Light controllable siRNAs regulate gene suppression and phenotypes in cells
Quan N. Nguyena, Rajesh V. Chavlia, Joao T. Marquesb, Peter G. Conrad IIa,
Die Wangb, Weihai Hea, Barbara E. Belislea, Aiguo Zhanga,
Larry M. Pastora, Frank R. Witneya, May Morrisc, Frederic Heitzc,
Gilles Divitac, Bryan R.G. Williamsb, Gary K. McMastera,⁎
aGenospectra, Inc., 6519 Dumbarton Circle, Fremont, CA 94555, USA
bCleveland Clinic Foundation, Department of Cancer Biology, NB40, The Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195, USA
cCRBM-CNRS-FRE-2593 Molecular Biophysics and Therapeutics, 1919 route de Mende 34293 Montpellier cedex 5, France
Received 26 September 2005; received in revised form 6 January 2006; accepted 9 January 2006
Available online 30 January 2006
Small interfering RNA (siRNA) is widely recognized as a powerful tool for targeted gene silencing. However, siRNA gene silencing occurs
during transfection, limiting its use is in kinetic studies, deciphering toxic and off-target effects and phenotypic assays requiring temporal, and/or
spatial regulation. We developed a novel controllable siRNA (csiRNA) that is activated by light. A single photo removable group is coupled
during oligonucleotide synthesis to the 5′ end of the antisense strand of the siRNA, which blocks the siRNA's activity. A low dose of light
activates the siRNA, independent of transfection resulting in knock down of specific target mRNAs and proteins (GAPDH, p53, survivin, hNuf2)
without stimulating non-specific effects such as regulated protein kinase PKR and induction of the interferon response. We demonstrate survivin
and hNuf2 csiRNAs temporally knockdown their mRNAs causing multinucleation and cell death by mitotic arrest, respectively. Furthermore, we
demonstrate a dose-dependent light regulation of hNuf2 csiRNA activity and resulting phenotype. The light controllable siRNAs are introduced
into cells using commercially available reagents including the MPG peptide based delivery system. The csiRNAs are comparable to standard
siRNAs in their transfection efficiency and potency of gene silencing. This technology should be of interest for phenotypic assays such as cell
survival, cell cycle regulation, and cell development.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Light controllable siRNAs to regulate gene expression; MPG delivery for difficult to transfect cell types; Branch DNA quantitation of mRNA expression;
Non-specific effects of the double-stranded RNA (dsRNA) regulated protein kinase PKR kinase and induction of the IFN response; Immunofluorescence detection of
protein expression; Western blot detection of protein expression; Apoptosis and multinucleation phenotypic assays
RNA-mediated interference (RNAi) is an evolutionarily
conserved mechanism to silence genes [1–4]. Basically, there
are two approaches to gene silencing using RNAi: (1)
Chemically synthesized double stranded RNA (21–27 nt)
known as “small interfering RNA” (siRNA) introduced into
cells as active molecules [1–4] and (2) Hairpin precursors
“shRNAs” (∼25–29 nt) transcribed endogenously and pro-
cessed within the cell to active siRNAs (∼21 nt) [5,6]. In the
cell, the siRNA assembles to the protein complex known as
“RNA-induced silencing complex” (RISC) which binds to the
complementary mRNA via base pairing interactions to the 5′
end of the siRNA antisense strand resulting in a sequence-
specific degradation of the mRNA and gene silencing .
Both siRNAs and shRNAs have been successfully applied for
“loss-of-function” assays with resulting phenotypes in cul-
tured cells and in vivo models [4,8,9]. Although RNAi is a
powerful approach for efficiently silencing genes, it does have
its limitations since constitutive expression of shRNAs or
delivery of active siRNAs cannot address phenotypic assays
that require temporal and/ or spatial regulation .
Biochimica et Biophysica Acta 1758 (2006) 394–403
E-mail address: email@example.com (G.K. McMaster).
