High potency silencing by single-stranded boranophosphate siRNA
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ABSTRACT
In RNA interference (RNAi), double-stranded short interfering RNA (ds-siRNA) inhibits expression from complementary
mRNAs. Recently, it was demonstrated that short, single-stranded antisense RNA (ss-siRNA) can also induce RNAi. While ss-
siRNA may offer several advantages in both clinical and research applications, its overall poor activity compared with ds-
siRNA has prevented its widespread use. In contrast to the poor gene silencing activity of native ss-siRNA, we found that the
silencing activity of boranophosphate-modified ss-siRNA is comparable with that of unmodified ds-siRNA. Boranophosphate ss-
siRNA has excellent maximum silencing activity and is highly effective at low concentrations. The silencing activity of
boranophosphate ss-siRNA is also durable, with significant silencing up to 1 week after transfection. Thus, we have
demonstrated that boranophosphate-modified ss-siRNA can silence gene expression as well as native ds-siRNA, suggesting that
boranophosphate-modified ss-siRNAs should be investigated as a potential new class of therapeutic agents.
INTRODUCTION
RNA interference (RNAi) is a form of post-transcriptional gene silencing in which double-stranded RNA (dsRNA) targets
complementary mRNAs for destruction [reviewed in (1–4)]. RNAi occurs in a wide variety of organisms, ranging from protozoa
to mammals. There has been considerable interest in harnessing the power of RNAi to treat human diseases such as viral
infections (5,6), cancer (7,8) and sepsis (9). While questions remain about the precise mechanism of RNAi, recent work has
provided a clearer understanding of the process. In the endogenous RNAi system, the effector molecule is a double-stranded
short interfering RNA (ds-siRNA) 21 bp in length with 3' dinucleotide overhangs (10). ds-siRNAs associate with Argonaute
family proteins to form an RNA induced silencing complex (RISC) (11,12). One of the strands of the ds-siRNA is removed or
destroyed and only a single strand remains in the mature RISC (13–16). The complex then targets mRNA complementary to the
incorporated strand (13). Thus, it is the antisense strand of the ds-siRNA that targets a particular mRNA for destruction.
Several studies have shown that it is possible to bypass the dsRNA stage by introducing single-stranded antisense RNA 21
bases long into RNAi-competent cells or cell lysates (13,17,18). These single-stranded siRNA (ss-siRNA) molecules have
several advantages over the standard, ds-siRNA, particularly in the context of potential clinical applications. First, as
only one RNA strand is needed, the cost and work of preparation is reduced by half. Single-stranded molecules may also avoid
one of the major obstacles to therapeutic use of ds-siRNA: unintended side effects. For example, a recent study showed that
both chemically and enzymatically synthesized ds-siRNA species induced interferon expression, whereas none of the ss-siRNA
species provoked a detectable interferon response (19). Off-target silencing of genes with similar but non-identical
sequences to the sense or antisense strand is another potential obstacle to the specificity of siRNA activity (20). By using
a single strand, the risk of off-target silencing guided by the sense strand is eliminated. Finally, ss-siRNAs may circumvent
the RNAi-suppressing activity of dsRNA-binding proteins (21,22).
Despite these potential advantages of ss-siRNAs, the single-stranded molecule has a significant shortcoming; ss-siRNAs do
not efficiently induce RNAi. ss-siRNA must be used at concentrations that are 6- to 100-fold higher than that of ds-siRNA to
achieve comparable levels of gene silencing (13,17,18). The reason for the poor silencing activity of the single-stranded
molecules is not yet known.
To investigate whether the low level of silencing by ss-siRNAs could be improved, we sought to examine the activity of
ss-siRNAs with the boranophosphate (BP) backbone modification. We have shown previously that BP-modified ds-siRNAs are more
active for gene silencing than the corresponding phosphodiester- or phosphorothioate-backbone ds-siRNAs (23). In the
boranophosphate (BP) linkage (Figure 1A), a non-bridging phosphodiester oxygen is replaced with an isoelectronic borane ()
moiety. BP nucleic acids maintain the ability to base pair with high specificity and affinity and BP nucleotides can be
readily incorporated into DNA and RNA molecules by DNA and RNA polymerases, permitting synthesis of stereoregular
boranophosphate oligonucleotides (23–28). The boranophosphate modification appears to be well tolerated in mammalian
systems; BP ds-siRNAs do not induce any appreciable toxicity in HeLa cells (23). Furthermore, BP DNA dinucleotides are
minimally toxic to rodents, and the degradation product of BP oligonucleotides (borate) is minimally toxic to humans
(23,29,30).
In this work, we demonstrate that partial BP-modification increases the silencing activity of ss-siRNA against green
fluorescent protein in HeLa cells. In our system single-stranded BP-siRNAs can silence gene expression as well as native ds-
siRNA.
MATERIALS AND METHODS
Synthesis of boranophosphate siRNAs
ss- and dsRNA molecules were synthesized as described previously (23). Briefly, DNA oligonucleotides were purchased from
Qiagen: T7 promoter primer, 5'-TAATACGACTCACTATAG-3'; EGFP1 templates: sense, 5'-AAGTTCACCTTGATGCCGTTCTATAGTGAGTCGTATTA-3'
and antisense, 5'-AAGAACGGCATCAAGGTGAACTATAGTGAGTCGTATTA-3'; control EGPF1 templates: sense, 5'-
AAGTTCACCGTAGTTCCGTTCTATAGTGAGTCGTATTA-3' and antisense, 5'-AAGAACGGAACTACGGTGAACTATAGTGAGTCGTATTA-3'; EGFP2 templates:
sense, 5'-AAGGACTTGAAGAAGTCGTGCTATAGTGAGTCGTATTA-3' and antisense, 5'-AAGCACGACTTCTTCAAGTCCTATAGTGAGTCGTATTA-3'; Control
EGFP2 templates: sense, 5'-AAGGACTTTGAAGAAGCGTGCTATAGTGAGTCGTATTA-3' and antisense, 5'-
AAGCACGCTTCTTCAAAGTCCTATAGTGAGTCGTATTA-3'. ssRNA molecules were synthesized using T7 RNA polymerase. To create
boranophosphate modified oligonucleotides, one or more ribonucleoside 5'-(-P-borano)triphosphates were added to the
transcription reaction in place of the corresponding rNTPs. RNAs were then purified as single strands using phenol extraction
and Microspin G-50 Micro Columns (Amersham) or PAGE and suspended in water. To make dsRNA, sense and antisense strands were
first annealed and then purified and concentrated using Microcon YM-10 centrifugal filters (Millipore). RNA concentration was
determined by UV absorbance at 260 nm.
Cell culture and transfection
EGFP-TRex HeLa cells, which express enhanced green fluorescent protein (EGFP) under control of a tetracycline responsive
promoter, were cultured as described previously (23). Twenty-four hours after plating, cells were transfected with siRNA
using Oligofectamine (Invitrogen) according to the manufacturer's instructions with 1.35 μl Oligofectamine per well of a 24
-well plate. The concentration of siRNA in the cell culture medium ranged from 3.1 to 50 nM, as described in figure legends.
Twenty-four hours after siRNA transfection, cells were induced to express EGFP with 1 μg/ml tetracycline. For the EGFP1
control sequence, the six central nucleotides of the active EGFP1 sequence were inverted. As the control sequence for EGFP2,
we used an siRNA in which the eight central nucleotides inverted because the sequence with six inverted nucleotides had an
unacceptable degree of homology to some human mRNAs.
Flow cytometry analysis
Cells were trypsinized and fixed in 1% formaldehyde in phosphate-buffered saline, 48 h post-transfection. Cells were
analyzed for EGFP expression using a FACScan (Becton-Dickinson) flow cytometer. Data were processed using CellQuest software
(Becton-Dickinson).
Cellular RNA analysis
Cells were transfected in culture medium at a final siRNA concentration of 12.5 nM. EGFP1 production was induced with 1
μg/ml tetracycline. Cells were trypsinized and total cytoplasmic RNA isolated using the RNeasy kit (Qiagen), 22 h after EGFP
induction. RNA from each sample (2 μg) was separated by formaldehyde–agarose gel electrophoresis, transferred to a
positively charged nylon membrane and crosslinked by UV irradiation. The immobilized RNA was hybridized with 32P-labeled RNA
probes complementary to the EGFP coding sequence and to human ?-actin (to normalize for signal). Hybridization was quantified
using a Molecular Dynamics phosphorimaging system.
Quantitative RT–PCR
Cells were transfected in culture medium at a final siRNA concentration of 25 nM or mock transfected with Oligofectamine
(Invitrogen) alone. Cells were trypsinized and total cytoplasmic RNA isolated using the RNeasy kit (Qiagen), 24 h after
transfection. RNA from each sample (1 μg) was used to make cDNA using the iScript kit (Bio-Rad). Quantitative PCR was
performed using SYBR Green I (Invitrogen), Platinum Taq (Invitrogen), 1.5 mM MgCl2 and primers at a concentration of 0.2 μM.
PCR was performed on a Roche Lightcycler and data were analyzed using Lightcycler software. The following primers were used:
human Ago2, 5'-TGGCTGTGCCTTGTAAAACGCT-3' and 5'-CGCGTCCGAAGGCTGCTCTA-3'; human GAPDH, 5'-CATGTTCGTCATGGGTGTGAACCA-3' and 5'-
AGTGATGGCATGGACTGTGGTCAT-3'.
RNase susceptibility analysis
EGFP1 and EGFP2 RNAs (10 pmol) were incubated with 0.5 and 2 ng RNase A (Roche), respectively, in 5 μl RNase reaction
buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA and 150 mM NaCl) at 37°C for specified lengths of time. The RNase reactions were
stopped by adding 2 vol of the gel-loading buffer (10 M urea, 50 mM EDTA, 0.1 bromophenol blue and 0.1% xylene cyanol), and
loaded on a 4% agarose gel. After SYBR Gold (Molecular Probes) staining, the gels were photographed under UV illumination and
analyzed using Fluorescent Image Analytical Software (Hitachi Software Engineering Co. Ltd).
RESULTS
ds-siRNAs and several species of ss-siRNAs, all targeting EGFP, were synthesized as described previously (23) (Figure 1).
Briefly, all RNA species were synthesized using T7 RNA polymerase and purified as single strands. To create BP-modified
siRNAs, one or more native ribonucleoside triphosphate was replaced by its BP counterpart in the transcription reaction.
Silencing activity of native and BP ss-siRNA
Native and BP ss-siRNAs targeted to EGFP were tested for silencing activity in HeLa cells that express EGFP under control
of a tetracycline-inducible promoter. For these studies, cells were transfected with ds- or ss-siRNA, induced to express
EGFP, and then, after 24 h, analyzed by flow cytometry to measure the level of EGFP protein expression. The fluorescence of
cells transfected with siRNA was compared with the fluorescence of mock-transfected cells (treated with Oligofectamine alone)
to determine the percent inhibition of EGFP expression. To control for non-specific effects of the RNA, we tested control
sequence ss-siRNAs with the central 6 or 8 nt inverted (Figure 1B). None of the control ss-siRNAs silenced EGFP expression
(Figure 2).
Initially, EGFP1 sequence ss-siRNAs were tested at a concentration of 25 nM because this is the concentration at which
silencing by native ds-siRNA approaches a maximum, thereby allowing comparison of the peak silencing activity of the
different siRNAs. Similar to earlier studies (13,17,18), we found that native ss-siRNA could inhibit EGFP expression, but was
significantly less active than native ds-siRNA (Figure 2A). When BP-modified siRNAs were studied, we found that ss-siRNA with
BP-modified cytidine residues () silenced EGFP expression with the same efficiency as the unmodified ds-siRNA. Furthermore,
an anti-EGFP ss-siRNA with modified adenosine and cytidine residues was significantly more active than native ds-siRNA (99%
versus 70% reduction in EGFP expression, respectively).
While BP modification of cytidine and adenosine residues significantly enhanced silencing activity of ss-siRNAs, ss-
siRNAs that included modified uridines ( and ) were notably less active than unmodified ss-siRNA. Therefore, we examined why
these species had reduced EGFP silencing activity. We have demonstrated previously in ds-siRNAs that a high proportion of BP
residues in the center of the antisense strand reduces siRNA silencing activity (23). As there are four uridine residues
among the central six nucleotides (those that surround the predicted target cleavage site, Figure 1B) in the EGFP1 sequence,
the reduced silencing activities of and were consistent with the pattern established previously with ds-siRNAs, and suggest
that modification of the central bases of ss-siRNAs reduces silencing activity in a manner analogous to the reduction seen
with modification of the central bases of double-stranded BP-siRNAs.
While we suspected that extensive modification of central nucleotides caused the reduction in siRNA silencing, the loss
of activity in ss-siRNAs with modified uridines could also have been due to modification of the two residues on the 3' end,
or simply due to the presence of modified uridines. To further examine the relationship between modification sites and ss-
siRNA activity, a different anti-EGFP sequence (EGFP2) was tested. This sequence has two uridines at the 3' end; however,
none of the six central nucleotides are uridines. Instead, there are four adenosines in this region (Figure 1B). Once again,
the native ss-siRNA had a much lower level of silencing activity than native ds-siRNA (Figure 2B). However, BP-modification
of the uridine residues (all peripheral) caused a dramatic increase in ss-siRNA activity, almost restoring it to the level of
ds-siRNA. In contrast, when adenosine residues were modified in the EGFP2 molecule (resulting in modification of 3 of the
central 6 nt), activity remained at a low level. These results demonstrate that BP-modified uridine residues and 3' overhangs
are compatible with ss-siRNA activity and provide additional support for the hypothesis that multiple central BP
modifications interfere with efficient silencing.
The overall pattern of increased activity with peripheral modification and reduced activity with central modification was
consistent for both anti-EGFP sequences. However, in contrast to the results for EGFP1 ss-siRNAs, increasing the degree of
peripheral BP modification in EGFP2 ss-siRNAs caused a slight decrease in silencing activity (Figure 2B, and ).
