环反义寡核苷酸的拓扑开关和β-半乳糖苷酶触发的开关通过CuAAC控制RNA切割。

IF 3.9 2区 化学 Q1 BIOCHEMICAL RESEARCH METHODS
Kento Miyaji*, Keita Takeuchi and Kohji Seio, 
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引用次数: 0

摘要

前药型反义寡核苷酸(ASOs)通过外部刺激或细胞内酶激活提供基因沉默的时空控制。然而,硫代磷酸酯(PS)修饰的间隙聚合物ASOs的强大的双关闭机制,特别是涉及拓扑约束,在很大程度上尚未被探索。本研究旨在设计和合成新的β-半乳糖苷酶响应环aso,通过抑制沃森-克里克碱基配对和拓扑约束来实现控制RNA裂解。通过优化的铜(I)催化叠氮化物-炔环加成(CuAAC)技术高效构建了环结构,为ps改性ASOs提供了高环收率和最小脱硫的条件。紫外熔化分析和RNase h介导的RNA切割实验证实,在环状ASOs中存在明显的双重关闭效应,这些环状ASOs具有三重交联或位于环中心的修饰的双重交联。在β-半乳糖苷酶处理后,半乳糖偶联的连接物被有效地去除,将双工稳定性和RNase h介导的RNA切割恢复到与天然ASOs相当的水平,显示出开关功能。在中性和酸性条件下都观察到这种活化。这一策略为开发下一代寡核苷酸疗法奠定了实用的化学基础,使基因沉默的精确时空控制成为可能。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Topological Switch-OFF and β-Galactosidase-Triggered Switch-ON of Cyclic Antisense Oligonucleotides via CuAAC for Controlled RNA Cleavage

Prodrug-type antisense oligonucleotides (ASOs) offer spatiotemporal control of gene silencing via external stimuli or intracellular enzyme activation. However, a robust dual switch-off mechanism for phosphorothioate (PS)-modified gapmer ASOs, particularly involving topological constraints, has been largely unexplored. This study aimed to design and synthesize novel β-galactosidase-responsive cyclic ASOs that achieve controlled RNA cleavage through both inhibited Watson–Crick base pairing and topological constraints. Cyclic structures were efficiently constructed via optimized copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC), tailored for PS-modified ASOs to ensure high cyclization yields and minimal desulfurization. UV-melting analyses and RNase H-mediated RNA cleavage assays confirmed a pronounced dual switch-off effect in the cyclic ASOs bearing triple cross-linking or double cross-linking with a modification positioned at the center of the loop. Upon β-galactosidase treatment, galactose-conjugated linkers were efficiently removed, restoring duplex stability and RNase H-mediated RNA cleavage to levels comparable to those of native ASOs, demonstrating switch-on functionality. This activation was observed under both neutral and acidic conditions. This strategy establishes a practical chemical foundation for developing next-generation oligonucleotide therapeutics, enabling precise spatiotemporal control of gene silencing.

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来源期刊
Bioconjugate Chemistry
Bioconjugate Chemistry 生物-化学综合
CiteScore
9.00
自引率
2.10%
发文量
236
审稿时长
1.4 months
期刊介绍: Bioconjugate Chemistry invites original contributions on all research at the interface between man-made and biological materials. The mission of the journal is to communicate to advances in fields including therapeutic delivery, imaging, bionanotechnology, and synthetic biology. Bioconjugate Chemistry is intended to provide a forum for presentation of research relevant to all aspects of bioconjugates, including the preparation, properties and applications of biomolecular conjugates.
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