0005-2736/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
2. Results and discussion
Previously, it was demonstrated that a 5′ end phosphate on
the antisense strand is required for a siRNA to function . We
have exploited this property by incorporating a single NPE
(nitrophenyl ethyl) photo removable “caging” group at the 5′
end of 21-mer siRNA antisense strand as the last step of a
conventional nucleotide synthesis (Fig. 1a). Exposure of the
csiRNA to light (365 nm), removes the caging group creating an
active siRNA. We demonstrated this principle using a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) csiRNA
(Fig. 1b, c). Increased light energy removed more caging groups
until the totally “uncaged” csiRNA electrophoresed to the same
position in the gel as the siRNA (Fig. 1b). When the same
csiRNA was transfected into HeLa cells and exposed to
increasing light energy, a dose-dependent knockdown of target
mRNA was demonstrated (Fig. 1c). The amount energy
required to fully uncage the GAPDH csiRNA for gel
electrophoresis (Fig. 1b) was between 2 and 4 J/cm2 and in
cells 1.4 J/cm2 (Fig. 1c). More energy is required to uncage in
vitro than in cells because the concentration of csiRNA is
significantly higher (Fig. 1b, 5 μM vs. Fig. 1c, 3 nM) and in a
vastly larger volume (microliters, Fig. 1b vs. picoliters, Fig. 1c).
The difference in energy required agrees with results previously
reported by Lin et al. to uncage β-ecdysone in solution and in
Highly purified csiRNA antisense strands are important for
successful controllable knockdown experiments. We investi-
gated the correlation between the purity of csiRNA and the
activity of csiRNA in cells, measured by HPLC chromatograms
of the antisense strand. Different lots GAPDH csiRNA of
various purities were transfected into HeLa cells and incubated
for 24 h at 37 °C. The cells were not exposed to light in order to
keep the csiRNA caged and unreactive. The cells were lysed
Fig. 1. (a) Schematic showing the principle of light activation of a csiRNA. csiRNA is transfected into cells as an “inactive caged” molecule. Light activation of the
csiRNA is made possible by the incorporation of a single NPE (nitrophenyl ethyl) photo removable (cleavable) or “caging” group blocking the access of RISC to the
phosphorylated 5′ end antisense strand of the 21-mer siRNA. Once the caging group is removed by a low dose of light (365 nm), the “uncaged active” siRNA
assembles with the RISC complex which binds to the complementary mRNAvia base pairing interactions to the siRNA antisense strand 5′ end resulting in a sequence-
specific degradation of the mRNA and gene silencing. (b) Structure of a GAPDH csiRNA antisense strand. A photo removable (cleavable) “caging” group
-biotinylated NPE (nitrophenyl ethyl) was covalently linked to the 5′ phosphate of the 21-mer GAPDH siRNA antisense strands using standard oligoribonucleotide
synthesis procedure. The dash line depicts the site of cleavage after light activation. 20 μl of a 5 μM csiRNA solution was pipetted into a clear bottom, black wall plate.
The 5 μM solution was exposed to 0–10 J/cm2of 365 nm using the UCOM. 6 μl of the solution was loaded into a 1× TBE PAGE gel and electrophoresis at 100 V for
2 h. (c) Dose-dependent gene suppression of GAPDH gene expression by light activated GAPDH csiRNA. HeLa cells were transfected using Lipofectamine for
4 h with 3 nM of the GAPDH csiRNA and scrambled negative control siRNA, and then exposed to 0–1.4 J/cm2using the UCOM Microplate Photo-Activator,
followed by analysis of mRNA of GAPDH and cyclophilin (QuantiGene) at 24 h post-transfection. GAPDH expression level was normalized to cyclophilin level to
correct for differences in cell numbers from well to well. Experiments were performed in triplicates.
395 Q.N. Nguyen et al. / Biochimica et Biophysica Acta 1758 (2006) 394–403
and GAPDH mRNA expression levels were measured.
Increased knockdown in GAPDH expression levels prior to
light activation is viewed as a less efficient csiRNA. According
to Table 1, there is increased knockdown of expression as the
caging efficiency decreases. This is most likely due to (n-1)
residues that make up the majority of impurities from
oligonucleotide syntheses. For csiRNA, (n-1) residues are
fully active, complete siRNA molecules. The purity of all of
csiRNAs are N95% as measured by HPLC, which is achieved
on a routine basis and results in maximum of ∼30–40%
knockdown without light and at least 80% knockdown after
exposure to light.
