ª 2003 Oxford University Press
`
`589–595
`Nucleic Acids Research, 2003, Vol. 31, No. 2
`DOI: 10.1093/nar/gkg147
`
`Tolerance for mutations and chemical modifications
`in a siRNA
`
`Mohammed Amarzguioui, Torgeir Holen, Eshrat Babaie and Hans Prydz*
`
`The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, N-0349, Oslo, Norway
`
`Received October 9, 2002; Revised and Accepted November 12, 2002
`
`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 phosphorothio-
`ates. We found that siRNA generally tolerated muta-
`tions in the 5¢ end, while the 3¢ end exhibited low
`tolerance. This observation may facilitate the design
`of siRNA for specific targeting of transcripts con-
`taining single nucleotide polymorphisms. We fur-
`ther demonstrate that
`in our system the single
`antisense strand of the wild-type siRNA is almost as
`effective as the siRNA duplex, while the correspond-
`ing 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.
`
`INTRODUCTION
`
`RNA interference (RNAi), the process of depleting RNA
`targets by the use of double-stranded RNA (dsRNA), was first
`reported in 1998 (1). Since the demonstration of the efficacy of
`short interfering RNA (siRNA) in mammalian cells (2–4), this
`new tool has been used to successfully target various
`infectious agents like HIV, poliovirus and RSV (5–9). In
`some cases, newly constructed vectors were used to produce
`short hairpin RNA (shRNA) that
`is processed to siRNA
`intracellularly (10–14).
`The mechanism of RNAi is not well understood (15–22).
`The triggering long dsRNA or aberrant RNA is assumed to be
`processed by the RNase III-like enzyme Dicer to approxi-
`mately 21–23 nt siRNA, which is then incorporated into the
`RNA-induced silencing complex (RISC). This step can be
`bypassed by transfection of chemically synthesised siRNA.
`
`the
`Dicer has also been implicated in processing of
`siRNA-related short
`temporal RNA let-7,
`shown in
`Caenorhabditis elegans to be involved in control of larval
`development, and widely conserved in other species, among
`them humans (23–25).
`is assumed to be
`The physiological function of RNAi
`defence against viral infections. This has been convincingly
`demonstrated in plants, where some viruses have evolved
`counter-measures against RNAi (26,27). An insect virus has
`recently been shown to both activate RNA silencing and
`express a suppressor protein in Drosophila cells (28). In
`C.elegans, mutations in RNAi genes have resulted in the
`activation of transposons (29,30), arguing for their involve-
`ment in the defence against these genomic parasites.
`The tolerance of the effect of siRNA for mutations is still
`unclear. Boutla et al. (31) reported that a mutated siRNA with
`a single centrally located mismatch relative to the mRNA
`target sequence retained substantial activity in Drosophila. In
`contrast, Elbashir et al. (32) found that a single mismatch was
`deleterious to activity in an in vitro Drosophila embryo lysate
`assay. In previous work (33) we tried to reconcile these
`conflicting results by depicting the RNAi process in vivo as a
`dynamic one where several
`factors influenced the final
`outcome, among them siRNA target position, siRNA concen-
`tration, mRNA concentration, mRNA synthesis rate and
`siRNA’s inherent cleavage activity, an activity that can be
`reduced gradually by mismatch mutations.
`In the present work we explore how various mutations and
`chemical modifications alter the efficacy and duration of our
`most effective siRNA (hTF167i) targeting the human Tissue
`Factor (hTF) mRNA. The objectives were, firstly, to find
`regions less tolerant in their siRNA effect for single mutations,
`thus possibly facilitating the design of siRNA for specific
`targeting of transcripts containing single nucleotide poly-
`morphisms. Secondly, we wished to improve the long-term
`activity of our siRNA by stabilising the RNA strands against
`nucleases through introducing phosphorothioates and modifi-
`cations of the 2¢-OH.
`We find that hTF167i has a general tolerance to mutations,
`with less tolerance for mutations at 3¢ end of the siRNA.
`Furthermore, with the exception of certain allyl-modifications,
`the backbone modifications did not reduce the activity of the
`siRNA to a significant degree. Extensive use of phosphoro-
`thioate modifications resulted in cytotoxicity. The 2¢-O-
`methylation modifications, on the other hand, showed promise
`
`*To whom correspondence should be addressed. Tel: +47 22840532; Fax: +47 22840501; Email: hans.prydz@biotek.uio.no
`
`The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
`
`Alnylam Exh. 1043
`
`

`

`590 Nucleic Acids Research, 2003, Vol. 31, No. 2
`
`as they resulted in increased persistence of activity with no
`toxicity to cells.
