Insertion of a methylene group into the backbone of an antisense oligonucleotide reveals the importance of deoxyribose recognition by RNase H
Insertion of a methylene group into the backbone of an antisense oligonucleotide reveals the importance of deoxyribose recognition by RNase H
复制标题
将亚甲基插入反义寡核苷酸的主链揭示了 RNase H 识别脱氧核糖的重要性
DOI：
10.1039/d2ob01667b
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发表时间：
2022
影响因子：
3.2
通讯作者：
Seio Kohji
中科院分区：
文献类型：
--
作者：
Masaki Yoshiaki;Tabira Ayano;Hattori Shihori;Wakatsuki Shunsuke;Seio Kohji
RNA酶H是反义寡核苷酸敲除基因的关键效应子。虽然已经开发了各种化学修饰来调节RNase H介导的切割，但尚未实现精确的控制。在本研究中，我们试图解决的问题，磷酸基团或脱氧核糖的相互作用是更重要的DNA/RNA双链体的识别RNase H。为了回答这个问题，我们研究了在5′-上游或3′-下游硫代磷酸酯基团处插入亚甲基对RNase H介导的切割的影响。通过在5′-上侧或3′-下侧插入亚甲基，磷酸或脱氧核糖之间的距离可以以不同的模式改变。当在核苷的5′-磷酸基团处插入亚甲基时，观察到切割反应的最大抑制，已知该亚甲基通过RNase H中W221残基的堆积来区分核糖和脱氧核糖。在不同的序列以及错配的双链体中观察到这种效应，表明脱氧核糖环与RNA酶H的相互作用比磷酸基团更重要。我们的研究结果将有助于设计进一步的分子修饰，提高RNase H介导的切割反应的选择性，这使得等位基因特异性ASO的发展。
RNase H acts as a key effector in gene knockdown by antisense oligonucleotides (ASOs). Although various chemical modifications have been developed to regulate RNase H-mediated cleavage, precise control is yet to be achieved. In this study, we tried to address the question of whether the interaction of phosphate groups or deoxyriboses is more important in the recognition of DNA/RNA duplex by RNase H. To answer this question, we investigated the effect of methylene group insertion at the 5′-upstream or 3′-downstream phosphorothioate groups on RNase H-mediated cleavage. By inserting a methylene group at the 5′-upside or 3′-downside, the distance between phosphates or deoxyriboses could be changed in a different pattern. Maximum suppression of the cleavage reaction was observed when a methylene group was inserted at the 5′-phosphate group of the nucleoside which is known to distinguish ribose and deoxyribose via stacking of the W221 residue in RNase H. This effect was observed in a different sequence as well as mismatched duplexes, suggesting the interaction of deoxyribose rings with RNase H is more important than that of phosphate groups. Our results will contribute to the designing of further molecular modifications that improve the selectivity of RNase H-mediated cleavage reactions which allows for the development of allele-specific ASOs.