{"title":"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。","authors":"Kento Miyaji*, Keita Takeuchi and Kohji Seio, ","doi":"10.1021/acs.bioconjchem.5c00295","DOIUrl":null,"url":null,"abstract":"<p >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.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":"36 8","pages":"1820–1837"},"PeriodicalIF":3.9000,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acs.bioconjchem.5c00295","citationCount":"0","resultStr":"{\"title\":\"Topological Switch-OFF and β-Galactosidase-Triggered Switch-ON of Cyclic Antisense Oligonucleotides via CuAAC for Controlled RNA Cleavage\",\"authors\":\"Kento Miyaji*, Keita Takeuchi and Kohji Seio, \",\"doi\":\"10.1021/acs.bioconjchem.5c00295\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >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.</p>\",\"PeriodicalId\":29,\"journal\":{\"name\":\"Bioconjugate Chemistry\",\"volume\":\"36 8\",\"pages\":\"1820–1837\"},\"PeriodicalIF\":3.9000,\"publicationDate\":\"2025-07-31\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://pubs.acs.org/doi/pdf/10.1021/acs.bioconjchem.5c00295\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bioconjugate Chemistry\",\"FirstCategoryId\":\"1\",\"ListUrlMain\":\"https://pubs.acs.org/doi/10.1021/acs.bioconjchem.5c00295\",\"RegionNum\":2,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bioconjugate Chemistry","FirstCategoryId":"1","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acs.bioconjchem.5c00295","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
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.
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.