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.

• MeSH
• adenosintrifosfát analogy a deriváty   metabolismus   MeSH
• bakteriofág T7 enzymologie   MeSH
• cytidintrifosfát analogy a deriváty   metabolismus   MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• purinové nukleosidy chemie   metabolismus   MeSH
• puriny chemie   metabolismus   MeSH
• pyrimidinové nukleosidy chemie   metabolismus   MeSH
• RNA chemie   metabolismus   MeSH
• substrátová specifita MeSH
• uridintrifosfát analogy a deriváty   metabolismus   MeSH
• virové proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Molecular dynamics simulations of complexes between Norwalk virus RNA dependent RNA polymerase and its natural CTP and 2dCTP (both containing the O5'-C5'-C4'-O4' sequence of atoms bridging the triphosphate and sugar moiety) or modified coCTP (C5'-O5'-C4'-O4'), cocCTP (C5'-O5'-C4'-C4'') substrates were produced by means of CUDA programmable graphical processing units and the ACEMD software package. It enabled us to gain microsecond MD trajectories clearly showing that similar nucleoside triphosphates can bind surprisingly differently into the active site of the Norwalk virus RNA dependent RNA polymerase. It corresponds to their different modes of action (CTP-substrate, 2dCTP-poor substrate, coCTP-chain terminator, cocCTP-inhibitor). Moreover, extremely rare events-as repetitive pervasion of Arg182 into a potentially reaction promoting arrangement-were captured.

• MeSH
• cytidintrifosfát analogy a deriváty   metabolismus   MeSH
• infekce viry z čeledi Caliciviridae virologie   MeSH
• lidé MeSH
• Norovirus enzymologie   metabolismus   MeSH
• RNA-dependentní RNA-polymerasa metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• substrátová specifita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH