Protocol for Controlling the Strand Selectivity of siRNA Using Acyclic Artificial Nucleic Acids

Abstract Small interfering RNA (siRNA) has emerged as a promising therapeutic candidate against previously intractable diseases. An effective siRNA must have high on‐target activity while off‐target effects are minimized. This balance can be achieved by enhancing the selectivity of the antisense strand through sequence optimization and appropriate chemical modifications. Acyclic artificial nucleic acids such as serinol nucleic acids (SNA) have demonstrated on‐target activity while suppressing off‐target effects. This article provides guidelines for designing SNA‐modified siRNA and outlines a method for the experimental evaluation of the on‐target and off‐target activities of siRNAs, ensuring accurate functional validation in cell systems. These protocols benefit researchers developing siRNA‐based therapeutics to optimize siRNA selectivity and efficacy while minimizing off‐target effects through innovative design strategies. © 2025 Wiley Periodicals LLC. Basic Protocol 1 : Design of SNA‐modified siRNA Basic Protocol 2 : Design and preparation of vector plasmids using inverse PCR Alternate Protocol : Design and preparation of vector plasmid using restriction enzymes and ligase Basic Protocol 3 : Evaluation of the on‐ and off‐target effects of siRNAs using the dual‐luciferase assay Support Protocol 1 : Agarose gel electrophoresis and protocol for purifying DNA from gels Support Protocol 2 : Transformation and amplification of plasmids