Next, we compared the GAPDH csiRNA light activated
before and after transfecting into HeLa cells to a GAPDH
siRNA of the same sequence and molarity (Fig. 2a). The
siRNA and the in vitro uncaged csiRNA were equally potent
in knocking down GAPDH mRNA expression at all times
measured. If the csiRNA was not exposed to light, 30–40%
suppression of mRNA at 4 h and 24 h post-transfection was
detected when compared to the negative control. This 30–40%
suppression of mRNA is the result of csiRNA n-1 residues not
caged (∼5%) and thus are fully active siRNA molecules (as
described above and Table 1). At 4 h post-transfection, cells
from the same transfection were exposed to light [in vivo
csiRNA light activated (T=0)] and cells were immediately
lysed. As can be observed the T=0 in vivo uncaged was
comparable to the caged csiRNA control without light, since
the in vivo uncaged csiRNA was only active for seconds.
However, by 24 h and 48 h post-transfection (T=20 h and
T=44 h post light activation of cells in vivo, respectively),
there was significant mRNA suppression, comparable to the
siRNA and the in vitro light activated csiRNA. These results
were confirmed at the protein expression level by immuno-
fluorescent analysis (Fig. 2b) and suggested that caged
GAPDH csiRNA remained quiescent in the cells until exposed
We then tested light activated GAPDH csiRNA cotrans-
fected into HeLa cells in the presence of a β-actin siRNA
and measured the expression of each mRNA (Fig. 2c). Only
cells exposed to light showed suppression of GAPDH
mRNA and were equal to cells transfected with either a
standard GAPDH siRNA or csiRNA exposed to light.
Overall, the β-actin siRNA did not interfere with the light
activation of the GAPDH csiRNA and its gene silencing.
Using β-actin siRNA in combination with light controllable
GAPDH csiRNA, we demonstrated that gene silencing was
We also tested whether the csiRNA could stimulate non-
specific effects such as activation of the double-stranded
RNA (dsRNA) regulated protein kinase PKR and induction
of the IFN response. Although the csiRNAs were able to
bind to recombinant PKR in vitro whether activated or not
(Fig. 3a), they did not induce any detectable activation of
PKR as measured by an in vitro kinase assay (Fig. 3b). In
accord with this, HT1080 cells transfected with the csiRNA
exhibited no evidence of PKR activation as measured by
phosphorylation of its downstream target eIF2α (data not
shown). Moreover, there was no induction of dsRNA and
IFN stimulated protein 56 by the csiRNAs whether caged or
uncaged, indicating that they do not activate the IFN system
(Fig. 3c). In contrast, a T7 synthesized RNA was able to
induce the interferon system as previously reported (Fig. 3c,
). The csiRNAs used specifically knocked down the
target genes p53 or GAPDH only when exposed to light (Fig.
3c) although the HT1080 cells exposed to light also showed
a small but reproducible increase in p53 levels (Fig. 3c and
data not shown). Thus, we conclude that csiRNA can
specifically induce knockdown of the target genes without
activating the IFN system.
We also evaluated the transfection of csiRNA using the
novel MPG peptide-based delivery system. We have previ-
ously demonstrated that MPG efficiently delivers siRNAs into
many different cell types including difficult-to-transfect cells
such as differentiated 3T3-L1 cells ([14,15] and data not
shown). Fig. 4 shows the photo-activation of GAPDH csiRNA
transfected HeLa cells using MPG, where the knock down of
GAPDH between normal siRNA and csiRNA are very similar,
suggesting that the 5′ photocaging group on csiRNA did not
affect the MPG peptide transfection efficiency. We found
similar results using lipofectamine transfection reagent
Next, we tested survivin and hNuf2 csiRNAs to induce
phenotypes in HeLa cells [16,17]. When compared to the
negative scrambled control with light and survivin csiRNA
control without light, the light activated survivin csiRNA
significantly reduced its expression and increased the number of
multinucleated cells, which agrees with previously reported
data for HeLa cells under similar conditions (Fig. 5, ).
