Example 2
Serum stability assay of GalNAc-siRNA conjugates containing two PS (STS12009L4), one PS (STS12009V21L4), two PS2 (STS12009V16L4) and one PS2 (STS12009V15L4) at terminal positions. GalNAc was conjugated to the 5′-end of the second strand and is internally stabilized by four PS. Phosphorothioates and phosphorodithioates, respectively were placed at the 5′-end of the first strand, 3′-end of the first strand and 3′-end of the second strand. 5 μM GalNAc-siRNA conjugates were incubated with 50% FBS for 3 d at 37° C. RNA was extracted and analysed on 20% TBE polyacrylamide gels and results are shown in FIG. 1. “UT” indicates untreated samples, “FBS” indicates FBS treatment. “Control” indicates a less stabilized GalNAc-siRNA conjugate of different sequence. The sequences are set out in Table 3.
TABLE 3
GalNAc-siRNA conjugates containing two PS, one PS, two PS2 and one PS2 at
all non-conjugated ends.
sequence and chemistry
duplex ID(top: first strand, bottom: second strand, both 5′-3′)
STS12009L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA
[ST23(ps)]3 ST41(ps)fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU
STS12009V21L4mA(ps)fAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG(ps)mA
[ST23(ps)]3 ST41(ps)fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU(ps)fU
STS12009V16L4mA(ps2)fA(ps2)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps2)fG(ps2)mA
[ST23(ps)]3 ST41(ps)fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps2)mU(ps2)fU
STS12009V15L4mA(ps2)fAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG(ps2)mA
[ST23(ps)]3 ST41(ps)fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU(ps2)fU
mA, mU, mC, mG-2′-OMe RNA
fA, fU, fC, fG - 2′-F RNA
(ps)- phosphorothioate
(ps2)- phosphorodithioate
