Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, United States.
J Am Chem Soc. 2022 Mar 2;144(8):3487-3496. doi: 10.1021/jacs.1c11985. Epub 2022 Feb 16.
-Acetylcytidine (ac4C) is a post-transcriptional modification of RNA that is conserved across all domains of life. All characterized sites of ac4C in eukaryotic RNA occur in the central nucleotide of a 5'-CCG-3' consensus sequence. However, the thermodynamic consequences of cytidine acetylation in this context have never been assessed due to its challenging synthesis. Here, we report the synthesis and biophysical characterization of ac4C in its endogenous eukaryotic sequence context. First, we develop a synthetic route to homogeneous RNAs containing electrophilic acetyl groups. Next, we use thermal denaturation to interrogate the biochemical effects of ac4C on duplex stability and mismatch discrimination in a native sequence found in human rRNA. Finally, we demonstrate the ability of this chemistry to incorporate ac4C into the complex modification landscape of human tRNA and use duplex melting to highlight an enforcing role for ac4C in this unique sequence context. By enabling ex vivo biophysical analyses of nucleic acid acetylation in its physiological sequence context, these studies establish a chemical foundation for understanding the function of a universally conserved nucleobase in biology and disease.
乙酰胞苷（ac4C）是 RNA 的一种转录后修饰，在所有生命领域中都保守存在。真核生物 RNA 中所有已被确定的 ac4C 位点都出现在 5' - CCG - 3' 保守序列的中央核苷酸处。然而，由于其合成具有挑战性，该修饰在这种情况下的核苷酸碱基乙酰化的热力学后果从未被评估过。在这里，我们报告了在其内源性真核序列环境中 ac4C 的合成和生物物理特性。首先，我们开发了一种合成方法，可得到含有亲电乙酰基的均相 RNA。接下来，我们使用热变性来研究 ac4C 对双链体稳定性和错配识别的生化影响，这是在人类 rRNA 中发现的天然序列。最后，我们证明了这种化学方法能够将 ac4C 整合到人类 tRNA 的复杂修饰景观中，并使用双链体熔解来突出 ac4C 在这种独特序列环境中的强制作用。通过在生理序列环境中对核酸乙酰化进行体外生物物理分析，这些研究为理解这种在生物学和疾病中普遍存在的碱基在生物学和疾病中的功能奠定了化学基础。
