Existing antisense oligonucleotide therapeutics (ASO) — such as mipomersen — consist of numerous stereoisomers, which can limit efficacy. Iwamoto et al. now report a novel method termed SOSICS (stereocontrolled oligonucleotide synthesis with iterative capping and sulfurization) to produce ASOs with high stereochemical purity. SOSICS was applied to synthesize stereochemically pure components of mipomersen, which revealed that phosphorothioate (PS) stereochemistry affects the physicochemical and pharmacological properties of ASOs. A specific stereochemical code in the DNA core of the PS ASO that is recognized by RNase H1 and that enhanced activity in vitro and in mice was identified.