Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

Abstract

A series of nucleobase-modified siRNA duplexes containing ''rare'' nucleosides, 2-thiouridine (s 2 U), pseudouridine (C), and dihydrouridine (D), were evaluated for their thermodynamic stability and gene silencing activity. The duplexes with modified units at terminal positions exhibited similar stability as the nonmodified reference. Introduction of the s 2 U or C units into the central part of the antisense strand resulted in duplexes with higher melting temperatures (Tm). In contrary, D unit similarly like wobble base pair led to the less stable duplexes (DTm 3.9 and 6.6°C, respectively). Gene-silencing activity of siRNA duplexes directed toward enhanced green fluorescent protein or beta-site APP cleaving enzyme was tested in a dual fluorescence assay. The duplexes with s 2 U and C units at their 39-ends and with a D unit at their 59-ends (with respect to the guide strands) were the most potent gene expression inhibitors. Duplexes with s 2 U and C units at their 59-ends were by 50% less active than the nonmodified counterpart. Those containing a D unit or wobble base pair in the central domain had the lowest Tm, disturbed the A-type helical structure, and had more than three times lower activity than their nonmodified congener. Activity of siRNA containing the wobble base pair could be rescued by placing the thio-nucleoside at the position 39-adjacent to the mutation site. Thermally stable siRNA molecules containing several s 2 U units in the antisense strand were biologically as potent as their native counterparts. The present results provide a new chemical tool for modulation of siRNA gene-silencing activity.

Structure - Functions Relations in Small Interfering RNAs

“…The increase of the RNAi efficacy was also observed after stabilization of the 3'-end of the antisense strand of siRNA by s 2 U or ψ analogs and destabilization of the 5'-end by introduction of D at the 3'-end of the sense strand [120]. It should be noted that the decrease of the thermostability of the duplex at the 3'-end of the sense strand is more preferable in comparison with the stabilization of the duplex at the 5'-end of the sense strand if the duplex stabilization is not needed [99].…”

“…Dihydrouridine (D) contains non-aromatic ring that does not participate in staking interactions ( fig. 7) which resulted in the destabilization of siRNA [120], but one substitution with D at the 3'-end of the sense or the antisense strand does not change siRNA silencing activity. The replacement of U by Ψ and s 2 U at the 3'-end of the sense strand reduces the silencing activity, whereas introduction of the same modification at the 3'-end of the antisense strand creates favorable thermodynamic asymmetry and provides siRNAs with higher activities than the parent siRNA.…”

“…The replacement of U by Ψ and s 2 U at the 3'-end of the sense strand reduces the silencing activity, whereas introduction of the same modification at the 3'-end of the antisense strand creates favorable thermodynamic asymmetry and provides siRNAs with higher activities than the parent siRNA. Thus, these modifications could be applied for the design of thermoasymmetric siRNAs [120] (see section 3.3).…”

“…To increase the thermostability of siRNA by providing the optimal conformation of nucleotides for base pairing 65,99,120 .…”

“…To increase the thermoasymmetry of the duplex by the decrease of thermostability at the 3'-flank of the sense strand or/and the increase of the thermostability at the 5'-flank of the sense strand of the duplex 99,105,107,120 .…”