Enzyme-Responsive Cyclic Gapmer Antisense Oligonucleotides via Thymidine <i>C</i> 5– <i>C</i> 5 Cross-Linking within the Gap Region Using a Galactosylated Branched Self-Immolative Linker

Enzyme-responsive prodrug-type antisense oligonucleotides (ASOs) are selectively activated only in target cells where specific enzymes are highly expressed, exerting therapeutic effects. Specifically, ASOs that are cyclized through enzyme-responsive linkers can impose topological constraints, inhibiting the formation of productive ASO/RNA duplexes. This mechanism facilitates spatiotemporal regulation of antisense activity. However, most reported cyclic ASOs are constructed by tethering the 5' and 3' termini, and examples of gapmer ASOs bearing topological constraints within the gap region, which is critical for ribonuclease H (RNase H) recognition, are limited. Here, we developed a β-galactosidase-responsive cyclic gapmer ASO by cyclizing two thymidine <i>C</i>5 positions within the gap region using a galactosylated branched self-immolative linker. Cyclization was achieved by reacting linear ASOs bearing <i>C</i>5-aminoalkyl-modified thymidines with an <i>N</i>-hydroxysuccinimide (NHS)-activated branched linker. Cyclic ASOs destabilized the ASO/RNA duplex and suppressed RNase H-mediated RNA cleavage compared to that of the corresponding linear ASOs. Moreover, β-galactosidase treatment removed these modifications, regenerating linear ASOs and restoring duplex stability and RNase H-mediated cleavage. This strategy should be extensible to various enzyme-responsive cyclic ASOs by replacing the galactose moiety with other responsive triggers, providing a general molecular design platform for prodrug-type ASOs based on topological constraints within the gap region.