`
`MATERIALS AND METHODS
`
`SiRNA preparation
`
`RNAs (21 nt) were chemically synthesised using phosphor-
`amidites
`(Glen Research and PerSeptive Biosystems).
`Deprotected and desilylated synthetic oligoribonucleotides
`were purified by reverse-phase HPLC. Ribonucleotides were
`annealed at 10 mM in 200 ml 10 mM Tris–HCl pH 7.5 by
`boiling and gradual cooling in a water bath. Successful
`annealing was confirmed by non-denaturing (15%) poly-
`acrylamide gel electrophoresis.
`
`Cell culture and transfections
`
`The human keratinocyte cell line HaCaT was cultured in
`serum-free keratinocyte medium (Gibco BRL) supplemented
`with 2.5 ng/ml epidermal growth factor and 25 mg/ml bovine
`pituitary extract. The cell line was regularly passaged at
`sub-confluence and plated 1 or 2 days before transfection.
`HaCaT cells in 6-well plates were transfected at
`low
`confluency (<40%) with 1.0 ml 100 nM siRNA in serum-
`free medium, using Lipofectamine 2000. For complexation,
`10 mM stock solution of siRNA was diluted with 103 vol. of
`serum-free medium and mixed with an equal volume
`of medium-diluted Lipofectamine 2000, at a v/w ratio of
`liposome to siRNA of 5:2. Batch dilutions of liposomes were
`performed for each 6-well plate and allowed to pre-incubate at
`room temperature for 5–7 min before addition to the medium-
`diluted siRNA. Complexes were replaced with full medium 5 h
`after initiation of transfection. For standard assays of activity,
`cells were harvested the day after transfection. For longer
`incubations and time-course experiments, medium was
`replaced every second day after transfection.
`
`Northern analysis
`
`isolated using Dynabeads
`Polyadenylated mRNA was
`oligo(dT)25
`(Dynal).
`Isolated mRNA was
`fractionated
`by electrophoresis for 16–18 h on 1.3% agarose/formalde-
`hyde (0.8 M) gels and blotted on to nylon membranes
`(MagnaCharge, Micron Separations). Membranes were
`hybridised with random-primed Tissue Factor (TF) (position
`61–1217 in cDNA) and GAPDH (1.2 kb) cDNA probes in
`PerfectHyb hybridisation buffer (Sigma).
`
`RESULTS
`
`Mutational scanning of siRNA targeting hTF167
`
`We have previously demonstrated that one and two central
`mutations in siRNA targeting position 167 in hTF did not
`abolish its ability to deplete endogenous TF mRNA levels
`(33). We decided to map this siRNA more systematically in
`order to determine whether mutations were tolerated equally
`well within the whole siRNA. To simplify analysis, only GC
`pairs were mutated by inversion of the pairs. A total of eight
`different single-mutant siRNAs were designed and named
`according to the position (starting from the 5¢ of the sense
`strand) of the mutation (s1, s2, s3, s4, s7, s11, s13, s16) (Fig. 1).
`
`wt
`
`sl
`
`s2
`
`s3
`
`N
`
`s7
`
`s10
`
`s11
`
`s13
`
`s16
`
`5'-gcgcuucaggcacuacaaaua-3'
`3'-gccgcgaaguccgugauguuu-s•
`
`5'-ccgcuucaggcacuacaaaua-3'
`3'-gcggcgaaguccgugauguuu-5'
`
`5'-gggcuucaggcacuacaaaua-3'
`3'-gccccgaaguccgugauguuu-5'
`
`5'-gcccuucaggcacuacaaaua-3'
`3'-gccgggaaguccgugauguuu-5'
`
`5'-gcgguucaggcacuacaaaua-3'
`3'-gccgccaaguccgugauguuu-s•
`
`5'-gcgcuugaggcacuacaaaua-3'
`3'-gccgcgaacuccgugauguuu-s•
`
`5'-gcgcuucagccacuacaaaua-3'
`3'-gccgcgaagucggugauguuu-5'
`
`5'-gcgcuucagggacuacaaaua-3'
`3'-gccgcgaagucccugauguuu-5'
`
`5'-gcgcuucaggcaguacaaaua-3'
`3'-gccgcgaaguccgucauguuu-5'
`
`5'-gcgcuucaggcacuagaaaua-3'
`3'-gccgcgaaguccgugaucuuu-5'
`
`ds7/10
`
`5'-gcgcuugagccacuacaaaua-3'
`3'-gccgcgaacucggugauguuu-5'
`
`dsl0/11
`
`s•-gcgcuucagcgacuacaaaua-3'
`3'-gccgcgaagucgcugauguuu-5'
`
`dsl0/13
`
`5'-gcgcuucagccaguacaaaua-3'
`3'-gccgcgaagucggucauguuu-5'
`
`dsl0/16
`
`5'-gcgcuucagccacuagaaaua-3'
`3'-gccgcgaagucggugaucuuu-5'
`
`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.
`
`The previously described centrally located single-mutant M1
`(33), was included in this analysis and renamed s10.
`These mutant siRNAs were analysed for their ability to
`deplete endogenous TF mRNA in HaCaT cells (Fig. 2). The
`wild-type siRNA, and the mutant s10, included as positive
`controls, depleted TF mRNA to 10 and 20% residual levels, as
`reported previously (33). The activities of the other mutants
`could be categorised into three groups depending on their
`position along the siRNA. Mutations in the extreme 5¢ end of
`the siRNA were well tolerated, exhibiting essentially the same
`activity as the wild-type. Mutations localised towards the 3¢
`end, and up to the approximate midpoint of the siRNA (s4, s7,
`s10, s11), were slightly impaired in their activity, resulting in
`depletion of mRNA to 20–30% residual levels. Both of the
`mutations in the 3¢ half of the siRNA (s13, s16), however,
`exhibited severely impaired activity, suggesting a bias in the
`tolerance for mutations in the reaction(s) involving siRNA.
`The activities of four double mutants, in which the central
`position (s10) was mutated in conjunction with one additional
`position (s7, s11, s13, s16), were also analysed. The bias in
`mutation tolerance was also evident for these double mutants,
`as the rank order of their activity mirrored that of the activity
`
`

`

`Nucleic Acids Research, 2003, Vol. 31, No. 2
`
`591
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`<( z
`a::
`E
`:i::
`a
`Cl..
`<(
`t!)
`i:i::
`I-
`]i
`'a
`§
`z
`
`0
`,I''+-
`
`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.
`
`of the single mutants of the variant position. This observation
`strengthens the proposition that the differential activity of
`mutants is due to an intrinsic bias in the tolerance for target
`mismatches along the sequence of the siRNA.
`
`Chemical modification of siRNAs for increased
`persistence of silencing
`
`The effect of siRNA in human cells is transient and typically
`decreases during a few days in cell culture (18,33). The ability
`to extend the period of effective silencing would be of
`importance for the possible use of siRNA in therapy. To
`increase the intracellular stability of siRNA, we introduced a
`gradual increase of various chemical modifications to both
`ends of the siRNA. We have previously used phosphorothioate
`linkages and 2¢-O-modifications in the form of methylation
`and allylation to stabilise hammerhead ribozymes (34), and
`decided to try a similar strategy for siRNAs. In all modified
`siRNA species, the same modifications were introduced in
`both strands. It has been reported that siRNA with a general
`2¢-O-methylation in either strand have no activity (32). We
`therefore started with less extensive modification. Initially,
`siRNAs with one modification in each of the extreme 5¢ and 3¢
`ends of their strands (P1+1, M1+1, A1+1, Fig. 3) were
`synthesised. The 5¢ end of the siRNAs might be more sensitive
`to modification since it must be phosphorylated in order to be
`active in vivo (22). We therefore included siRNAs with
`modifications only in the non-basepairing 3¢ overhangs (P0+2,
`M0+2 and A0+2, Fig. 3), which were known to tolerate
`various types of modifications (2,3,32,33).
`Northern analysis of transfected HaCaT cells demonstrated
`essentially undiminished activity of all the chemically modi-
`fied siRNAs, with the exception of the siRNA with allylation
`at both ends (Fig. 3). Allyl-modification in the 3¢ end only had
`no effect on activity. The presence of a bulky substituent in the
`2¢-hydroxyl of the 5¢ terminal nucleotide might interfere with
`the in vivo phosphorylation of the siRNA necessary for its
`activity (22). Further modification of the siRNA by methyla-
`tion resulted in gradual attenuation of activity. One day after
`transfection with siRNAs carrying 4+4, 4+6, 6+6 or 6+8
`
`the cells exhibited residual
`methyl groups in their ends,
`reporter gene expression levels of 28, 42, 68 and 75%,
`respectively.
`We next wanted to know if any of these modifications
`increased the persistence of siRNA-mediated silencing. The
`level of endogenous TF mRNA recovers gradually 3–5 days
`after transfection with wild-type siRNA targeting hTF167
`(33). Transfecting HaCaT cells with unmodified and chemic-
`ally modified siRNAs in parallel did not result
`in any
`significant differences in their silencing activities at 3 and
`5 days post-transfection until a rather high degree of
`modification was used. Allylated siRNAs were not tested in
`this experiment, since they showed reduced effectivity even
`with only one substituent in the 5¢ end (Fig. 3). SiRNAs
`methylated in the 2¢-OH in up to 2+4 positions showed well
`conserved activity at 24 h [16–18% residual levels compared
`with 11% in cells transfected with the wild-type (Fig. 3)]. The
`most extensively phosphorothioated siRNA proved to be
`cytotoxic, resulting in ~70% cell death compared with mock-
`transfected cells (measured as the expression level of the
`control mRNA GAPDH). This siRNA species was therefore
`not included in the further analysis. The remaining siRNA
`species were evaluated for increased persistence of silencing
`by analysing TF mRNA expression 5 days after a single
`transfection of 100 nM siRNA. At this point, TF expression in
`cells transfected with wild-type siRNA had recovered almost
`completely (80% residual expression compared with mock-
`transfected cells) (data not shown). In cells transfected with
`the most extensively 2¢-O-methylated siRNA (M2+4), how-
`ever, strong silencing was still evident (32% residual expres-
`sion). The less extensively modified siRNA species (P2+2,
`M2+2), although less effective than M2+4, consistently
`resulted in lower TF expression 5 days post-transfection
`(55–60%) than the wild-type. Fewer modifications (P1+1,
`P0+2, M1+1, M0+2, A0+2) did not improve the silencing
`effect 5 days post-transfection. Time-course experiments
`demonstrated that the wild-type siRNA was still the most
`effective 3 days post-transfection, when silencing was
`relatively unimpaired. However, silencing drops off at a
`
`

`

`592 Nucleic Acids Research, 2003, Vol. 31, No. 2
`
`P l +l
`
`P0+2
`
`P2+2
`
`P2+4
`
`5' -g • c -gcuucaggcacuaca-a-a-u*a - 3'
`3' -g• c -c-g-cgaaguccgugaugu-u• u-5'
`
`S' -g-c -gcuucaggcacuaca-a - a •u• a-3'
`3'-g*c *c-g- cgaaguccgugaugu-u- u-5 '
`
`5 ' -g• c • gcuucaggcacuaca-a-a• u• a - 3'
`3'-g*c*c-g-cgaaguccgugaugu• u• u - 5 '
`
`s •-g • c • gcuucaggcacuaca• a • a • u• a - 3'
`3'-g*c *c *g*cgaaguccguga ugu• u • u-5'
`
`120
`
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`M0 + 2
`
`M2 +2
`
`M2 +4
`
`Al+l
`
`A0+2
`
`5 ' - Qcgcuuca ggcacuacaaauA- 3'
`3 ' - Gccgcgaaguccgugauguu0-5'
`
`5 ' - gcgcuucaggcacuacaaaUA-3 '
`3 ' -GCcgcgaaguccgugauguuu - 5'
`
`5'-QCgcuucaggcacuacaaaUA- 3'
`3 ' -QCcgcgaaguccgugaugutJU-5'
`
`5 '-GCgcuucaggcacuacaAAUA-3 '
`3' -GCCGcgaaguccgugauguUU-5 '
`
`5'-QcgcuucaggcacuacaaauA- 3'
`3 ' -~c~gcgaaguccgugauguu~-5 1
`
`5'-gcgcuucaggcacuacaaa~ -3 '
`3' - ~ cgcgaaguccguga uguuu - 5 '
`
`mock wt
`
`Pl+l P0+2 Ml +l M0+2 Al+l A0+2
`
`P2+2 P2+4 M2+2 M2+ 4
`
`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.
`
`p
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`<( z
`Ct'.
`E
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`Cl
`a.
`<(
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`al V)
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`§
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`· ·O · wt
`
`- %- P2+2
`
`-11112+2
`--+- 11112+4
`
`~- -··· ···
`
`l d
`
`3d
`
`Sd
`
`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.
`
`8 I z a z
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`TF
`
`GAPDH
`
`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).
`
`much higher rate thereafter for the unmodified siRNA (Fig. 4),
`possibly due to a faster depletion of the intracellular siRNA
`pool.
`
`Comparison of the effect of duplex and single-stranded
`antisense siRNA in whole cells
`
`The antisense strand of siRNA is incorporated into RISC in
`HeLa cell extracts and supports RISC-specific target RNA
`cleavage, although at lower efficiency than the siRNA duplex
`(35,36). The antisense RNA was shown to silence transgene
`expression in a dose-dependent manner (36). We investigated
`whether antisense siRNA could silence endogenous gene
`
`expression with an efficiency comparable to duplex siRNA at
`moderate concentrations of the antisense oligo. HaCaT cells
`were transfected in parallel with 100 nM siRNA or 200 nM of
`the antisense strand, with or without pre-phosphorylation of
`the oligo (Fig. 5). In our quantitative assay the antisense RNA
`was as efficient as the siRNA duplex in depleting endogenous
`TF mRNA. A clear cleavage product was seen for both agents.
`The observation that antisense RNA exhibits almost full
`activity most likely reflects the high efficiency and excess
`capacity of this particular siRNA species. For the slightly less
`efficient chemically modified siRNA M2+4, on the other hand,
`there is a clear difference in depletion rate between the duplex
`
`

`

`siRNA and antisense siRNA (Fig. 5), suggesting that higher
`concentrations are required for maximum effect of this
`antisense RNA. Phosphorylation prior to transfection did not
`improve the efficacy of either antisense strand, consistent with
`recent observations that single-stranded RNA can be phos-
`phorylated in vivo (36).
`
`DISCUSSION
`
`This work has demonstrated tolerance of a highly effective
`siRNA, hTF167i, for a wide range of mutational and chemical
`modifications, as well as for removal of the sense strand from
`the duplex. Although allyl-modification of both terminal
`nucleotides resulted in some loss of activity, a total of six
`modifications in the form of either methylation of the sugar
`moiety or thiolation of the backbone were well tolerated,
`causing only a marginal reduction in the maximal silencing
`observed with the unmodified siRNA. Toxicity was, however,
`observed with longer stretches of phosphorothioates, but not
`with the same level of methyl-modification. SiRNA with 2+4
`2¢-O-methylated terminal nucleotides (M2+4) demonstrated
`both conserved initial activity and its increased duration in
`time-course
`experiments. These
`active
`end-methylated
`siRNAs are in possible contrast
`to the observations by
`Elbashir et al. (32) that fully 2¢-O-methylated siRNAs are
`inactive. While full substitution of either strand with 2¢-
`deoxynucleotides destroys siRNA activity (31–33), up to four
`deoxynucleotide modifications in the 3¢ ends were found to
`support good activity in an in vitro cleavage assay (32). Taken
`together, the above data suggest the existence of different
`degrees of tolerance for chemical modification of siRNAs.
`We find that single-stranded antisense siRNA can be highly
`effective in depleting endogenous gene expression at rela-
`tively moderate concentrations. Although antisense siRNA is
`still less effective than the corresponding siRNA duplex, this
`observation should help reduce the cost of selecting the best
`siRNA site, as the initial screening can be accomplished
`through the synthesis of the antisense strand only.
`We have demonstrated a lower tolerance to mutations in the
`3¢ end of our most active siRNA. This bias cannot be due to
`differences in siRNA duplex stability as all mutations were
`G-C inversions and thus to a first approximation energetically
`equal. The stability of the duplex between a mutated siRNA
`antisense strand and the mRNA should be more severely
`affected by a central
`than a peripheral
`target mismatch.
`Consistent with this, we observe that 5¢ end mutations have a
`negligible effect on activity compared with more centrally
`located mutations. The observed bias might be linked to the
`proposed existence of a ‘ruler’ region in the siRNA which is
`primarily used by the RISC complex to ‘measure’ the target
`mRNA for cleavage (32). It was demonstrated that the 5¢ end
`of the antisense strand of siRNA sets the ruler for target RNA
`cleavage. This is likely to occur by a physical interaction of
`RISC with this region of the siRNA, which should therefore be
`more sensitive to mismatches with the target RNA. The
`universality of this observation is currently being investigated,
`using siRNAs targeting other sites in TF. The identification of
`a region of generally increased sensitivity to the effect of
`mismatches with the target mRNA, would be of great
`importance for the potential use of siRNAs to specifically
`
`Nucleic Acids Research, 2003, Vol. 31, No. 2
`
`593
`
`transcripts of disease-associated alleles in various
`target
`dominant-negative disorders.
