N³-methylcytidine (m³C) is present in both eukaryotic tRNA and mRNA and plays critical roles in many biological processes. We report the synthesis of the m³C phosphoramidite building block and its containing RNA oligonucleotides. The base-pairing stability and specificity studies show that the m³C modification significantly disrupts the stability of the Watson–Crick C:G pair. Further m³C decreases the base pairing discrimination between C:G and the other mismatched C:A, C:U, and C:C pairs. Our molecular dynamic simulation study further reveals the detailed structural insights into the m³C:G base pairing pattern in an RNA duplex. More importantly, the biochemical investigation of m³C using reverse transcription in vitro shows that N³-methylation specifies the C:A pair and induces a G to A change using HIV-1-RT, MMLV-RT, and MutiScribe-RT enzymes, all with relatively low replication fidelity. For other reverse transcriptases with higher fidelity like AMV-RT, the methylation could completely shut down DNA synthesis. Our work provides detailed insights into the thermostability of m³C in RNA and a foundation for developing new molecular tools for mapping m³C in different RNA contexts and exploring the biochemical and biomedical potentials of m³C in the design and development of RNA based therapeutics.

中文翻译：

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RNA中N ^3-甲基胞嘧啶（m ³ C）的碱基配对和功能洞察

N ^3-甲基胞嘧啶核苷（m ³ C）存在于真核生物tRNA和mRNA中，并且在许多生物学过程中都起着至关重要的作用。我们报告了m ³ C亚磷酰胺结构单元及其包含的RNA寡核苷酸的合成。碱基对的稳定性和特异性研究表明，m ³ C修饰显着破坏了Watson-Crick C：G对的稳定性。另外，m ³ C降低了C：G和其他错配的C：A，C：U和C：C对之间的碱基配对辨别力。我们的分子动力学模拟研究进一步揭示了RNA双链体中m ³ C：G碱基配对模式的详细结构见解。更重要的是，微生物的生化研究使用体外逆转录的³ C显示N ^3-甲基化指定了C：A对，并使用HIV-1-RT，MMLV-RT和MutiScribe-RT酶诱导了G到A的变化，所有这些酶的复制保真度都相对较低。对于其他具有较高保真度的逆转录酶，例如AMV-RT，甲基化作用可能会完全阻止DNA合成。我们的工作提供了有关RNA中m ³ C的热稳定性的详细见解，并为开发新的分子工具以在不同RNA环境中定位m ³ C以及探索基于RNA的设计和开发中m ³ C的生化和生物医学潜力奠定了基础疗法。