosphates. The fluorophores showed solvatochromic fluorescence with strong fluorescence at 433-457 nm in non-polar solvents and very weak fluorescence at 567 nm in alcohols. The nucleosides and nucleotides also showed only negligible fluorescence in alcohols or water. The triphosphates were substrates for DNA polymerase in the enzymatic synthesis of modified DNA probes that showed only very weak fluorescence in aqueous buffer but a significant light-up and blue shift were observed when they interacted with proteins (histone H3.1 or p53 for double-stranded DNA probes or single-strand binding protein for single-stranded oligonucleotide probes). Hence, nucleotides have good potential in the construction of DNA sensors for studying protein-DNA interactions. The modified dNTPs were also transported into cells using a cyclodextrin-based transporter but they were not incorporated into the genomic DNA.
The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is labeled by incorporation of a metabolically-active fluorescent (Cy3) deoxyuridine triphosphate derivative, which is delivered into the cells by a synthetic transporter (SNTT1). The cells are then fixed, stained with DAPI and analyzed by flow cytometry. Thus, this protocol obviates post-labeling steps, which are indispensable in currently used incorporation assays (BrdU, EdU). The applicability of the protocol is demonstrated in analyses of cell cycles of adherent (U-2 OS, HeLa S3, RAW 264.7, J774 A.1, Chem-1, U-87 MG) and suspension (CCRF-CEM, MOLT-4, THP-1, HL-60, JURKAT) cell cultures, including those affected by a DNA polymerase inhibitor (aphidicolin). Owing to a short incorporation time (5-60 min) and reduced number of steps, the protocol can be completed within 1-2 h with a minimal cell loss and with excellent reproducibility.
- MeSH
- barvení a značení metody MeSH
- bromodeoxyuridin aplikace a dávkování MeSH
- buněčný cyklus * MeSH
- DNA analýza MeSH
- fluorescenční barviva aplikace a dávkování MeSH
- HeLa buňky MeSH
- HL-60 buňky MeSH
- Jurkat buňky MeSH
- karbocyaniny aplikace a dávkování MeSH
- lidé MeSH
- průtoková cytometrie metody MeSH
- replikace DNA * MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytosine 2'-deoxyribonucleoside dCTBdp and its triphosphate (dCTBdpTP) bearing tetramethylated thiophene-bodipy fluorophore attached at position 5 were designed and synthesized. The green fluorescent nucleoside dCTBdp showed a perfect dependence of fluorescence lifetime on the viscosity. The modified triphosphate dCTBdpTP was substrate to several DNA polymerases and was used for in vitro enzymatic synthesis of labeled oligonucleotides (ONs) or DNA by primer extension. The labeled single-stranded ONs showed a significant decrease in mean fluorescence lifetime when hybridized to the complementary strand of DNA or RNA and were also sensitive to mismatches. The labeled dsDNA sensed protein binding (p53), which resulted in the increase of its fluorescence lifetime. The triphosphate dCTBdpTP was transported to live cells where its interactions could be detected by FLIM but it did not show incorporation to genomic DNA in cellulo.
- MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- DNA metabolismus MeSH
- fluorescenční spektrometrie MeSH
- hybridizace nukleových kyselin * MeSH
- kationty MeSH
- lidé MeSH
- lipidy chemie MeSH
- nádorové buněčné linie MeSH
- nukleotidy chemická syntéza chemie MeSH
- oligonukleotidové sondy metabolismus MeSH
- rozpouštědla chemie MeSH
- sekvence nukleotidů MeSH
- sloučeniny boru chemie MeSH
- teplota MeSH
- thiofeny chemie MeSH
- vazba proteinů MeSH
- viskozita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Synthesis of cytosine, uracil, and 7-deazaadenine 2'-deoxyribonucleosides and triphosphates (dNTPs) bearing hexamethylated phenyl-bodipy fluorophore attached at position 5 of pyrimidines or at position 7 of 7-deazapurine was developed. All the title labeled nucleosides and dNTPs displayed bright green fluorescence with very high quantum yields. The modified dNmBdpTPs were good substrates to diverse DNA polymerases and were used for in vitro enzymatic synthesis of labeled DNA by primer extension or PCR. In combination with cationic cyclodextrin-peptide-based dNTP transporter, the dNmBdpTPs were successfully used for staining of genomic DNA in living cells for applications in confocal microscopy and in flow cytometry. The best performing cytosine nucleotide dCmBdpTP was used to monitor mitosis in live cells.
