Development and Comparison of 4-Thiouridine to Cytidine Base Conversion Reaction.

To study transcriptome dynamics without harming cells, it is essential to convert chemical bases. 4-Thiouridine (4sU) is a biocompatible uridine analogue that can be converted into a cytidine analogue. Although several reactions can convert 4sU into a cytidine analogue, few studies have compared the features of these reactions. In this study, we performed three reported base conversion reactions, including osmium tetroxide, iodoacetamide, and sodium periodate treatment, as well as a new reaction using 2,4-dinitrofluorobenzene. We compared the reaction time, conversion efficacy, and effects on reverse transcription. These reactions successfully converted 4sU into a cytidine analogue quantitatively using trinucleotides. However, the conversion efficacy and effect on reverse transcription vary depending on the reaction with the RNA transcript. OsO4 treatment followed by NH4Cl treatment showed the best base-conversion efficiency. Nevertheless, each reaction has its own advantages and disadvantages as a tool for studying the transcriptome. Therefore, it is crucial to select the appropriate reaction for the target of interest.

Variety of Nucleotide Polymerase Mutants Aiming to Synthesize Modified RNA.

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.