2’,3’‐Protected Nucleotides as Building Blocks for Enzymatic de novo RNA Synthesis

Abstract

Besides being a key player in numerous, fundamental biological process, RNA also represents a versatile platform for the creation of therapeutic agents and efficient vaccines. The production of RNA oligonucleotides, especially those decorated with chemical modifications, cannot meet the exponential demand. Due to the inherent limits of solid‐phase synthesis and in vitro transcription, alternative, biocatalytic approaches are in dire need to facilitate the production of RNA oligonucleotides. Here, we present a first step towards the controlled enzymatic synthesis of RNA oligonucleotides. We have explored the possibility of a simple protection step of the vicinal cis‐diol moiety to temporarily block ribonucleotides. We demonstrate that pyrimidine nucleotides protected with acetals, particularly 2',3'‐O‐isopropylidene, are well‐tolerated by the template‐independent RNA polymerase PUP (polyU polymerase) and highly efficient coupling reactions can be achieved within minutes – an important feature for the development of enzymatic de novo synthesis protocols. Even though purines are not equally well‐tolerated, these findings clearly demonstrate the possibility of using cis‐diol‐protected ribonucleotides combined with template‐independent polymerases for the stepwise construction of RNA oligonucleotides.

Controlled enzymatic synthesis of oligonucleotides

“…However, this protection scheme abrogates the possibility of introducing therapeutically relevant, 2’-altered nucleotides. Concomitantly, our laboratory has identified a number of potential cis -diol protecting groups for the design of RNA reversible terminators 98 . Pyrimidine nucleotides equipped with a 2′,3′-O-isopropylidene masking group (structure 10 in Fig.…”