bc300642u_si_001.pdf (224.85 kB)
Effects of Conformational Alteration Induced by d‑/l‑Isonucleoside Incorporation in siRNA on Their Stability in Serum and Silencing Activity
journal contribution
posted on 2013-06-19, 00:00 authored by Ye Huang, Zhuo Chen, Yue Chen, Hao Zhang, Yichao Zhang, Yilei Zhao, Zhenjun Yang, Lihe ZhangWe
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.