Tuning siRNA Specificity through Seed Region Incorporation of Deoxyribonucleotide Stereoisomers

ABSTRACT Precise chemical design continues to drive advances in RNA-based therapeutics. Here, we report the synthesis and site-specific incorporation of four canonical phosphoramidites (U, C, A, and G), each bearing non-natural nucleoside configurations: β-D-2′-deoxyxylonucleosides and α-L-2′-deoxyribonucleosides. These stereochemically distinct nucleoside analogs were introduced at positions 6 and 7 within siRNA seed regions. When applied to siRNAs targeting Ttr , ACTN1 , and Marc1 , these modifications reduced off-target gene repression in functional assays and, in several cases, in transcriptome-wide differential expression analyses, while preserving robust on-target activity. In vivo , Marc1 -targeting siRNAs containing these modified nucleosides showed decreased hepatotoxicity, as evidenced by reduced serum ALT and AST levels. Collectively, these findings establish β-D-2′-deoxyxylonucleoside and α-L-2′-deoxyribonucleoside analogs as promising chemical tools for enhancing the specificity and safety of siRNA therapeutics. This work underscores the power of integrating rational nucleoside design with comprehensive functional and in vivo evaluation to advance drug development based on RNA interference (RNAi). GRAPHICAL ABSTRACT