Dose response of native and BP ss-siRNA
We determined silencing dose response curves for several anti-EGFP1 siRNAs (Figure 3). As we had observed when a single
siRNA concentration was used, the double-stranded native AnSn siRNA exhibited moderate silencing activity. AnSn induced an
80% reduction in EGFP fluorescence at 50 nM siRNA and had half maximal silencing activity in the range of 2 nM. In contrast,
An silences EGFP expression only modestly, inducing only 60% silencing at 50 mM with half maximal silencing at 15 nM. When BP
cytidine residues are introduced to the EGFP1 ss-siRNA, its activity improves such that the dose response curve of is almost
identical to that of AnSn. When modified cytidine and adenosine residues are used, silencing activity of the ss-siRNA becomes
highly active; induces close to 100% EGFP silencing at 25 nM, with half-maximal silencing induced at 1 nM siRNA.
Kinetics of silencing by native and BP ss-siRNA
Next, we examined the effect of BP modification on ss-siRNA silencing over time. HeLa cells were transfected with siRNA,
induced to express EGFP after 5 h or after 1, 3, 5 or 7 days and fixed 24 h after induction. To assess the duration of
silencing relative to each interfering RNA's activity, the level of silencing after 1 day was normalized to 100. While the
activity of all RNA species starts to decline after 3 days, the activity of native ss-siRNA (An) declines much more quickly
than that of native ds-siRNA (AnSn) or BP ss-siRNAs (, ) (Figure 4). It should be noted that the raw data reveal that,
consistent with our other results, native ss-siRNA was significantly less active than native ds-siRNA and that modified ss-
siRNAs were as active () or more active () than native ds-siRNA at all times tested (data not shown). Notably, the raw data
also reveal that still reduced EGFP expression by 50% 1 week after transfection. Finally, while other investigators have
reported that ss-siRNA achieves maximum silencing more quickly than ds-siRNA (18), we did not observe such a pattern.
BP ss-siRNA silences by reducing target mRNA levels
siRNAs inhibit protein expression primarily by inducing cleavage of target mRNA. However, it has also been shown that
some RNA species, known as miRNAs, can silence genes by inhibiting translation of their targets (31). Previous work suggests
that the presence of a base mismatch or a bulge when the siRNA pairs with its target can cause it to function through the
miRNA pathway rather than by the RNAi mechanism (32). Because miRNAs have been shown to function as single strands (33), we
investigated whether BP ss-siRNAs might act by inhibiting translation of target mRNA. The effects of BP ss-siRNA and native
ds-siRNA on EGFP mRNA levels were examined by northern blot and were normalized to ?-actin expression. Similar to ds-siRNA,
BP-modified ss-siRNA caused a reduction in the level of EGFP mRNA (Figure 5A). To compare the relative change in protein and
mRNA levels, samples of the cells used for northern analysis were analyzed by flow cytometry. If the BP ss-siRNA inhibited
translation in addition to reducing mRNA levels, it should cause a relatively greater protein inhibition than mRNA
inhibition, compared with unmodified ds-siRNA. Instead, for both native ds-siRNA and BP ss-siRNA, the percent inhibition of
protein expression closely matches the percent inhibition of mRNA expression (Figure 5B). Furthermore, the ratios of the
reductions in protein levels to the reductions in mRNA levels are the same for BP ss-siRNA and native ds-siRNA. We therefore
conclude that modified ss-siRNAs inhibit protein expression primarily by reducing EGFP mRNA levels, rather than by inhibiting
translation.
BP ss-siRNA activity requires Argonaute2 expression
Having determined that BP ss-siRNA silence protein expression by mRNA cleavage, we further examined the process of
silencing by these molecules. Previous work has suggested that native ss-siRNA act through the RISC to induce RNAi
(13,17,18). However, in all of these studies, the concentrations of ss-siRNA required to achieve gene silencing comparable
with that induced by ds-siRNA were 6- to 100-fold higher. Therefore, it is important to determine whether the highly
efficient silencing observed with BP ss-siRNA is due to RISC-related activity or to engagement of a different gene silencing
mechanism, such as induction of degradation by a double-stranded RNase (34). To determine the RISC-dependence of BP ssRNA
silencing, we examined the effect of siRNAs that have been shown to knock-down expression of Argonaute2 (Ago2), a key
component of the RISC (35). For these studies, EGFP-HeLa cells were transfected with Ago2 siRNA or with control, non-
silencing siRNA. The cells were transfected with native ds-siRNA (which is known to interact with the RISC) or BP ss-siRNA
targeted to EGFP, 24 h later. Cells were induced to express EGFP 24 h after the second transfection and analyzed by flow
cytometry 24 h after induction. It has been demonstrated previously that when RISC is disrupted by knocking-down Ago2
expression, silencing by ds-siRNA is eliminated (35). If BP ss-siRNA acts through a RISC-dependent process, its silencing
activity should be also dramatically reduced when Ago2 is silenced.
The siRNA targeted to Ago2 caused a notable reduction in Ago2 expression as measured by quantitative RT–PCR (Figure 6A). We
found that when Ago2 expression was suppressed, the ability of native ds-siRNA to silence EGFP expression was reduced
dramatically, compared with cells initially transfected with control siRNA (Figure 6B). The silencing activity of BP ss-siRNA
was abrogated to a similar extent by Ago2 silencing, indicating that BP ss-siRNA acts primarily through a RISC-dependent
process (Figure 6B). To ensure that inhibiting Ago2 expression does not, by itself, affect EGFP expression, we also examined
cells transfected with Ago2 siRNA, followed by control siRNA. The Ago2 siRNA did not appreciably affect EGFP expression; mean
fluorescence for mock transfected and Ago2 transfected cells were 537.1 and 536.1, respectively (in arbitrary units). The
combination of the northern blot and the Ago2 knock-down data suggests that BP ss-siRNA acts primarily through the standard
RNAi pathway used by ds-siRNA.
BP-modification has variable effects on ss-siRNA nuclease resistance
Having demonstrated that the BP modification dramatically improves the silencing activity of ss-siRNAs, we sought to examine
the reason for this change in activity. Given the susceptibility of ssRNA to RNases and the enhanced nuclease stability of
BP-modified dsRNA (23), we initially speculated that the enhanced silencing activity of BP ss-siRNAs was at least partially
due to their greater resistance to RNase degradation. To test this hypothesis, we examined the susceptibility of native and
BP ss-siRNAs to degradation by RNase A. As expected, unmodified ss-siRNA was very susceptible to nuclease digestion while ds
-siRNA was relatively resistant to degradation (Figure 7). Boranophosphate modification did enhance nuclease resistance of
some ss-siRNA species. However, BP modification did not result in a consistent improvement in nuclease stability, nor did
resistance to RNase degradation correlate with greater silencing activity. Notably, the most active siRNA we tested, the
EGFP1 ss-siRNA with modified adenosine and cytosine residues, was degraded at essentially the same rate as the native ss-
siRNA. This result suggests that nuclease resistance may not be the sole reason for the enhanced silencing activity of BP ss
-siRNAs.
DISCUSSION
ssRNA can efficiently induce RNAi
While testing the silencing activity of native and boranophosphate modified ss- and ds-siRNAs, we discovered that BP ss-
siRNA can be a highly effective gene silencing tool. As seen in several previous studies (13,17,18), we found that the gene
silencing activity of unmodified ss-siRNA was substantially less than that of ds-siRNA. In contrast, we found that the most
effective BP-modified ss-siRNA species (EGFP1 , with modified adenosine and cytidine residues) is even more active than
native ds-siRNA, implying that ss-siRNA can be efficiently incorporated into the RISC (Figures 2 and 3). The maximum activity
of is also greater than that of any BP-modified ds-siRNA that we have examined (data not shown) (23). To our knowledge, this
is the first demonstration that the silencing activity of ssRNA can match or exceed that of dsRNA. Also, given our findings
that BP ss-siRNA silencing is due to a reduction in mRNA levels and is Argonaute2-dependent, it seems likely that BP ss-siRNA
acts through the standard RNAi pathway.
