Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases

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 mo...

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Bibliographic Details
Published in:Nucleic acids research 2016-04, Vol.44 (7), p.3000-3012
Main Authors: Raindlová, Veronika, Janoušková, Martina, Slavíčková, Michaela, Perlíková, Pavla, Boháčová, Soňa, Milisavljevič, Nemanja, Šanderová, Hana, Benda, Martin, Barvík, Ivan, Krásný, Libor, Hocek, Michal
Format: Article
Language:English
Subjects:
Bacillus subtilis
Bacillus subtilis - enzymology
Chemical Biology and Nucleic Acid Chemistry
Deoxyribonucleotides - biosynthesis
Deoxyribonucleotides - chemistry
DNA - chemistry
DNA - metabolism
DNA-Directed RNA Polymerases - metabolism
Escherichia coli
Escherichia coli - enzymology
Nucleic Acid Conformation
Templates, Genetic
Transcription, Genetic
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Summary: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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkw171