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

0492887 - ÚOCHB 2019 RIV GB eng J - Journal Article
Milisavljevič, Nemanja - Perlíková, Pavla - Pohl, Radek - Hocek, Michal
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.
Organic & Biomolecular Chemistry. Roč. 16, č. 32 (2018), s. 5800-5807. ISSN 1477-0520. E-ISSN 1477-0539
R&D Projects: GA ČR GBP206/12/G151; GA MŠMT(CZ) EF16_019/0000729
Grant - others:AV ČR(CZ) AP1501
Program: Akademická prémie - Praemium Academiae
Institutional support: RVO:61388963
Keywords : in vitro selection * unnatural base * nucleic acids
OECD category: Biochemistry and molecular biology
Impact factor: 3.490, year: 2018
Method of publishing: Open access
http://pubs.rsc.org/en/content/articlehtml/2018/ob/c8ob01498a

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.
Permanent Link: http://hdl.handle.net/11104/0286349