Structure-activity relationships in BP ss-siRNA
We found that position effects of BP modifications followed the same general rule we discerned when testing BP-modified
dsRNA (23): peripheral BP modification is well tolerated, while central modifications cause a significant loss of activity.
However, the magnitude of the benefit of peripheral BP modification was greater when applied to single-stranded molecules. At
25 nM, peripheral modification of ss-siRNA caused up to a 2.4-fold increase in silencing activity (Figure 2, ), compared with
a maximum increase in ds-siRNA activity of 1.3-fold (23).
Because peripheral modifications dramatically increase ss-siRNA silencing activity, we initially hypothesized that the
positive attributes conferred by central modifications would offset their inhibitory effects, such that even central
modifications would enhance activity. Instead, ss-siRNA species with central modifications have activity levels that are as
low as or lower than those of native ss-siRNA (Figure 2). Apparently, the unfavorable effects of central modification are
still significant in single-stranded molecules. The poor activity of centrally modified ds-siRNA and ss-siRNA may reflect a
disruption of the enzymatic activity of the RISC, which cleaves the target directly across from the center of the interfering
RNA (36). Previous studies have demonstrated that central sequence mismatches or ribose modifications inhibit ds-siRNA
activity (36,37), although the regions that are sensitive to such changes appears to be more extensive than the area that is
sensitive to BP modification.
Mechanisms of enhanced activity in boranophosphate ss-siRNA
We initially hypothesized that BP-modified ss-siRNAs would be more nuclease resistant that native ss-siRNA and that this
would be a major reason for their enhanced silencing activity. However, we found that BP modification did not consistently
result in greater resistance to RNase A digestion for the sequences tested here. Indeed, the BP ss-siRNA species with the
greatest silencing activity (EGFP1 ) was degraded at essentially the same rate as native ss-siRNA (Figure 7). It is still
possible that BP modification could confer greater stability within the cell or in the culture medium; RNase A resistance may
not directly correlate with resistance to other nucleases. However, the absence of an observable increase in RNAse stability
with BP modification suggests that the enhanced activity of BP ss-siRNAs may not be solely due to improved nuclease
resistance. This hypothesis is supported by other work showing that modifications that confer nuclease resistance do not
necessarily enhance the activity of ds- or ss-siRNAs (37,38).
Instead, we suspect that BP ss-siRNAs have other advantageous properties that confer efficient silencing activity. Borane
substitution of a non-bridging oxygen results in changes in size, charge, polarity and stereochemistry (39). It can be
imagined that the presence of a borane group instead of oxygen in the phosphate backbone of RNA would change their
interaction with other molecules, e.g. mRNA target and RISC protein components. Indeed, we have found recently that BP
modification raises the melting temperature of siRNAs, suggesting that a modified siRNA might have a greater affinity for the
target mRNA than a native siRNA (40). Another characteristic of BP siRNA that may enhance its silencing activity is its
tendency to make an A-form helix (40). The results of several different studies suggest that efficient cleavage of the target
RNA requires the A-form structure between the guide strand of the ds-siRNA and the target (37,41,42). Investigation of the
interactions between BP ss-siRNA and RISC should facilitate mechanistic understanding of RNAi and may suggest generally
applicable strategies for enhancing siRNA activity.
Therapeutic potential of boranophosphate ss-siRNAs
Our examination of silencing by boranophosphate-modified ss-siRNA suggests that BP ss-siRNA could have significant
therapeutic potential. The high maximum activity, potency at low concentrations and duration of silencing by EGFP1 suggest
that relatively low and infrequent dosing of BP ss-siRNA could be effective, even in rapidly dividing cells. Indeed, by
accounting for dilution by cell division and comparing the activity over time to dose response curves, it appears that
essentially all of the decline in activity can be accounted for by this dilution. Therefore, we expect that silencing would
last even longer in non-dividing cells or when the siRNA blocks cell growth (7).
In addition to its excellent silencing activity, BP ss-siRNA has advantages unique to ssRNA: simplified synthesis and no
risk of off-target silencing by the sense strand. Furthermore, the smaller size and reduced negative charge of ss-siRNA may
facilitate delivery into cells. We also believe that ssRNA is less likely to induce an interferon response than dsRNA. Most
examinations of interferon induction by siRNAs have shown that ss-siRNAs do not provoke an interferon response (19). However,
a recent publication reported that ssRNA synthesized by phage polymerases (the method used here) can induce interferon
expression due to the presence of 5' triphosphate groups (43). In the context of potential clinical applications, this should
not pose a significant problem because the terminal phosphates can be easily removed by calf intestinal alkaline phosphatase
treatment (43). Because control sequence siRNAs (also synthesized by T7 polymerase) did not show any silencing activity
(Figure 2), we do not believe that our analysis of silencing activity was affected by the presence of 5' triphosphate groups
on the siRNAs we tested.
The use of single-stranded molecules may be particularly desirable in specific situations. For example, BP ss-siRNAs may
permit the use of sequences whose sense strand sequence would be nearly complementary to other (off-target) human genes. In
addition, the ability to use single-stranded molecules should enhance silencing by sequences that would otherwise have poor
activity due to incorporation of the inactive sense strand into the RISC [it should be noted that other strategies to favor
incorporation of the active strand have also been developed (44)]. This relaxation of sequence requirements is likely to be
especially important when there are relatively few potential sequences, such as when the target is a particular splice
variant or mRNA with a specific point mutation. ss-siRNAs are also likely to be useful in treating viral infections because
many human viruses express dsRNA-binding proteins, presumably to evade the host cell interferon response (21,22).
CONCLUSION
We have shown that boranophosphate modified ss-siRNA efficiently induces silencing at high and low concentrations and for
extended periods of time. This finding is significant, because it demonstrates that, when appropriately modified, ss-siRNA
can induce RNAi at least as effectively as ds-siRNA. It also introduces a new category of molecules for potential use as
RNAi-based therapeutics.
CKNOWLEDGEMENTS
We would like to thank Danuta Gasinski and the Duke University Cancer Center Cell Culture and Flow Cytometry facilities
for excellent technical assistance. This work was supported by a grant from The Children's Miracle Network (K.A.A.), NIH R01
CA081214 (K.A.A.), NIH R21 CA104604-01 (K.A.A. and B.R.S.), Department of Defense Grant DAMD 17-02-1-0376, sponsored by the
Department of the Army (B.R.S.), NIH R01 GM57693 (B.R.S.) and the Duke University Medical Scientist Training Program
(A.H.S.H). The content of the information does not necessarily reflect the position or the policy of the Government, and no
official endorsement should be inferred. Funding to pay the Open Access publication charges for this article was provided by
the Section of Pediatric Infectious Diseases, the University of Chicago.
Conflict of interest statement. None declared.