- MeSH
- adenin analogy a deriváty chemie MeSH
- barvení a značení MeSH
- cytosin chemie MeSH
- deoxyribonukleosidy chemie MeSH
- DNA analýza MeSH
- fluorescenční barviva chemie MeSH
- lidé MeSH
- metylace MeSH
- nádorové buněčné linie MeSH
- optické zobrazování MeSH
- polyfosfáty chemie MeSH
- průtoková cytometrie MeSH
- puriny chemie MeSH
- sloučeniny boru chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Per(2,3,6-tri-O-benzyl)-γ-cyclodextrin was debenzylated by DIBAL-H to produce a mixture of C6(I),C6(IV) and C6(I),C6(V) isomeric diols, which were separated and isolated. The C2-symmetrical C6(I),C6(V) diol was transformed into dithiol and dimerized to produce a γ-cyclodextrin duplex structure. A crystal structure revealed tubular cavity whose peripheries are slightly elliptically distorted. The solvent accessible volume of the cavity of the γ-CD duplex is about 740 Å(3). Due to this large inner space the duplex forms very stable inclusion complexes with steroids; bile acids examined in this study show binding affinities to the γ-cyclodextrin duplex in the range of 5.3 × 10(7) M(-1)-1.9 × 10(8) M(-1).
- MeSH
- chemie farmaceutická metody MeSH
- dimerizace MeSH
- disulfidy chemie MeSH
- gama-cyklodextriny chemická syntéza chemie MeSH
- imatinib mesylát chemie MeSH
- kalorimetrie MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- krystalografie rentgenová MeSH
- kyselina lithocholová chemie MeSH
- kyslík chemie MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární konformace MeSH
- rozpouštědla chemie MeSH
- steroidy chemie MeSH
- sulfhydrylové sloučeniny chemie MeSH
- termodynamika MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Apart from its role in insulin receptor (IR) activation, the C terminus of the B-chain of insulin is also responsible for the formation of insulin dimers. The dimerization of insulin plays an important role in the endogenous delivery of the hormone and in the administration of insulin to patients. Here, we investigated insulin analogues with selective N-methylations of peptide bond amides at positions B24, B25, or B26 to delineate their structural and functional contribution to the dimer interface. All N-methylated analogues showed impaired binding affinities to IR, which suggests a direct IR-interacting role for the respective amide hydrogens. The dimerization capabilities of analogues were investigated by isothermal microcalorimetry. Selective N-methylations of B24, B25, or B26 amides resulted in reduced dimerization abilities compared with native insulin (K(d) = 8.8 μM). Interestingly, although the N-methylation in [NMeTyrB26]-insulin or [NMePheB24]-insulin resulted in K(d) values of 142 and 587 μM, respectively, the [NMePheB25]-insulin did not form dimers even at high concentrations. This effect may be attributed to the loss of intramolecular hydrogen bonding between NHB25 and COA19, which connects the B-chain β-strand to the core of the molecule. The release of the B-chain β-strand from this hydrogen bond lock may result in its higher mobility, thereby shifting solution equilibrium toward the monomeric state of the hormone. The study was complemented by analyses of two novel analogue crystal structures. All examined analogues crystallized only in the most stable R(6) form of insulin oligomers (even if the dimer interface was totally disrupted), confirming the role of R(6)-specific intra/intermolecular interactions for hexamer stability.
- MeSH
- inzulin prasečí chemie MeSH
- krystalografie rentgenová MeSH
- kvarterní struktura proteinů MeSH
- metylace MeSH
- multimerizace proteinu MeSH
- prasata MeSH
- sekundární struktura proteinů MeSH
- stabilita proteinů MeSH
- vodíková vazba MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Four structurally different alloxazine-cyclodextrin conjugates were prepared and tested as catalysts for the enantioselective oxidation of prochiral sulfides to sulfoxides by hydrogen peroxide in aqueous solutions. The alloxazinium unit was appended to the primary face of α- and β-cyclodextrins via a linker with variable length. A series of sulfides was used as substrates: n-alkyl methyl sulfides (n-alkyl = hexyl, octyl, decyl, dodecyl), cyclohexyl methyl sulfide, tert-butyl methyl sulfide, benzyl methyl sulfide and thioanisol. α-Cyclodextrin conjugate having alloxazinium unit attached via a short linker proved to be a suitable catalyst for oxidations of n-alkyl methyl sulfides, displaying conversions up to 98% and enantioselectivities up to 77% ee. β-Cyclodextrin conjugates were optimal catalysts for the oxidation of sulfides carrying bulkier substituents; e.g. tert-butyl methyl sulfide was oxidized with quantitative conversion and 91% ee. Low loadings (0.3-5 mol%) of the catalysts were used. No overoxidation to sulfones was observed in this study.
- MeSH
- alfa-cyklodextriny chemie MeSH
- aminokyseliny chemie MeSH
- chemické modely MeSH
- chemie organická metody MeSH
- krystalografie rentgenová metody MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- nanotechnologie metody MeSH
- nanotrubičky chemie MeSH
- peptidy chemie MeSH
- vodíková vazba MeSH
- Publikační typ
- práce podpořená grantem MeSH
