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.