Substrate properties of C′‐methyl UTP derivatives in T7 RNA polymerase reactions. Evidence for N‐type NTP conformation

Abstract

The number of synthetic UTP analogues containing methyl groups in different positions of the ribose moiety were tested as substrates for T7 RNA polymerase (T7 RNAP). Two of these compounds (containing substituents in the 5' position) were shown to be weak substrates of T7 RNAP. 3'Me-UTP was neither substrate nor inhibitor of T7 RNAP while 2'Me-UTP was shown to terminate RNA chain synthesis. Conformational analysis of the analogues and parent nucleotide using the forcefield method indicates that the allowed conformation of UTP during its incorporation into the growing RNA chain by T7 RNAP is limited to the X angle range of 192-256° of N-type conformation.

Antiviral and Antimicrobial Nucleoside Derivatives: Structural Features and Mechanisms of Action

“…As a result, the synthesis of the nucleic acid chain stops. This phenomenon was first described in the late 1980s into the 90s [37][38][39]. Under the conditions of a single-substrate reaction, when the D-allo-diastereomer or L-talo-diastereomer of 5'-methyl-UTP (5'-aMe-UTP and 5'-tMe-UTP) was added to the presynthesized primer RNA together with natural UTP, 5'-aMe-UTP was incorporated approximately 100 times less efficiently, and 5'-tMe-UTP was incorporated 1000 times less efficiently than the natural substrate of UTP [38,39].…”

“…For example, 3'-methyl-UTP, which is the termination substrate of Escherichia coli RNA polymerase, can be used for nucleic acid sequencing. In this compound, the 3'-methyl group, which replaces the proton, significantly reduces the reactivity of the 3'-hydroxyl group [ 36 , 37 ].…”

Synthesis of 2′‐ and 3′‐C‐Methylribonucleosides

“…This unit describes details for preparation of 2 -and 3 -C-methylribonucleosides starting from common 2-and 3-C-methylribofuranose precursors. These conformationally restricted analogs of natural ribonucleosides proved to be valuable tools (Mikhailov et al, 1999) for the elucidation of the mechanism of action for several enzymes operating with nucleic acids and their components, including adenosine deaminase (Kalinitchenko et al, 1988), uridine and purine nucleoside phosphorylases (Zinchenko et al, 1987), various nucleases (Mikhailov et al, 1992;Moiseyev et al, 1997), and RNA polymerases (Aivazashvili et al, 1986;Savochkina et al, 1989;Pravdina et al, 1990;Mikhailov et al, 1991;Tunitskaya et al, 1997). Another application, developed by Piccirilli's group, is an advanced strategy to probe the conformation of specific residues within functional RNA (ribozymes, aptamers) based on a strong preference for 3 -endo conformation in 2 -C-methylribonucleosides (Tang et al, 1999;Ye et al, 2005).…”