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et al. (2005) Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality Nucleic Acids Res, . 33,
439–447(Allison H. S. Hall1, Jing)
In RNA interference (RNAi), double-stranded short interfering RNA (ds-siRNA) inhibits expression from complementary
mRNAs. Recently, it was demonstrated that short, single-stranded antisense RNA (ss-siRNA) can also induce RNAi. While ss-
siRNA may offer several advantages in both clinical and research applications, its overall poor activity compared with ds-
siRNA has prevented its widespread use. In contrast to the poor gene silencing activity of native ss-siRNA, we found that the
silencing activity of boranophosphate-modified ss-siRNA is comparable with that of unmodified ds-siRNA. Boranophosphate ss-
siRNA has excellent maximum silencing activity and is highly effective at low concentrations. The silencing activity of
boranophosphate ss-siRNA is also durable, with significant silencing up to 1 week after transfection. Thus, we have
demonstrated that boranophosphate-modified ss-siRNA can silence gene expression as well as native ds-siRNA, suggesting that
boranophosphate-modified ss-siRNAs should be investigated as a potential new class of therapeutic agents.
INTRODUCTION
RNA interference (RNAi) is a form of post-transcriptional gene silencing in which double-stranded RNA (dsRNA) targets
complementary mRNAs for destruction [reviewed in (1–4)]. RNAi occurs in a wide variety of organisms, ranging from protozoa
to mammals. There has been considerable interest in harnessing the power of RNAi to treat human diseases such as viral
infections (5,6), cancer (7,8) and sepsis (9). While questions remain about the precise mechanism of RNAi, recent work has
provided a clearer understanding of the process. In the endogenous RNAi system, the effector molecule is a double-stranded
short interfering RNA (ds-siRNA) 21 bp in length with 3' dinucleotide overhangs (10). ds-siRNAs associate with Argonaute
family proteins to form an RNA induced silencing complex (RISC) (11,12). One of the strands of the ds-siRNA is removed or
destroyed and only a single strand remains in the mature RISC (13–16). The complex then targets mRNA complementary to the
incorporated strand (13). Thus, it is the antisense strand of the ds-siRNA that targets a particular mRNA for destruction.
Several studies have shown that it is possible to bypass the dsRNA stage by introducing single-stranded antisense RNA 21
bases long into RNAi-competent cells or cell lysates (13,17,18). These single-stranded siRNA (ss-siRNA) molecules have
several advantages over the standard, ds-siRNA, particularly in the context of potential clinical applications. First, as
only one RNA strand is needed, the cost and work of preparation is reduced by half. Single-stranded molecules may also avoid
one of the major obstacles to therapeutic use of ds-siRNA: unintended side effects. For example, a recent study showed that
both chemically and enzymatically synthesized ds-siRNA species induced interferon expression, whereas none of the ss-siRNA
species provoked a detectable interferon response (19). Off-target silencing of genes with similar but non-identical
sequences to the sense or antisense strand is another potential obstacle to the specificity of siRNA activity (20). By using
a single strand, the risk of off-target silencing guided by the sense strand is eliminated. Finally, ss-siRNAs may circumvent
the RNAi-suppressing activity of dsRNA-binding proteins (21,22).
Despite these potential advantages of ss-siRNAs, the single-stranded molecule has a significant shortcoming; ss-siRNAs do
not efficiently induce RNAi. ss-siRNA must be used at concentrations that are 6- to 100-fold higher than that of ds-siRNA to
achieve comparable levels of gene silencing (13,17,18). The reason for the poor silencing activity of the single-stranded
molecules is not yet known.
To investigate whether the low level of silencing by ss-siRNAs could be improved, we sought to examine the activity of
ss-siRNAs with the boranophosphate (BP) backbone modification. We have shown previously that BP-modified ds-siRNAs are more
active for gene silencing than the corresponding phosphodiester- or phosphorothioate-backbone ds-siRNAs (23). In the
boranophosphate (BP) linkage (Figure 1A), a non-bridging phosphodiester oxygen is replaced with an isoelectronic borane ()
moiety. BP nucleic acids maintain the ability to base pair with high specificity and affinity and BP nucleotides can be
readily incorporated into DNA and RNA molecules by DNA and RNA polymerases, permitting synthesis of stereoregular
boranophosphate oligonucleotides (23–28). The boranophosphate modification appears to be well tolerated in mammalian
systems; BP ds-siRNAs do not induce any appreciable toxicity in HeLa cells (23). Furthermore, BP DNA dinucleotides are
minimally toxic to rodents, and the degradation product of BP oligonucleotides (borate) is minimally toxic to humans
(23,29,30).
In this work, we demonstrate that partial BP-modification increases the silencing activity of ss-siRNA against green
fluorescent protein in HeLa cells. In our system single-stranded BP-siRNAs can silence gene expression as well as native ds-
siRNA.
MATERIALS AND METHODS
Synthesis of boranophosphate siRNAs
ss- and dsRNA molecules were synthesized as described previously (23). Briefly, DNA oligonucleotides were purchased from
Qiagen: T7 promoter primer, 5'-TAATACGACTCACTATAG-3'; EGFP1 templates: sense, 5'-AAGTTCACCTTGATGCCGTTCTATAGTGAGTCGTATTA-3'
and antisense, 5'-AAGAACGGCATCAAGGTGAACTATAGTGAGTCGTATTA-3'; control EGPF1 templates: sense, 5'-
AAGTTCACCGTAGTTCCGTTCTATAGTGAGTCGTATTA-3' and antisense, 5'-AAGAACGGAACTACGGTGAACTATAGTGAGTCGTATTA-3'; EGFP2 templates:
sense, 5'-AAGGACTTGAAGAAGTCGTGCTATAGTGAGTCGTATTA-3' and antisense, 5'-AAGCACGACTTCTTCAAGTCCTATAGTGAGTCGTATTA-3'; Control
EGFP2 templates: sense, 5'-AAGGACTTTGAAGAAGCGTGCTATAGTGAGTCGTATTA-3' and antisense, 5'-
AAGCACGCTTCTTCAAAGTCCTATAGTGAGTCGTATTA-3'. ssRNA molecules were synthesized using T7 RNA polymerase. To create
boranophosphate modified oligonucleotides, one or more ribonucleoside 5'-(-P-borano)triphosphates were added to the
transcription reaction in place of the corresponding rNTPs. RNAs were then purified as single strands using phenol extraction
and Microspin G-50 Micro Columns (Amersham) or PAGE and suspended in water. To make dsRNA, sense and antisense strands were
first annealed and then purified and concentrated using Microcon YM-10 centrifugal filters (Millipore). RNA concentration was
determined by UV absorbance at 260 nm.
Cell culture and transfection
EGFP-TRex HeLa cells, which express enhanced green fluorescent protein (EGFP) under control of a tetracycline responsive
promoter, were cultured as described previously (23). Twenty-four hours after plating, cells were transfected with siRNA
using Oligofectamine (Invitrogen) according to the manufacturer's instructions with 1.35 μl Oligofectamine per well of a 24
-well plate. The concentration of siRNA in the cell culture medium ranged from 3.1 to 50 nM, as described in figure legends.
