Topological Switch-OFF and β-Galactosidase-Triggered Switch-ON of Cyclic Antisense Oligonucleotides via CuAAC for...

Area of Science:

• Oligonucleotide chemistry
• Molecular biology
• Drug delivery

Background:

• Antisense oligonucleotides (ASOs) offer targeted gene silencing but lack precise spatiotemporal control.
• Phosphorothioate (PS)-modified gapmer ASOs require robust dual switch-off mechanisms for enhanced therapeutic applications.
• Topological constraints in ASOs for controlled gene silencing remain underexplored.

Purpose of the Study:

• To design and synthesize novel β-galactosidase-responsive cyclic ASOs.
• To achieve controlled RNA cleavage via inhibited Watson-Crick base pairing and topological constraints.
• To establish a dual switch-off mechanism for PS-modified gapmer ASOs.

Main Methods:

• Synthesis of cyclic ASOs using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).
• Optimization of CuAAC for PS-modified ASOs to maximize cyclization yields and minimize desulfurization.
• Evaluation of duplex stability using UV-melting analyses.
• Assessment of RNase H-mediated RNA cleavage assays.
• Demonstration of β-galactosidase-triggered linker removal and gene silencing switch-on functionality.

Main Results:

• Efficient synthesis of cyclic ASOs with high yields and minimal desulfurization.
• Demonstrated pronounced dual switch-off effect in cyclic ASOs with triple or double cross-linking.
• Successful restoration of duplex stability and RNA cleavage upon β-galactosidase treatment (switch-on).
• Consistent switch-on functionality observed under both neutral and acidic conditions.

Conclusions:

• Developed a novel strategy for β-galactosidase-responsive cyclic ASOs.
• Established a robust dual switch-off mechanism leveraging topological constraints and enzyme-cleavable linkers.
• This approach provides a practical chemical foundation for next-generation oligonucleotide therapeutics with precise spatiotemporal gene silencing control.