Table 2.

Chemical modifications of siRNA and their impact on stability, immunogenicity and in vivo behavior

Modification	Advantage	Note
Backbone modifications (phosphorothioate (PS) and phosphoester (PO) modifications)	Nuclease resistance, prolonged tissue retention.	PS modifications reduce binding affinity of oligo to its target and inhibit RNAi when in the center of the antisense strand.
Nucleobase modifications: 5‐ methylcytidine 5‐methyluridine	Increased melting temperature (T m ) by 0.5 °C persubstitution.
Abasic RNA	Decreased off target activity.
Ribose sugar modifications: 2′O‐methyl (2′O‐Me)	Increased nuclease resistance at 5–30% 2′O‐Me modification (in vitro and in vivo), improved plasma stability.	Two or more consecutive 2′O‐Me inhibit RNAi. Stabilizes 3′‐endo ribose conformation. 2′O‐Me A, G, and U reduce immune response.
2′Fluoro (2′F)	Barely reduced RNAi if 2′F in all positions. Increased nuclease resistance at >50% modification.	2′F A reduces immune response.
2′O‐methoxyethyl (2′O‐MOE)	Stabilized 3′‐endo ribose conformation. Increased nuclease resistance with 2′‐MOE in terminal positions.	Replacement of the 9th or 10th nucleotide from the 5′‐end with 2′‐MOE increases probability of entry into RISC.
Locked nucleic acids (LNA)	Reduced conformational flexibility of nucleotides fixing the C3′‐endo conformation of the ribose. Increased nuclease resistance in vitro with ≥10–20% LNA.	>20% LNA in the antisense chain or the first LNA nt at 5′ end completely inhibit RNAi LNA can change the thermal asymmetry of the duplex, increasing siRNA efficiency