Twenty-four hours after siRNA transfection, cells were induced to express EGFP with 1 μg/ml tetracycline. For the EGFP1
control sequence, the six central nucleotides of the active EGFP1 sequence were inverted. As the control sequence for EGFP2,
we used an siRNA in which the eight central nucleotides inverted because the sequence with six inverted nucleotides had an
unacceptable degree of homology to some human mRNAs.
Flow cytometry analysis
Cells were trypsinized and fixed in 1% formaldehyde in phosphate-buffered saline, 48 h post-transfection. Cells were
analyzed for EGFP expression using a FACScan (Becton-Dickinson) flow cytometer. Data were processed using CellQuest software
(Becton-Dickinson).
Cellular RNA analysis
Cells were transfected in culture medium at a final siRNA concentration of 12.5 nM. EGFP1 production was induced with 1
μg/ml tetracycline. Cells were trypsinized and total cytoplasmic RNA isolated using the RNeasy kit (Qiagen), 22 h after EGFP
induction. RNA from each sample (2 μg) was separated by formaldehyde–agarose gel electrophoresis, transferred to a
positively charged nylon membrane and crosslinked by UV irradiation. The immobilized RNA was hybridized with 32P-labeled RNA
probes complementary to the EGFP coding sequence and to human ?-actin (to normalize for signal). Hybridization was quantified
using a Molecular Dynamics phosphorimaging system.
Quantitative RT–PCR
Cells were transfected in culture medium at a final siRNA concentration of 25 nM or mock transfected with Oligofectamine
(Invitrogen) alone. Cells were trypsinized and total cytoplasmic RNA isolated using the RNeasy kit (Qiagen), 24 h after
transfection. RNA from each sample (1 μg) was used to make cDNA using the iScript kit (Bio-Rad). Quantitative PCR was
performed using SYBR Green I (Invitrogen), Platinum Taq (Invitrogen), 1.5 mM MgCl2 and primers at a concentration of 0.2 μM.
PCR was performed on a Roche Lightcycler and data were analyzed using Lightcycler software. The following primers were used:
human Ago2, 5'-TGGCTGTGCCTTGTAAAACGCT-3' and 5'-CGCGTCCGAAGGCTGCTCTA-3'; human GAPDH, 5'-CATGTTCGTCATGGGTGTGAACCA-3' and 5'-
AGTGATGGCATGGACTGTGGTCAT-3'.
RNase susceptibility analysis
EGFP1 and EGFP2 RNAs (10 pmol) were incubated with 0.5 and 2 ng RNase A (Roche), respectively, in 5 μl RNase reaction
buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA and 150 mM NaCl) at 37°C for specified lengths of time. The RNase reactions were
stopped by adding 2 vol of the gel-loading buffer (10 M urea, 50 mM EDTA, 0.1 bromophenol blue and 0.1% xylene cyanol), and
loaded on a 4% agarose gel. After SYBR Gold (Molecular Probes) staining, the gels were photographed under UV illumination and
analyzed using Fluorescent Image Analytical Software (Hitachi Software Engineering Co. Ltd).
RESULTS
ds-siRNAs and several species of ss-siRNAs, all targeting EGFP, were synthesized as described previously (23) (Figure 1).
Briefly, all RNA species were synthesized using T7 RNA polymerase and purified as single strands. To create BP-modified
siRNAs, one or more native ribonucleoside triphosphate was replaced by its BP counterpart in the transcription reaction.
Silencing activity of native and BP ss-siRNA
Native and BP ss-siRNAs targeted to EGFP were tested for silencing activity in HeLa cells that express EGFP under control
of a tetracycline-inducible promoter. For these studies, cells were transfected with ds- or ss-siRNA, induced to express
EGFP, and then, after 24 h, analyzed by flow cytometry to measure the level of EGFP protein expression. The fluorescence of
cells transfected with siRNA was compared with the fluorescence of mock-transfected cells (treated with Oligofectamine alone)
to determine the percent inhibition of EGFP expression. To control for non-specific effects of the RNA, we tested control
sequence ss-siRNAs with the central 6 or 8 nt inverted (Figure 1B). None of the control ss-siRNAs silenced EGFP expression
(Figure 2).
Initially, EGFP1 sequence ss-siRNAs were tested at a concentration of 25 nM because this is the concentration at which
silencing by native ds-siRNA approaches a maximum, thereby allowing comparison of the peak silencing activity of the
different siRNAs. Similar to earlier studies (13,17,18), we found that native ss-siRNA could inhibit EGFP expression, but was
significantly less active than native ds-siRNA (Figure 2A). When BP-modified siRNAs were studied, we found that ss-siRNA with
BP-modified cytidine residues () silenced EGFP expression with the same efficiency as the unmodified ds-siRNA. Furthermore,
an anti-EGFP ss-siRNA with modified adenosine and cytidine residues was significantly more active than native ds-siRNA (99%
versus 70% reduction in EGFP expression, respectively).
While BP modification of cytidine and adenosine residues significantly enhanced silencing activity of ss-siRNAs, ss-
siRNAs that included modified uridines ( and ) were notably less active than unmodified ss-siRNA. Therefore, we examined why
these species had reduced EGFP silencing activity. We have demonstrated previously in ds-siRNAs that a high proportion of BP
residues in the center of the antisense strand reduces siRNA silencing activity (23). As there are four uridine residues
among the central six nucleotides (those that surround the predicted target cleavage site, Figure 1B) in the EGFP1 sequence,
the reduced silencing activities of and were consistent with the pattern established previously with ds-siRNAs, and suggest
that modification of the central bases of ss-siRNAs reduces silencing activity in a manner analogous to the reduction seen
with modification of the central bases of double-stranded BP-siRNAs.
While we suspected that extensive modification of central nucleotides caused the reduction in siRNA silencing, the loss
of activity in ss-siRNAs with modified uridines could also have been due to modification of the two residues on the 3' end,
or simply due to the presence of modified uridines. To further examine the relationship between modification sites and ss-
siRNA activity, a different anti-EGFP sequence (EGFP2) was tested. This sequence has two uridines at the 3' end; however,
none of the six central nucleotides are uridines. Instead, there are four adenosines in this region (Figure 1B). Once again,
the native ss-siRNA had a much lower level of silencing activity than native ds-siRNA (Figure 2B). However, BP-modification
of the uridine residues (all peripheral) caused a dramatic increase in ss-siRNA activity, almost restoring it to the level of
ds-siRNA. In contrast, when adenosine residues were modified in the EGFP2 molecule (resulting in modification of 3 of the
central 6 nt), activity remained at a low level. These results demonstrate that BP-modified uridine residues and 3' overhangs
are compatible with ss-siRNA activity and provide additional support for the hypothesis that multiple central BP
modifications interfere with efficient silencing.
The overall pattern of increased activity with peripheral modification and reduced activity with central modification was
consistent for both anti-EGFP sequences. However, in contrast to the results for EGFP1 ss-siRNAs, increasing the degree of
peripheral BP modification in EGFP2 ss-siRNAs caused a slight decrease in silencing activity (Figure 2B, and ).
