Ohashi Sana, Nakamura Mayu, Acharyya Susit, Inagaki Masahito, Abe Naoko, Kimura Yasuaki, Hashiya Fumitaka, Abe Hiroshi
Graduate School of Science, Nagoya University, Nagoya, Aichi 464-8602, Japan.
Research Center for Material Science, Nagoya University, Nagoya, Aichi 464-8602, Japan.
ACS Omega. 2024 Feb 14;9(8):9300-9308. doi: 10.1021/acsomega.3c08516. eCollection 2024 Feb 27.
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. OsO treatment followed by NHCl 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.
为了在不损伤细胞的情况下研究转录组动态，对化学碱基进行转换至关重要。4-硫尿苷（4sU）是一种生物相容性尿苷类似物，可被转化为胞苷类似物。尽管有几种反应可将4sU转化为胞苷类似物，但很少有研究比较这些反应的特点。在本研究中，我们进行了三种已报道的碱基转换反应，包括四氧化锇、碘乙酰胺和高碘酸钠处理，以及一种使用2,4-二硝基氟苯的新反应。我们比较了反应时间、转换效率以及对逆转录的影响。这些反应使用三核苷酸成功地将4sU定量转化为胞苷类似物。然而，转换效率和对逆转录的影响因与RNA转录本的反应而异。OsO处理后再进行NHCl处理显示出最佳的碱基转换效率。尽管如此，作为研究转录组的工具，每种反应都有其自身的优缺点。因此，为感兴趣的目标选择合适的反应至关重要。
