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
• Adenosine Triphosphate analogs & derivatives   metabolism   MeSH
• Bacteriophage T7 enzymology   MeSH
• Cytidine Triphosphate analogs & derivatives   metabolism   MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• Purine Nucleosides chemistry   metabolism   MeSH
• Purines chemistry   metabolism   MeSH
• Pyrimidine Nucleosides chemistry   metabolism   MeSH
• RNA chemistry   metabolism   MeSH
• Substrate Specificity MeSH
• Uridine Triphosphate analogs & derivatives   metabolism   MeSH
• Viral Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study 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
• Cytidine Triphosphate analogs & derivatives   metabolism   MeSH
• Caliciviridae Infections virology   MeSH
• Humans MeSH
• Norovirus enzymology   metabolism   MeSH
• RNA-Dependent RNA Polymerase metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Molecular Docking Simulation MeSH
• Substrate Specificity MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Adenosine Triphosphate analogs & derivatives   analysis   chemistry   MeSH
• Cytidine Triphosphate analogs & derivatives   analysis   chemistry   MeSH
• DNA-Directed DNA Polymerase chemistry   MeSH
• Electrochemistry MeSH
• Financing, Organized MeSH
• Boronic Acids chemistry   MeSH
• Molecular Structure MeSH
• Oligonucleotides chemical synthesis   chemistry   MeSH
• Uridine Triphosphate analogs & derivatives   analysis   chemistry   MeSH