Dose response of native and BP ss-siRNA
We determined silencing dose response curves for several anti-EGFP1 siRNAs (Figure 3). As we had observed when a single
siRNA concentration was used, the double-stranded native AnSn siRNA exhibited moderate silencing activity. AnSn induced an
80% reduction in EGFP fluorescence at 50 nM siRNA and had half maximal silencing activity in the range of 2 nM. In contrast,
An silences EGFP expression only modestly, inducing only 60% silencing at 50 mM with half maximal silencing at 15 nM. When BP
cytidine residues are introduced to the EGFP1 ss-siRNA, its activity improves such that the dose response curve of is almost
identical to that of AnSn. When modified cytidine and adenosine residues are used, silencing activity of the ss-siRNA becomes
highly active; induces close to 100% EGFP silencing at 25 nM, with half-maximal silencing induced at 1 nM siRNA.
Kinetics of silencing by native and BP ss-siRNA
Next, we examined the effect of BP modification on ss-siRNA silencing over time. HeLa cells were transfected with siRNA,
induced to express EGFP after 5 h or after 1, 3, 5 or 7 days and fixed 24 h after induction. To assess the duration of
silencing relative to each interfering RNA's activity, the level of silencing after 1 day was normalized to 100. While the
activity of all RNA species starts to decline after 3 days, the activity of native ss-siRNA (An) declines much more quickly
than that of native ds-siRNA (AnSn) or BP ss-siRNAs (, ) (Figure 4). It should be noted that the raw data reveal that,
consistent with our other results, native ss-siRNA was significantly less active than native ds-siRNA and that modified ss-
siRNAs were as active () or more active () than native ds-siRNA at all times tested (data not shown). Notably, the raw data
also reveal that still reduced EGFP expression by 50% 1 week after transfection. Finally, while other investigators have
reported that ss-siRNA achieves maximum silencing more quickly than ds-siRNA (18), we did not observe such a pattern.
BP ss-siRNA silences by reducing target mRNA levels
siRNAs inhibit protein expression primarily by inducing cleavage of target mRNA. However, it has also been shown that
some RNA species, known as miRNAs, can silence genes by inhibiting translation of their targets (31). Previous work suggests
that the presence of a base mismatch or a bulge when the siRNA pairs with its target can cause it to function through the
miRNA pathway rather than by the RNAi mechanism (32). Because miRNAs have been shown to function as single strands (33), we
investigated whether BP ss-siRNAs might act by inhibiting translation of target mRNA. The effects of BP ss-siRNA and native
ds-siRNA on EGFP mRNA levels were examined by northern blot and were normalized to ?-actin expression. Similar to ds-siRNA,
BP-modified ss-siRNA caused a reduction in the level of EGFP mRNA (Figure 5A). To compare the relative change in protein and
mRNA levels, samples of the cells used for northern analysis were analyzed by flow cytometry. If the BP ss-siRNA inhibited
translation in addition to reducing mRNA levels, it should cause a relatively greater protein inhibition than mRNA
inhibition, compared with unmodified ds-siRNA. Instead, for both native ds-siRNA and BP ss-siRNA, the percent inhibition of
protein expression closely matches the percent inhibition of mRNA expression (Figure 5B). Furthermore, the ratios of the
reductions in protein levels to the reductions in mRNA levels are the same for BP ss-siRNA and native ds-siRNA. We therefore
conclude that modified ss-siRNAs inhibit protein expression primarily by reducing EGFP mRNA levels, rather than by inhibiting
translation.
BP ss-siRNA activity requires Argonaute2 expression
Having determined that BP ss-siRNA silence protein expression by mRNA cleavage, we further examined the process of
silencing by these molecules. Previous work has suggested that native ss-siRNA act through the RISC to induce RNAi
(13,17,18). However, in all of these studies, the concentrations of ss-siRNA required to achieve gene silencing comparable
with that induced by ds-siRNA were 6- to 100-fold higher. Therefore, it is important to determine whether the highly
efficient silencing observed with BP ss-siRNA is due to RISC-related activity or to engagement of a different gene silencing
mechanism, such as induction of degradation by a double-stranded RNase (34). To determine the RISC-dependence of BP ssRNA
silencing, we examined the effect of siRNAs that have been shown to knock-down expression of Argonaute2 (Ago2), a key
component of the RISC (35). For these studies, EGFP-HeLa cells were transfected with Ago2 siRNA or with control, non-
silencing siRNA. The cells were transfected with native ds-siRNA (which is known to interact with the RISC) or BP ss-siRNA
targeted to EGFP, 24 h later. Cells were induced to express EGFP 24 h after the second transfection and analyzed by flow
cytometry 24 h after induction. It has been demonstrated previously that when RISC is disrupted by knocking-down Ago2
expression, silencing by ds-siRNA is eliminated (35). If BP ss-siRNA acts through a RISC-dependent process, its silencing
activity should be also dramatically reduced when Ago2 is silenced.
The siRNA targeted to Ago2 caused a notable reduction in Ago2 expression as measured by quantitative RT–PCR (Figure 6A). We
found that when Ago2 expression was suppressed, the ability of native ds-siRNA to silence EGFP expression was reduced
dramatically, compared with cells initially transfected with control siRNA (Figure 6B). The silencing activity of BP ss-siRNA
was abrogated to a similar extent by Ago2 silencing, indicating that BP ss-siRNA acts primarily through a RISC-dependent
process (Figure 6B). To ensure that inhibiting Ago2 expression does not, by itself, affect EGFP expression, we also examined
cells transfected with Ago2 siRNA, followed by control siRNA. The Ago2 siRNA did not appreciably affect EGFP expression; mean
fluorescence for mock transfected and Ago2 transfected cells were 537.1 and 536.1, respectively (in arbitrary units). The
combination of the northern blot and the Ago2 knock-down data suggests that BP ss-siRNA acts primarily through the standard
RNAi pathway used by ds-siRNA.
BP-modification has variable effects on ss-siRNA nuclease resistance
Having demonstrated that the BP modification dramatically improves the silencing activity of ss-siRNAs, we sought to examine
the reason for this change in activity. Given the susceptibility of ssRNA to RNases and the enhanced nuclease stability of
BP-modified dsRNA (23), we initially speculated that the enhanced silencing activity of BP ss-siRNAs was at least partially
due to their greater resistance to RNase degradation. To test this hypothesis, we examined the susceptibility of native and
BP ss-siRNAs to degradation by RNase A. As expected, unmodified ss-siRNA was very susceptible to nuclease digestion while ds
-siRNA was relatively resistant to degradation (Figure 7). Boranophosphate modification did enhance nuclease resistance of
some ss-siRNA species. However, BP modification did not result in a consistent improvement in nuclease stability, nor did
resistance to RNase degradation correlate with greater silencing activity. Notably, the most active siRNA we tested, the
EGFP1 ss-siRNA with modified adenosine and cytosine residues, was degraded at essentially the same rate as the native ss-
siRNA. This result suggests that nuclease resistance may not be the sole reason for the enhanced silencing activity of BP ss
-siRNAs.
