Variety of Nucleotide Polymerase Mutants Aiming to Synthesize Modified RNA

• Published in: Chembiochem : a European journal of chemical biology Vol. 22; no. 14; pp. 2398 - 2406
• Main Authors: Ohashi, Sana, Hashiya, Fumitaka, Abe, Hiroshi
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 15.07.2021
• Subjects: 2’ modified ribonucleotides; aptamer; Aptamers; Aptamers, Nucleotide - chemistry; Aptamers, Nucleotide - metabolism; Chemical synthesis; DNA-Directed RNA Polymerases - metabolism; Mutants; Mutation; Nuclease; Nucleoside triphosphates; Nucleotides; polymerase mutant; protein engineering; Ribonucleic acid; RNA; RNA - chemistry; RNA - metabolism; RNA therapeutics; Substrates; Sugar; Transcription; polymerase mutant; protein engineering; RNA therapeutics; 2’ modified ribonucleotides; aptamer
• ISSN: 1439-4227; 1439-7633; 1439-7633
• DOI: 10.1002/cbic.202100004

Significant efforts have been made to develop therapeutic RNA aptamers that exploit synthetic RNA to capture target molecules. However, ensuring RNA aptamers are resistant against intrinsic nucleases remains an issue and restricts their use as therapeutics. Introduction of chemical modifications to the 2′ sugar moiety of RNA improves their stability effectively and can be achieved by chemical synthesis using modified phosphoramidites; however, this approach is not suitable for preparing long RNA molecules. Although recombinant nucleotide polymerases can transcribe RNA, these polymerases cannot synthesize modified RNA because they do not recognize 2′ modified nucleoside triphosphates. In this review, we focus on several polymerase mutants that tolerate substrates containing modifications of the 2′ sugar moiety to synthesize RNA, and the problems that must be overcome to prepare chemically modified RNA with high efficacy by in vitro transcription. This review introduces several nucleotide polymerase mutants that can incorporate chemically modified nucleotides. Altering amino acid residues around the active sites effectively renders both DNA and RNA polymerases tolerant to ribose 2’ modified ribonucleotide substrates. Introduction of these modifications makes RNA stable and beneficial for therapeutics.