Depletion of hNuf2 mRNA in HeLa cells results in mitotic
arrest and eventually leads to cell death. Mitotic arrested cells
Purity of csiRNA as function of residual activity
Purity of antisense
csiRNA residual activity (% gene
knock down with no exposure to light)a
Highly purified csiRNA antisense strands are important for successful
controllable knockdown experiments. The table shows the correlation between
the purity of csiRNA and the activity of csiRNA in cells, measured by HPLC
chromatograms of the antisense strand. Different lots GAPDH csiRNA of
various purities were transfected into HeLa cells and incubated for 24 h at 37
°C. The cells were not exposed to light in order to keep the csiRNA caged
and unreactive. The cells were lysed and GAPDH mRNA expression levels
were measured. Increased knockdown in GAPDH expression levels prior to
light activation is viewed as a less efficient csiRNA. There is increased
knockdown of expression as the caging efficiency decreases. This is most
likely due to (n-1) residues that make up the majority of impurities from
oligonucleotide syntheses. For csiRNA, (n-1) residues are fully active,
complete siRNA molecules. These data are consistent with other csiRNAs
used in this study.
a% gene knockdown=(1−ratio of normalized mRNA levels of positive/
negative csiRNA)×100. All mRNA levels are normalized to cyclophilin level
to correct for the differences in cell number from well to well.
396 Q.N. Nguyen et al. / Biochimica et Biophysica Acta 1758 (2006) 394–403
exhibited a round cell phenotype and apoptotic cells showed
shrinkage and collapse of both the cell and nuclear membranes
. We evaluated hNuf2 csiRNA's capability to photo initiate
cell cycle arrest and cell death in HeLa cells and found only
light activated hNuf2 csiRNA suppressed its target mRNA
expression and exhibited significant numbers of cells of mitotic
arrest and apoptotic phenotypes (Fig. 6). In addition, the
csiRNA light exposed cells showed considerable reduction of
ATP level, suggesting that the cells' energy metabolism was
suppressed. On the other hand, higher ATP levels and healthy
cells were observed using the negative control siRNAwith light
or csiRNA without exposure to light. These results agree with
those found by Deluca et al. using a standard siRNA .
However, we did detect some residual gene suppression (30–
40%) in hNuf2 csiRNA transfected cells not exposed to light
(Fig. 6), which may be due to csiRNA molecules not blocked
during oligosynthesis and not completely removed by our
purification procedure (b5%) as described above. The hNuf2
mRNA is expressed at only 10–20 copies/cell and thus
picomolar concentrations (5% of 3 nM transfected) may be
sufficient to cause the 30–40% knockdown. Nonetheless, we
did not detect the phenotype. As can be observed in Fig. 7,using
the hNuf2 csiRNA and increasing the light energy, we
demonstrated a dose-dependent induction of the expected
phenotypes, where 0.3 J/cm2was enough energy to fully
induce cell cycle arrest and cause apoptosis.
Temporal and/or spatial regulation of gene expression is
important for many phenotypic assays . Recently,
inducible promoter systems driving expression of shRNAs in
mammals and plants have been reported [10,18]. However,
these systems require more manipulation, making them more
applicable for in vivo experimentation than for in vitro testing
in cultured cell assays. In addition, stable cell lines can have
basal expression in absence of the inducer, making them
Fig. 2. (a) Photo-activation of GAPDH csiRNA in HeLa cells. HeLa cells transfected with 3 nM of GAPDH siRNA, GAPDH csiRNA or negative control siRNA for
4 h exposedto 1.4 J/cm2of light (365 nm) and analyzedforgene expressionat 4 h, 24 h and 48 h post-transfection. In vitro=photo-activationof siRNAin solution prior
to the start of transfection procedure, In vivo=photo-activation of siRNA in cell after 4 h transfection procedure. (b) Immunofluorescent detection of GAPDH protein.
