Effects of Conformational Alteration Induced by d‑/l‑Isonucleoside Incorporation in siRNA on Their Stability in Serum and Silencing Activity

We report here that all of the d- or l-isonucleoside (isoNA) modified siRNAs investigated showed the characteristic A-form conformation in the circular dichroism (CD) spectra compared to native siRNA. The d-isoNA modification had less influence on the thermal stability of siRNAs, but all l-isoNA modification displayed a significant tendency to decrease the thermal stability of siRNA. It was also found that the stabilities of d-/l-isoNA modified siMek1 in serum were different and d-isoNA modification was more potent, i.e., increase of serum stability of siRNA, than l-isoNA modification. When d-isoNA incorporated at position 4 and position 5 at antisense strand of siMek1 showed obvious improvement on serum stability, however, l-isoNA incorporated at positions 11 and 12 at antisense strand and position 9 at sense strand made the siMek1 duplex formed very unstable in serum. The silencing activities of modified siMek1s with d-/l-isoNA at position 1 of antisense strand also dropped dramatically; however, the modification at 3′-terminal of the sense strand with d- or l-isoNA significantly enhanced the silencing activity targeting the antisense strand as reporter and minimized the passenger strand-specific off-target effect. IsoNA modified in the seed area of siMek1, siMek1 A04D and siMek1 A05L, showed similar activity to the native one and better target selectivity. In the case of modification at the position near the cleavage area, it was found that d- or l-isoNA modified sense strand at position 8, 9, or 15 of siMek1 could retain the silencing activities targeting the antisense strand as reporter. Especially, both siMek1 S15D and siMek1 S15L showed good silencing activity and high target selectivity compared to native siMek1. The effects of conformational alteration of such isoNA modification of siRNA on their stability in serum and silencing activity are discussed based on computer simulation. Systematic investigation of the relationship between modified siRNA conformation and their physical and biological properties should provide a useful guideline for chemical modification and optimization of siRNA for further clinical application.