- MeSH
- bakteriální RNA MeSH
- biochemie MeSH
- dinukleosidfosfáty MeSH
- Escherichia coli MeSH
- výzkum MeSH
- Publikační typ
- populární práce MeSH
- rozhovory MeSH
- MeSH
- inhibitory enzymů chemická syntéza chemie farmakologie MeSH
- molekulární struktura MeSH
- organické sloučeniny křemíku chemická syntéza chemie farmakologie MeSH
- restrikční endonukleasy typu II antagonisté a inhibitory metabolismus MeSH
- štěpení DNA účinky léků MeSH
- stereoizomerie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A series of six pyrimidine-modified dNTPs--5-ethynyl-, 5-phenyl-, and 5-(3-nitrophenyl)deoxycitidine and -deoxyuridine triphosphates--were prepared and incorporated by primer extension with Vent (exo-)polymerase to specific DNA sequences within or next to the recognition sequences of selected restriction endonucleases. The cleavage of these pyrimidine-modified DNA sequences by 13 restriction enzymes was then studied. Whereas the presence of any modified C within the target sequence completely prevented any restriction cleavage, most enzymes tolerated the presence of 5-ethynylU and two of them even the presence of 5-phenyl- and 5-(3-nitrophenyl)U. Modifications outside the recognition sequence were tolerated except in the case of phenyl derivatives with the PvuII enzyme. 5-EthynylC was used for protection of the recognition sequence from cleavage in the presence of the second unmodified copy of the same sequence that was cleaved.
A simple approach to DNA tail-labelling using terminal deoxynucleotidyl transferase and modified deoxynucleoside triphosphates is presented. Amino- and nitrophenyl-modified dNTPs were found to be good substrates for this enzyme giving 3'-end stretches of different lengths depending on the nucleotide and concentration. 3-Nitrophenyl-7-deazaG was selected as the most useful label because its dNTP was efficiently incorporated by the transferase to form long tail-labels at any oligonucleotide. Accumulation of many nitrophenyl tags per oligonucleotide resulted in a considerable enhancement of voltammetric signals due to the nitro group reduction, thus improving the sensitivity of electrochemical detection of the tail-labelled probes. We demonstrate a perfect discrimination between complementary and non-complementary target DNAs sequences by tail-labelled hybridization probes as well as the ability of tumour suppressor p53 protein to recognize a specific binding site within tail-labelled DNA substrates, making the methodology useful in electrochemical DNA hybridization and DNA-protein interaction assays.
- MeSH
- DNA sondy analýza chemie MeSH
- DNA vazebné proteiny chemie MeSH
- DNA-nukleotidylexotransferasa chemie MeSH
- elektrochemické techniky metody MeSH
- hybridizace nukleových kyselin metody MeSH
- molekulární struktura MeSH
- purinové nukleotidy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Aqueous Sonogashira cross-coupling reactions of 5-iodopyrimidine or 7-iodo-7-deazaadenine nucleosides with bile acid-derived terminal acetylenes linked via an ester or amide tether gave the corresponding bile acid-nucleoside conjugates. Analogous reactions of halogenated nucleoside triphosphates gave directly bile acid-modified dNTPs. Enzymatic incorporation of these modified nucleotides to DNA was successfully performed using Phusion polymerase for primer extension. One of the dNTPs (dCTP bearing cholic acid) was also efficient for PCR amplification.
- MeSH
- denaturace nukleových kyselin MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- DNA chemie metabolismus MeSH
- nukleosidy chemická syntéza chemie MeSH
- nukleotidy chemická syntéza chemie metabolismus MeSH
- Thermococcaceae enzymologie MeSH
- žlučové kyseliny a soli chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Modified 2'-deoxynucleoside triphosphates (dNTPs) bearing [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes attached via an acetylene linker to the 5-position of pyrimidines (C and U) or to the 7-position of 7-deazapurines (7-deaza-A and 7-deaza-G) have been prepared in one step by aqueous cross-couplings of halogenated dNTPs with the corresponding terminal acetylenes. Polymerase incorporation by primer extension using Vent (exo-) or Pwo polymerases gave DNA labeled in specific positions with Ru(2+) or Os(2+) complexes. Square-wave voltammetry could be efficiently used to detect these labeled nucleic acids by reversible oxidations of Ru(2+/3+) or Os(2+/3+). The redox potentials of the Ru(2+) complexes (1.1-1.25 V) are very close to that of G oxidation (1.1 V), while the potentials of Os(2+) complexes (0.75 V) are sufficiently different to enable their independent detection. On the other hand, Ru(2+)-labeled DNA can be independently analyzed by luminescence. In combination with previously reported dNTPs bearing ferrocene, aminophenyl, and nitrophenyl tags, the Os-labeled dATP has been successfully used for "multicolor" redox labeling of DNA and for DNA minisequencing.
- MeSH
- barva MeSH
- barvení a značení metody MeSH
- DNA-dependentní DNA-polymerasy chemie MeSH
- DNA chemie MeSH
- elektrochemie MeSH
- luminiscence MeSH
- oligonukleotidy chemie MeSH
- osmium chemie MeSH
- oxidace-redukce MeSH
- reagencia zkříženě vázaná chemie MeSH
- ruthenium chemie MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- adenosintrifosfát analogy a deriváty analýza chemie MeSH
- cytidintrifosfát analogy a deriváty analýza chemie MeSH
- DNA-dependentní DNA-polymerasy chemie MeSH
- elektrochemie MeSH
- financování organizované MeSH
- kyseliny boronové chemie MeSH
- molekulární struktura MeSH
- oligonukleotidy chemická syntéza chemie MeSH
- uridintrifosfát analogy a deriváty analýza chemie MeSH
dATP derivatives bearing Br, Me or Ph groups in position 8 were prepared and tested as substrates for DNA polymerases to show that 8-Br-dATP and 8-Me-dATP were efficiently incorporated, while 8-Ph-dATP was a poor substrate due to its bulky Ph group.