siRNA transfected cells were fixed and probed for expression of GAPDH protein expression using mouse anti-human GAPDH antibody, 48 h post-transfection. (c)
Sequential silencing of two genes using a GAPDH csiRNA and a β-actin siRNA. csiRNA GAPDH (2.5 nM) (csi-GAPDH) and siRNA β-actin (62.5 nM) (si-actin),
and the negative control (65 nM) siRNA (si-negative) were either co-transfected or individually transfected for 4 h as described in the Materials and methods
section. After 4 h transfection, wells were exposed to 2 J/cm2(+Light) or 0 J/cm2(−Light) followed by mRNA analysis (QuantiGene) at 24 h post-transfection. All
mRNA expression levels were normalized to cyclophilin mRNA level to correct for differences in cell number from well to well.
397Q.N. Nguyen et al. / Biochimica et Biophysica Acta 1758 (2006) 394–403
“leaky” and some inducers may cause toxic or off target
effects on cells, making them not desirable for many
phenotypic assays .
A variety of light activated or “caged” molecules have been
used extensively in cell biology and pharmacology including
secondary messengers, RNA, DNA, peptides and proteins
[12,19–25]. Recently, a siRNAwas caged post-synthesis using
4, 5 dimethyoxy-2-nitrophenylethyl (DMNPE) to create a diazo
compound that reacts randomly with multiple internal phos-
phate groups of the siRNA . Depending on the caging group
equivalents per siRNA molecule, either the siRNA's knock-
down activity was partially blocked (∼50%) or only partially
activated (∼50%) by light, even when increasing the exposure
times, where longer irradiation resulted in phototoxicity. In this
study, we have demonstrated an alternative approach to control
the activity of a siRNA resulting in regulated gene silencing
with phenotypes. We found incorporating a single NPE
(nitrophenyl ethyl) caging group at the 5′ end of a siRNA
antisense strand was sufficient to block enough of its activity in
cells and was totally removed using a 2 J/cm2of light (365 nm)
energy to induce phenotypes. At this energy level, we have not
detected any effects on cell viability (ATP levels, caspase 3
activity, membrane integrity, morphology), including light
sensitive cells (e.g., Jurkat cells), which is in agreement with
previous results ; however, we did observe small increases
of p53 in HT1080 cells. We observed a minimum of 12 J/cm2to
induce apoptosis, which is in accordance with the findings that
Fig. 3. (a) The ability of the siRNAs, caged or uncaged, to bind PKR was analyzed by electrophoresis mobility shift assays (EMSA). The siRNAs were incubated with
recombinant PKR (K296R), separated on a nativePAGE andstainedwith SYBR Goldto visualizethe nucleic acids.(b) Cagedor uncagedsiRNAswere testedfor their
capacity to activate PKR in vitro. The kinase assay was performed with recombinant PKR and activity was determined by phosphorylation of B56 alpha as a substrate
(30). poly(I:C) was used as a positive control for activation of PKR. (c) Caged or uncaged csiRNAs, were tested to determine whether they would activate the IFN
system in cell culture. The csiRNAs, targeting GAPDH or p53, were transfected into HT1080 cells at a final concentration of 30 nM using Lipofectamine 2000.
After 4 h, the transfection mix was discarded and new media added. The cells were exposed to 1.4 J/cm2of light (365 nm) where indicated (+Light). After 40 h, whole
cell lysates were prepared and analyzed by Western blot with the indicated antibodies.
