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Enzymatic synthesis of base-modified RNA by T7 RNA polymerase. A systematic study and comparison of 5-substituted pyrimidine and 7-substituted 7-deazapurine nucleoside triphosphates as substrates.
Milisavljevic, Nemanja; Perlíková, Pavla; Pohl, Radek; Hocek, Michal.
Afiliação
  • Milisavljevic N; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16610, Prague 6, Czech Republic. hocek@uochb.cas.cz.
Org Biomol Chem ; 16(32): 5800-5807, 2018 08 15.
Article em En | MEDLINE | ID: mdl-30063056
ABSTRACT
We synthesized a small library of eighteen 5-substituted pyrimidine or 7-substituted 7-deazapurine nucleoside triphosphates bearing methyl, ethynyl, phenyl, benzofuryl or dibenzofuryl groups through cross-coupling reactions of nucleosides followed by triphosphorylation or through direct cross-coupling reactions of halogenated nucleoside triphosphates. We systematically studied the influence of the modification on the efficiency of T7 RNA polymerase catalyzed synthesis of modified RNA and found that modified ATP, UTP and CTP analogues bearing smaller modifications were good substrates and building blocks for the RNA synthesis even in difficult sequences incorporating multiple modified nucleotides. Bulky dibenzofuryl derivatives of ATP and GTP were not substrates for the RNA polymerase. In the case of modified GTP analogues, a modified procedure using a special promoter and GMP as initiator needed to be used to obtain efficient RNA synthesis. The T7 RNA polymerase synthesis of modified RNA can be very efficiently used for synthesis of modified RNA but the method has constraints in the sequence of the first three nucleotides of the transcript, which must contain a non-modified G in the +1 position.
Assuntos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Nucleosídeos de Purina / Purinas / Nucleosídeos de Pirimidina / Proteínas Virais / RNA Polimerases Dirigidas por DNA / RNA / Bacteriófago T7 Idioma: En Ano de publicação: 2018 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Nucleosídeos de Purina / Purinas / Nucleosídeos de Pirimidina / Proteínas Virais / RNA Polimerases Dirigidas por DNA / RNA / Bacteriófago T7 Idioma: En Ano de publicação: 2018 Tipo de documento: Article