`Highly diverging effects of mutations on siRNA activity
`have been reported. Jacque et al. (6) found that a single
`mismatch in siRNA targeting the HIV LTR resulted in partial
`loss of activity, while another siRNA targeting the HIV VIF
`exhibited almost
`full activity. Four mutations, however,
`abolished activity completely. Complete abolishment of
`activity has been reported by Gitlin et al. (8), Klahre et al.
`(37) and Garrus et al. (38), for siRNAs with 5, 6 and 7
`mutations, respectively. Kisielow et al. (39) reported that a
`siRNA resulting in essentially complete knockdown of the
`expression of its target gene, was unable to inhibit
`the
`expression of a transgene with two non-contiguous mutations
`in the recognition sequence of the siRNA. The positions of
`these mutations correspond to our ds10/13 double mutant,
`which also exhibited low activity in our assay. A central
`double mutation, reported by Boutla et al. (31) and ourselves
`(33) to support partial activity, led to severe loss of activity for
`Yu et al. (40) and Wilda et al. (41), the latter using a siRNA
`with only 17 base pairs. On the other hand, abolishment of
`in vivo activity by a single mutation has been reported.
`Brummelkamp et al. (10), using a shRNA assumed to be
`processed to siRNA by Dicer (13), achieved inactivation by
`single mutations in either the second or ninth position from the
`putative 5¢ end of the shRNA. Gitlin et al. (8), argued the case
`for single mutation inactivation more strongly by isolating
`siRNA-resistant poliovirus strains containing single mutations
`in the target site on the genomic RNA, corresponding to the
`sixth or ninth nucleotide of the siRNA, counted from the 5¢
`end of the sense strand. On balance, different siRNAs seem to
`be inactivated by mutations to different degrees, the outcome
`being at least in part target-sequence dependent.
`In previous work (33) we established that the siRNA effect
`depends at
`least
`in some instances on the siRNA target
`position, mirroring earlier observations for ribozymes and
`antisense oligos (34,42,43). Further support
`for siRNA
`position effects has now emerged from several different
`sources. Several examples of inactive siRNAs in mammalian
`cells have recently been described (11,44,45), while a weak
`positional effect in Drosophila lysates can be inferred from
`published data (32). In C.elegans, Simmer et al. (46) managed
`to activate dozens of previously inactive dsRNA stretches,
`using the RNAi-sensitive rrf-3 negative strain, and in some
`cases created RNAi knockout phenotypes from genes that had
`hitherto not been responsive to dsRNA. Finally, Yang et al.
`(47) recently reported some inactive chemically synthesised
`and in vitro transcribed siRNA, as well as inactive shRNA.
`The authors demonstrated that the new technique of esiRNA,
`in which an overlapping set of siRNAs are produced in vitro
`by partial digestion with Escherichia coli RNase III, can be
`superior even to dsRNA. Processing of dsRNA by Dicer starts
`from a fixed end and proceeds in a sequential manner
`(3,48,49), producing a largely non-overlapping set of siRNAs.
`Cleavage of a dsRNA by E.coli RNase III will thus create a
`larger and more complete set of siRNAs than the non-
`overlapping set produced by Dicer from dsRNA. The higher
`activity of the esiRNA supports the argument that different
`siRNAs have different activities. Otherwise any set of siRNA
`from a dsRNA would have the same activity.
`
`

`

`594 Nucleic Acids Research, 2003, Vol. 31, No. 2
`
`The inactivation of siRNA by mismatches has implications
`for the proposed function of RNAi as a defence system against
`retro-transposons and viruses. It is unclear why a viral defence
`mechanism should allow escape of a virus by a single
`mismatch. A differentiated population of siRNA with widely
`differing activities seems more likely. Some siRNAs with an
`intermediate activity can thus be more vulnerable to
`siRNA:target mismatch, while intrinsically stronger siRNAs
`have excess capacity and tolerate a single mismatch, as clearly
`exemplified by the tolerance exhibited to chemical and
`mutational modifications of hTF167i in the present study.
`This tolerance in highly active siRNAs should make viral
`escape more difficult, and our model is therefore consistent
`with both the published data and the proposed biological role
`of RNAi as a viral defence.
`
`ACKNOWLEDGEMENTS
`
`This work was supported by grants from the Norwegian
`Cancer Society, Health and Rehabilitation, and the Research
`Council of Norway (RCN) to H.P. T.H. is a fellow of RCN.
`
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