Fig. 4. MPG delivery of GAPDH csiRNA into HeLa cells. HeLa cells
transfected with 3 nM of GAPDH siRNA, GAPDH csiRNA or negative control
siRNA for 4 h exposed to 1.4 J/cm2of light (365 nm) and analyzed for gene
expression at 24 h post-transfection. In vitro=photo-activation of siRNA in
solution prior to the start of transfection procedure, In vivo=photo-activation of
siRNA in cell after 4 h transfection procedure. Note: The in vivo photo-
activation represents 20 h knockdown, since the activity was released 4 h post-
398 Q.N. Nguyen et al. / Biochimica et Biophysica Acta 1758 (2006) 394–403
27 J/cm2of light (365 nm) induced apoptosis in keratinocytes
 and intracellular free Ca2+was released in Jurkat cells
exposed to 10 J/cm2of light . Using commercially available
transfection reagents, we found that csiRNAs are comparable to
a standard siRNAs in their transfection efficiency and potency
of gene silencing. To date, we have successfully photo-activated
Fig. 5. Light activation of survivin csiRNA in HeLa cells. (Left) HeLa cells were transfected with 3 nM of survivin csiRNA, exposed to 2 J/cm2of light 4 h post-
transfection,lysed24 h post-transfectionand mRNAlevelquantitated.In vitro=photo-activation ofsiRNA in solutionprior totransfection,Invivo=photo-activation of
siRNA in cells after 4 h post-transfection. (Right) Nuclear stain of HeLa cells was performed using DAPI at 24 h post-transfection. Multinucleated cells (arrow) are
observedin wells transfected withsurvivin csiRNAand exposedto light, whereasround nuclei (line) are observedin wells transfectedwith csiRNAand not exposedto
light or with the negative control siRNA exposed to light.
Fig. 6. Activation of hNuf2 csiRNA and mitotic arrest in HeLa cells. HeLa cells were transfected with 3 nM of hNuf2 csiRNA, exposed to 2 J/cm2of light at 4 h post-
transfection and bright field images were taken at 48 h and 72 h post-transfection (top). mRNA (left) level and ATP/Cell Viability Assay (right) were performed at
48 h or 72 h post-transfection, respectively.
399Q.N. Nguyen et al. / Biochimica et Biophysica Acta 1758 (2006) 394–403
Fig. 7. Dose response of hNuf2 csiRNA using light. HeLa cells were transfected with 3 nM of hNuf2 csiRNA, exposed to 0, 0.1, 0.3, 0.5 and 2 J/cm2of light at 4 h post-transfection. Bright field images (bottom) and
intracellular Calcein AM esterase activity of cells (top) were obtained at 72 h post-transfection.
Q.N. Nguyen et al. / Biochimica et Biophysica Acta 1758 (2006) 394–403
csiRNAs 24 h post-transfection and achieved knockdown of
target mRNAs (data not shown). The half-life of a csiRNA is
equivalent to that of a siRNA and siRNA half-life depends on
the number of cell divisions (dilution effect) and overall
ribonuclease activity of transfected cells. To achieve longer-
term stability against ribonucleases, internal ribonucleotides of
the csiRNAs can be modified as previously demonstrated for
The data presented here demonstrated that light could
precisely control csiRNA gene silencing and the resulting
phenotypes in living cells. The technology enabled a T=0 time
point and the csiRNA served as its own control. In addition, by
controlling the light energy, csiRNA was activated such that a
dose-dependent response of mRNA suppression was achieved.
Controllable siRNA (csiRNA) opens the door to studying toxic
and off-target events independently of cellular transfection,
enabling kinetic measurements in cells and the induction or
modulation of phenotypes. Finally, csiRNA could be used in
combination with GFP fused proteins to demonstrate protein
suppression and associated phenotype in living cells.
3. Materials and methods
3.1. csiRNA and siRNA synthesis
The following human csiRNAs and siRNAs were synthesized using
commercially available phosphoramidite monomers:
GAPDH: 5′-CAUCAUCCCUGCCUCUACUTT-3′ (sense strand)
survivin: 5′-GGAACAUAAAAAGCAUUCGTT-3′ (sense strand)
hNuf2: 5′-AAGCATGCCGTGAAACGTATT-3′ (sense strand)
p53: 5′-GGAAAUUUGCGUGUGGAGUTT-3′ (sense strand)
β-actin and GAPDH negative control siRNAs were obtained from Ambion.
