The oxidation of RNA has been implicated in the development of many diseases. Among the four ribonucleotides, guanosine is the most susceptible to oxidation, resulting in the formation of 8-oxo-7,8-dihydroguanosine (8-oxoG). Despite the limited knowledge about how cells regulate the detrimental effects of oxidized RNA, cellular factors involved in its regulation have begun to be identified. One of these factors is polynucleotide phosphorylase (PNPase), a multifunctional enzyme implicated in RNA turnover. In the present study, we have examined the interaction of PNPase with 8-oxoG in atomic detail to provide insights into the mechanism of 8-oxoG discrimination. We hypothesized that PNPase subunits cooperate to form a binding site using the dynamic SFF loop within the central channel of the PNPase homotrimer. We evolved this site using a novel approach that initially screened mutants from a library of beneficial mutations and assessed their interactions using multi-nanosecond Molecular Dynamics simulations. We found that evolving this single site resulted in a fold change increase in 8-oxoG affinity between 1.2 and 1.5 and/or selectivity between 1.5 and 1.9. In addition to the improvement in 8-oxoG binding, complementation of K12 Δpnp with plasmids expressing mutant PNPases caused increased cell tolerance to H₂O₂. This observation provides a clear link between molecular discrimination of RNA oxidation and cell survival. Moreover, this study provides a framework for the manipulation of modified-RNA protein readers, which has potential application in synthetic biology and epitranscriptomics.

RNA的氧化与许多疾病的发展有关。在这四个核糖核苷酸中，鸟苷最易被氧化，导致形成8-oxo-7,8-dihydroguanosine（8-oxoG）。尽管关于细胞如何调节氧化的RNA的有害作用的知识有限，但是已经开始鉴定参与其调节的细胞因子。这些因素之一是多核苷酸磷酸化酶（PNPase），这是一种涉及RNA周转的多功能酶。在本研究中，我们已经详细地研究了PNPase与8-oxoG的相互作用，以提供对8-oxoG识别机制的见解。我们假设PNPase亚基合作使用PNPase同三聚体的中央通道内的动态SFF环形成结合位点。我们使用一种新颖的方法演化了该位点，该方法最初从有益突变库中筛选了突变体，并使用多纳秒分子动力学模拟评估了它们之间的相互作用。我们发现，进化该单个位点导致8-oxoG亲和力在1.2和1.5之间和/或选择性在1.5和1.9之间发生倍数变化。除了改善8-oxoG结合外，还补充了K12Δ带有表达突变PNPase的质粒的pnp引起细胞对H ₂ O _2的耐受性增加。该观察结果为RNA氧化的分子识别与细胞存活之间提供了明确的联系。此外，该研究为修饰RNA蛋白质阅读器的操作提供了一个框架，该框架在合成生物学和转录组学中具有潜在的应用。