RESUMO
Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction in the major groove of DNA containing 5-ethynyluracil (UE ) with azides was used for turning off sequence-specific protein-DNA interactions. The concept was first demonstrated on switching off cleavage of short modified DNA by restriction endonuclease BamHI-HF. Finally, DNA template containing UE was used for in vitro transcription with E.â coli RNA polymerase and the transcription was turned off by CuAAC with 3-azidopropane-1,2-diol or 3-azido-7-hydroxycoumarin.
Assuntos
Alcinos/química , Azidas/química , Cumarínicos/química , RNA Polimerases Dirigidas por DNA/química , DNA/química , Escherichia coli/química , RNA Bacteriano/química , Catálise , Química Click , Cobre , Reação de Cicloadição , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Bacteriano/metabolismo , Uracila/análogos & derivadosRESUMO
DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2'-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. colienzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , DNA/química , Transcrição Gênica , Bacillus subtilis/enzimologia , DNA/metabolismo , Desoxirribonucleotídeos/biossíntese , Desoxirribonucleotídeos/química , Escherichia coli/enzimologia , Conformação de Ácido Nucleico , Moldes GenéticosRESUMO
Additions of alkyl- or arylthiols to 7-vinyl-7-deaza-2'-deoxyadenosine gave a series of 7-[2-(alkyl- or arylsulfanyl)ethyl]-7-deaza-2'-deoxyadenosines in 45-85% yields. The nucleosides were converted to 5'-O-mono-(dASRMP) or triphosphates (dASRTP) by phosphorylation. The modified triphosphates were also prepared by thiol addition to 7-vinyl-7-deaza-dATP. The triphosphates dASRTP were good substrates for DNA polymerases useful in the enzymatic synthesis of base-modified oligonucleotides (ONs) or DNA containing flexibly linked hydrophobic substituents in the major groove. Primer extension was used for the synthesis of ONs with one or several modifications, PCR was used for the synthesis of heavily modified DNA, whereas terminal deoxynucleotidyl transferase was used for a single-nucleotide labeling of the 3'-end.
Assuntos
DNA/química , Nucleotídeos de Desoxiadenina/química , Desoxiadenosinas/química , Nucleosídeos/química , Compostos de Sulfidrila/química , Compostos de Vinila/química , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética/métodos , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The syntheses of 5-arylsulfanyl- or 5-arylselanylpyrimidine and 7-arylsulfanyl- or 7-arylselanyl-7-deazapurine nucleosides and nucleotides were developed by the Cu-mediated sulfanylations or selanylations of the corresponding 5-iodopyrimidine or 7-iodo-7-deazapurine nucleosides or nucleotides with diaryldisulfides or -diselenides. The reactions were also applicable for direct modifications of 2'-deoxycytidine triphosphate and the resulting 5-arylsulfanyl or 5-arylselanyl-dCTP served as substrates for the polymerase synthesis of modified DNA bearing arylsulfanyl or arylselanyl groups in the major groove.