Photo removable (cleavable) phosphoraimidite, [1-N-(4, 4′-Dimethoxytrityl)-5-
diisopropyl)-phosphoramidite was obtained from Glenn Research. It was
incorporated into 5′ phosphate group of the antisense strand of a 21-mer siRNA
using the standard oligoribonucleotide synthesis procedure. The modified 21-
mer antisense strand was purified using RNAse-free HPLC and verified by gel
electrophoresis and mass spectrometry. The purity was routinely N95%. The
modified antisense strand was annealed to the sense strand by heating equal
molar of both strands in the annealing solution (250 mM Tris, pH 7.4, 500 mM
NaCl, 5 mM EDTA) at 90 °C for 5 min. The solution is allowed to cool for 4 h at
3.2. Transfection of csiRNAs
HeLa and HT1080 cells were transfected using Lipofectamine 2000
(Invitrogen), Oligofectamine (Invitrogen) and/or MPG “Express-si” (Genospec-
tra)accordingtothe manuals.Briefly,approximately2000–5000cells/well were
grown overnight in a clear bottom, black well 96-well microplate (Costar). Cells
were incubated with csiRNA or siRNA for 4 h and then replaced with complete
growthmedia. HT1080cells weretransfectedusingthe sameprotocol in 12-well
plates and whole cell lysates were prepared for Western blot analysis.
3.3. Sequential silencing of GAPDH csiRNA and β-actin siRNA
The csiRNA GAPDH (csi-GAPDH, Genospectra), siRNA β-actin (si-actin,
Ambion) and negative control GAPDH siRNA (si-negative, Ambion) were
thawed on ice and diluted to 5 μM stock concentrations in 1× dilution buffer
(5×siDilution Buffer, Genospectra). For each csiRNA or siRNA, 15 μl of stock
siRNA-Actin, 0.6 μl stock csiRNA-GAPDH or 15.6 μl of siRNA-negative
(Ambion) was diluted in 106 μl (final volume) of Opti-MEM (Invitrogen) at
room temperature and incubated for 5–10 min. For cotransfection, final
concentration per well was 62.5 nM siRNA β-actin and 2.5 nM csiRNA-
GAPDH. For single siRNA transfection, final concentration was 62.5 nM si-
actin, 2.5 nM csiRNA-GAPDH or siRNA-GAPDH, and 65 nM siRNA-
negative. Oligofectamine (Invitrogen) was warmed to room temperature and
mixed according to the manufacturer's instructions. For each well assayed 6 μl
were diluted in 30 μl Opti-MEM [final volume, (Invitrogen)] and incubated at
room temperature for 15–20 min. To form transfection complexes, 30 μl of
diluted Oligofectamine (Invitrogen) were added to each tube of diluted siRNA
and incubated for 30 min at room temperature. After the 30-min incubation
period, 64 μl of additional Opti-MEM (Invitrogen) were added to each tube and
the tube gently mixed by inverting. 20 μl of diluted complexes were added
directly to wells containing cells in complete growth medium (T=0). The final
volume in each well is 120 μl. At T=4 h post-transfection, complexes were
removed and replaced with 120 μl of fresh complete growth medium. The plates
were exposed to 2 J/cm2of 365 nm light using the UCOM Microplate
Photoactivator (Genospectra) and then returned to the incubator. Replicates of 3
wells were run for all conditions tested. At T=24 h post-transfection, cells were
lysed with 60 μl of QuantiGene lysis buffer and mRNA levels for GAPDH, β-
actin and cyclophilin (internal control gene) were measured using the
QuantiGene Reagent System (Genospectra). All mRNA expression levels
were normalized to cyclophilin mRNA levels to correct for differences in cell
number from well to well.
3.4. In vivo photo activation of csiRNA
After transfection, cells were exposed to 0.02–2.0 J/cm2(5–50 s) of
365 nm±20 nm light using a UCOM Microplate Photo-Activator (Genospectra)
according to the UCOM and csiRNA manuals. The UCOM is designed
specifically for photo-activation and photo-affinity applications. The UCOM
generates light that is uniformly distributed across a standard microplate. The
large area of illumination allows simultaneous and high throughput release of
photo-activated samples in a microplate format. To test potency of a new
csiRNA (N95% purity), we routinely transfect cells with increasing amounts of
csiRNA (1–30 nM) without light induction until 10–30% mRNA knockdown is
observed. Once the concentration of csiRNA is established to initiate
knockdown of mRNA without light, it is our experience that at least 80%
knockdown is observed with light induction.
