磷硫代酸酯(PS)已经证明其在治疗寡核苷酸领域非常有效，应用范围涵盖了癌症治疗到神经退行性疾病等。最初，PS取代是为了反义寡核苷酸（PS ASOs），因为它赋予了增加核酸酶的抗性，同时改善了细胞摄取和体内生物利用度。因此，PS寡核苷酸已经成为基因沉默治疗方法中的基本资产。但是，尽管它们被广泛使用，人们很少知道PS取代可能在DNA·RNA杂交体中引起可能不同的结构变化。此外，关于磷硫代酸酯手性在调节PS属性方面的作用，存在着有限的信息和显著的争议。通过全面的计算研究和实验测量，在这里，我们阐明了PS手性对基于DNA的反义寡核苷酸的影响。不同的磷硫代酸二面体异构体如何影响DNA拓扑、稳定性和灵活性，以最终揭示在DNA外切酶和人类核糖核酸酶H的催化中心的pro-Sp S和pro-Rp S角色;两个ASOs基础治疗的主要障碍。总的来说，我们的结果提供了关于PS取代引发的结构异常和解释PS链对DNA·RNA杂交物所产生的核酸酶抗性起源的全原子和机械性的洞察，这些都是提高当前的ASOs基础治疗的关键信息。© 作者（2023年）。由牛津大学出版社代表核酸研究出版。

Phosphorothioates (PS) have proven their effectiveness in the area of therapeutic oligonucleotides with applications spanning from cancer treatment to neurodegenerative disorders. Initially, PS substitution was introduced for the antisense oligonucleotides (PS ASOs) because it confers an increased nuclease resistance meanwhile ameliorates cellular uptake and in-vivo bioavailability. Thus, PS oligonucleotides have been elevated to a fundamental asset in the realm of gene silencing therapeutic methodologies. But, despite their wide use, little is known on the possibly different structural changes PS-substitutions may provoke in DNA·RNA hybrids. Additionally, scarce information and significant controversy exists on the role of phosphorothioate chirality in modulating PS properties. Here, through comprehensive computational investigations and experimental measurements, we shed light on the impact of PS chirality in DNA-based antisense oligonucleotides; how the different phosphorothioate diastereomers impact DNA topology, stability and flexibility to ultimately disclose pro-Sp S and pro-Rp S roles at the catalytic core of DNA Exonuclease and Human Ribonuclease H; two major obstacles in ASOs-based therapies. Altogether, our results provide full-atom and mechanistic insights on the structural aberrations PS-substitutions provoke and explain the origin of nuclease resistance PS-linkages confer to DNA·RNA hybrids; crucial information to improve current ASOs-based therapies.© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.