Structural and Physicochemical Optimization of Next-Generation Antisense Oligonucleotide with BROTHERS for Mitigating...

Antisense oligonucleotides (ASOs) are powerful and programmable therapeutic agents that silence gene expression via Watson-Crick base pairing. Despite advances in chemical modifications, ASO utility remains limited by off-target effects arising from both hybridization-dependent and -independent mechanisms. Here, we present BROTHERS (BRace On a THERapeutic aSo), a nanoarchitectural platform in which a single-stranded ASO is hybridized with a partially complementary peptide nucleic acid (PNA) strand. This duplex, termed BRO, reduces undesired protein binding by shielding ASO and dynamically discriminates target RNAs via toehold-mediated strand displacement (TMSD). PNA's unique properties-neutral backbone, high nuclease resistance, and low protein affinity-enable BRO to avoid off-target interactions while preserving on-target efficacy. This chapter outlines key steps in BRO design, including synthesis, purification, and evaluation strategies, providing a practical guide for applying this platform to improve the safety and selectivity of ASO therapeutics.