ArticleMethods in molecular biology (Clifton, N.J.)2026
Structural and Physicochemical Optimization of Next-Generation Antisense Oligonucleotide with BROTHERS for Mitigating Off-Target Effects.
Article in Methods in molecular biology (Clifton, N.J.), 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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.
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