Tolerance for mutations and chemical modifications in a siRNA

Abstract

Short interfering RNA (siRNA), the active agent of RNA interference, shows promise of becoming a valuable tool in both basic and clinical research. We explore the tolerance to mutations and chemical modifications in various parts of the two 21-nt strands of a siRNA targeting the blood clotting initiator Tissue Factor. The mutations were G/C transversions. The chemical modifications were 2'-O-methylation, 2'-O-allylation and phosphorothioates. We found that siRNA generally tolerated mutations in the 5' end, while the 3' end exhibited low tolerance. This observation may facilitate the design of siRNA for specific targeting of transcripts containing single nucleotide polymorphisms. We further demonstrate that in our system the single antisense strand of the wild-type siRNA is almost as effective as the siRNA duplex, while the corresponding methylated M2+4 version of the antisense had reduced activity. Most of the chemically modified versions tested had near-wild-type initial activity, while the long-term activity was increased for certain siRNA species. Our results may improve the design of siRNAs for in vivo experiments.

Figure 1

Mutated and wild-type (wt) versions of the siRNA hTF167i. The sequence of the sense strand of wild-type siRNA corresponds to position 167–187 in hTF (Acc.No. M16553). Single (s1, s2, s3, s4, s7, s10, s11, s13, s16) and double (ds7/10, ds10/11, ds10/13, ds10/16) mutants are all named according to the position of the mutation, counted from the 5′ end of the sense strand. All mutations (in bold) are GC inversions relative to the wild-type.

Figure 2

Activity of mutant siRNAs against endogenous hTF mRNA. HaCaT cells were harvested for mRNA isolation 24 h post-transfection. TF expression was normalised to that of GAPDH. Normalised expression in mock-transfected cells was set as 100%. Data are averages + s.d. of at least three independent experiments.

Figure 3

Activity of chemically modified versions of the siRNA hTF167i. Non-modified ribonucleotides are in lower case. Phosphorothioate linkages are indicated by asterisks (*), while 2′-O-methylated and 2′-O-allylated ribonucleotides are in normal and underlined bold upper case, respectively. Expression of TF and GAPDH mRNA was determined 24 h post-transfection of HaCaT cells. Experiments were performed and analysed as in Figure 2. Data are averages + s.d. of at least three independent experiments.

Figure 4

Persistence of TF silencing by chemically modified siRNAs in HaCaT cells harvested 1, 3 and 5 days after a single transfection of 100 nM siRNA. Medium was replaced every second day. Data are from one representative out of three independent experiments.

Figure 5

Levels of TF and control mRNA (GAPDH) in cells 24 h after transfection with various siRNA derivatives (see Figs 1 and 3). Cells were transfected with 100 nM siRNA duplex or 200 nM single-stranded RNA or DNA. RNA samples phosphorylated prior to transfection are indicated with ‘(+P)’. Phosphorylation was by polynucleotide kinase (New England Biolabs), followed by phenol/chloroform extraction and desalting on G25 Sephadex Quick-Spin columns (Roche).