3.5. Cell viability assay
Cell viability was determined by measuring the ATP levels using the Cell
Titer-Glo Assay (Promega) according to the manual.
3.6. Quantitation of mRNA
The cells were lysed and mRNA expression directly measured using the
Branch DNA Technology “QuantiGene Reagent System” (Genospectra)
according to the manual. No mRNA purification and amplification are required
for this assay. Duplicate or triplicate wells were run to obtain average and
standard deviation values. The difference in cell number per well was corrected
by calculating the ratio of siRNA-targeted gene to an internal control gene
3.7. Cells and reagents
HT1080 cells were grown in 10% FBS DMEM. Antibodies against human
p53 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and
GAPDH from Chemicon International (Temecula, CA). Antibodies against p56
were a gift from Ganes Sen (Cleveland Clinic Foundation).
3.8. Immunofluorescent detection of GAPDH
Cells were washed with PBS and fixed with 4% formaldehyde for 4 min.
Cells were washed with PBS, treated with 0.1% triton X-100 for 5 min and
blocked with 5% dry milk for 30 min and incubated in 1:2000 dilution of mouse
401 Q.N. Nguyen et al. / Biochimica et Biophysica Acta 1758 (2006) 394–403
anti-human GAPDH (Abcam) for 1 h. After a PBS wash, fixed cells were
incubated in 1:1000 dilution of goat anti-mouse Alexa-488 conjugate
(Molecular Probes) for 45 min. Finally, after a PBS wash, cells were viewed
under a fluorescent microscope.
3.9. Western blot analyses
Briefly, cells were lysed in 50 mM Tris buffer, pH 7.4 containing 150
mM of NaCl, 50 mM of NaF, 10 mM of β-glycerophosphate, 1% Triton X-
100, 0.1 mM of EDTA, 10% glycerol and protease/phosphatase inhibitors.
The samples were kept on ice for 10 min, vortexed and centrifuged for 15
min at 14000 rpm, the supernatant collected in a new tube and protein
concentrations determined using the Protein assay kit (Bio-Rad). 30 g of total
protein were separated on SDS-polyacrylamide gels (PAGE), transferred to
Immobilon™-PSQ membranes (Millipore) and probed with the indicated
3.10. Kinase activity assay
100 ng of purified PKR was incubated in kinase buffer (10 mM Tris–HCl
[pH 7.6], 50 mM KCl, 2 mM Mg acetate, 7 mM 2-mercaptoethanol, 20%
glycerol, 1 mM MnCl2) in the presence of 10 mM ATP and 0.1 μCi/μl [γ-32p]-
ATP. The siRNAs were added to the kinase assay at different concentrations and
the kinase activity of PKR was inspected by including 100 ng of recombinant
B56α prepared as described previously . 1 ng/μl of poly(I:C) was used as a
positive control. The reaction was incubated for 30 min at 30 °C, the proteins
resolved by SDS-PAGE and the gel autoradiographed.
3.11. Survivin phenotypic assay
HeLa cells were transfected with the human survivin csiRNA or in vitro
photo-activated csiRNA using Lipofectamine 2000 as described above. The
HeLa cells transfected with the csiRNA were photoactivated with light 4
h post-transfection, fixed using 4% paraformaldehyde at 24 h post transfection
and the nuclei were stained using DAPI dye (Calbiochem). Quantitation of
multinucleated cells was performed by counting the total number of
fragmented multi-nuclei (arrow) and round nuclei (line) from two fields per
3.12. hNuf2 phenotypic assay
HeLa cells were transfected with human hNuf2 csiRNA or in vitro photo
activated csiRNA for 4 h using Lipofectamine 2000 as described above. At 48
h and 72 h post-transfection, bright field images of transfected cells were taken.
To measure cell viability, Calcein AM (Molecular Probes) was used to detect
esterase activity in cells. Images were taken at 48 h post-transfection.
We would like to thank Melanie Mahtani for her input
and review of the manuscript. Thanks also to Kate Stankis
for graphic assistance. This work was supported in part by
a grant from the National Institutes of Health (NIH)
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