Substrate Specificity of T7 RNA Polymerase toward Hypophosphoric Analogues of ATP

Modified nucleotides are commonly used in molecular biology as substrates or inhibitors for several enzymes but also as tools for the synthesis of modified DNA and RNA fragments. Introduction of modification into RNA, such as phosphorothioate (PS), has been demonstrated to provide higher stability, more effective transport, and enhanced activity of potential therapeutic molecules. Hence, in order to achieve widespread use of RNA molecules in medicine, it is crucial to continuously refine the techniques that enable the effective introduction of modifications into RNA strands. Numerous analogues of nucleotides have been tested for their substrate activity with the T7 RNA polymerase and therefore in the context of their utility for use in in vitro transcription. In the present studies, the substrate preferences of the T7 RNA polymerase toward beta,gamma-hypophospho-modified ATP derivatives for the synthesis of unmodified RNA and phosphorothioate RNA (PS) are presented. The performed studies revealed the stereoselectivity of this enzyme for alpha-thio-beta,gamma-hypo-ATP derivatives, similar to that for alpha-thio-ATP. Additionally, it is demonstrated herein that hypodiphosphoric acid may inhibit in vitro transcription catalyzed by T7 RNA polymerase.