DISCUSSION
ssRNA can efficiently induce RNAi
While testing the silencing activity of native and boranophosphate modified ss- and ds-siRNAs, we discovered that BP ss-
siRNA can be a highly effective gene silencing tool. As seen in several previous studies (13,17,18), we found that the gene
silencing activity of unmodified ss-siRNA was substantially less than that of ds-siRNA. In contrast, we found that the most
effective BP-modified ss-siRNA species (EGFP1 , with modified adenosine and cytidine residues) is even more active than
native ds-siRNA, implying that ss-siRNA can be efficiently incorporated into the RISC (Figures 2 and 3). The maximum activity
of is also greater than that of any BP-modified ds-siRNA that we have examined (data not shown) (23). To our knowledge, this
is the first demonstration that the silencing activity of ssRNA can match or exceed that of dsRNA. Also, given our findings
that BP ss-siRNA silencing is due to a reduction in mRNA levels and is Argonaute2-dependent, it seems likely that BP ss-siRNA
acts through the standard RNAi pathway.
Structure-activity relationships in BP ss-siRNA
We found that position effects of BP modifications followed the same general rule we discerned when testing BP-modified
dsRNA (23): peripheral BP modification is well tolerated, while central modifications cause a significant loss of activity.
However, the magnitude of the benefit of peripheral BP modification was greater when applied to single-stranded molecules. At
25 nM, peripheral modification of ss-siRNA caused up to a 2.4-fold increase in silencing activity (Figure 2, ), compared with
a maximum increase in ds-siRNA activity of 1.3-fold (23).
Because peripheral modifications dramatically increase ss-siRNA silencing activity, we initially hypothesized that the
positive attributes conferred by central modifications would offset their inhibitory effects, such that even central
modifications would enhance activity. Instead, ss-siRNA species with central modifications have activity levels that are as
low as or lower than those of native ss-siRNA (Figure 2). Apparently, the unfavorable effects of central modification are
still significant in single-stranded molecules. The poor activity of centrally modified ds-siRNA and ss-siRNA may reflect a
disruption of the enzymatic activity of the RISC, which cleaves the target directly across from the center of the interfering
RNA (36). Previous studies have demonstrated that central sequence mismatches or ribose modifications inhibit ds-siRNA
activity (36,37), although the regions that are sensitive to such changes appears to be more extensive than the area that is
sensitive to BP modification.
Mechanisms of enhanced activity in boranophosphate ss-siRNA
We initially hypothesized that BP-modified ss-siRNAs would be more nuclease resistant that native ss-siRNA and that this
would be a major reason for their enhanced silencing activity. However, we found that BP modification did not consistently
result in greater resistance to RNase A digestion for the sequences tested here. Indeed, the BP ss-siRNA species with the
greatest silencing activity (EGFP1 ) was degraded at essentially the same rate as native ss-siRNA (Figure 7). It is still
possible that BP modification could confer greater stability within the cell or in the culture medium; RNase A resistance may
not directly correlate with resistance to other nucleases. However, the absence of an observable increase in RNAse stability
with BP modification suggests that the enhanced activity of BP ss-siRNAs may not be solely due to improved nuclease
resistance. This hypothesis is supported by other work showing that modifications that confer nuclease resistance do not
necessarily enhance the activity of ds- or ss-siRNAs (37,38).
Instead, we suspect that BP ss-siRNAs have other advantageous properties that confer efficient silencing activity. Borane
substitution of a non-bridging oxygen results in changes in size, charge, polarity and stereochemistry (39). It can be
imagined that the presence of a borane group instead of oxygen in the phosphate backbone of RNA would change their
interaction with other molecules, e.g. mRNA target and RISC protein components. Indeed, we have found recently that BP
modification raises the melting temperature of siRNAs, suggesting that a modified siRNA might have a greater affinity for the
target mRNA than a native siRNA (40). Another characteristic of BP siRNA that may enhance its silencing activity is its
tendency to make an A-form helix (40). The results of several different studies suggest that efficient cleavage of the target
RNA requires the A-form structure between the guide strand of the ds-siRNA and the target (37,41,42). Investigation of the
interactions between BP ss-siRNA and RISC should facilitate mechanistic understanding of RNAi and may suggest generally
applicable strategies for enhancing siRNA activity.
Therapeutic potential of boranophosphate ss-siRNAs
Our examination of silencing by boranophosphate-modified ss-siRNA suggests that BP ss-siRNA could have significant
therapeutic potential. The high maximum activity, potency at low concentrations and duration of silencing by EGFP1 suggest
that relatively low and infrequent dosing of BP ss-siRNA could be effective, even in rapidly dividing cells. Indeed, by
accounting for dilution by cell division and comparing the activity over time to dose response curves, it appears that
essentially all of the decline in activity can be accounted for by this dilution. Therefore, we expect that silencing would
last even longer in non-dividing cells or when the siRNA blocks cell growth (7).
In addition to its excellent silencing activity, BP ss-siRNA has advantages unique to ssRNA: simplified synthesis and no
risk of off-target silencing by the sense strand. Furthermore, the smaller size and reduced negative charge of ss-siRNA may
facilitate delivery into cells. We also believe that ssRNA is less likely to induce an interferon response than dsRNA. Most
examinations of interferon induction by siRNAs have shown that ss-siRNAs do not provoke an interferon response (19). However,
a recent publication reported that ssRNA synthesized by phage polymerases (the method used here) can induce interferon
expression due to the presence of 5' triphosphate groups (43). In the context of potential clinical applications, this should
not pose a significant problem because the terminal phosphates can be easily removed by calf intestinal alkaline phosphatase
treatment (43). Because control sequence siRNAs (also synthesized by T7 polymerase) did not show any silencing activity
(Figure 2), we do not believe that our analysis of silencing activity was affected by the presence of 5' triphosphate groups
on the siRNAs we tested.
The use of single-stranded molecules may be particularly desirable in specific situations. For example, BP ss-siRNAs may
permit the use of sequences whose sense strand sequence would be nearly complementary to other (off-target) human genes. In
addition, the ability to use single-stranded molecules should enhance silencing by sequences that would otherwise have poor
activity due to incorporation of the inactive sense strand into the RISC [it should be noted that other strategies to favor
incorporation of the active strand have also been developed (44)]. This relaxation of sequence requirements is likely to be
especially important when there are relatively few potential sequences, such as when the target is a particular splice
variant or mRNA with a specific point mutation. ss-siRNAs are also likely to be useful in treating viral infections because
many human viruses express dsRNA-binding proteins, presumably to evade the host cell interferon response (21,22).
CONCLUSION
We have shown that boranophosphate modified ss-siRNA efficiently induces silencing at high and low concentrations and for
extended periods of time. This finding is significant, because it demonstrates that, when appropriately modified, ss-siRNA
can induce RNAi at least as effectively as ds-siRNA. It also introduces a new category of molecules for potential use as
RNAi-based therapeutics.
CKNOWLEDGEMENTS
We would like to thank Danuta Gasinski and the Duke University Cancer Center Cell Culture and Flow Cytometry facilities
for excellent technical assistance. This work was supported by a grant from The Children's Miracle Network (K.A.A.), NIH R01
CA081214 (K.A.A.), NIH R21 CA104604-01 (K.A.A. and B.R.S.), Department of Defense Grant DAMD 17-02-1-0376, sponsored by the
Department of the Army (B.R.S.), NIH R01 GM57693 (B.R.S.) and the Duke University Medical Scientist Training Program
(A.H.S.H). The content of the information does not necessarily reflect the position or the policy of the Government, and no
official endorsement should be inferred. Funding to pay the Open Access publication charges for this article was provided by
the Section of Pediatric Infectious Diseases, the University of Chicago.
Conflict of interest statement. None declared.
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