Patterning of Gene Expression
A R T I C L E S
and the mixture was refluxed for 4 h at 90 °C. The resulting yellow
oily solution was evaporated to dryness and partitioned between
chloroform and water (2 mL each). The organic layer was washed
twice with water and the combined aqueous layers were washed
once with chloroform. The combined chloroform layers were
evaporated to dryness yielding a yellow solid. The resulting solid
was dissolved in dichloromethane (DCM; 2 mL) and then purified
using silica gel flash column chromatography with 5% methanol
in DCM. Fractions were collected, dried, and analyzed by LC-MS
and appropriate fractions were combined and used for the next
reaction. The crude contained a mixture of 2 isomers, E and Z.
Yield 32.4 mg (98.2% yield). TLC (EtOAc/MeOH, 75:25 v/v): Rf
approach, and so we have sought to address this through the
synthesis and incorporation of new photolabile groups into
dsRNA. The guiding hypothesis is that these groups, prior to
irradiation, block interaction with Dicer, and hence prevent
RNAi until irradiation removes them. The incomplete block of
RNAi prior to irradiation can be due to multiple factors: (1)
the groups, while lowering the affinity of Dicer for the modified
RNA, do not completely abrogate binding, or (2) the groups
can be cleaved by nucleases that remove terminal nucleotides
from the modified RNA.
For this work, we designed new photolabile groups that had
the potential to have a greater ability to block interactions with
Dicer and/or nucleases. This was achieved by incorporating a
carboxyl synthetic handle into DMNPE that allowed a range of
bulky amines to be incorporated. These bulkier photolabile
groups then have the potential to have greater steric clash with
Dicer and/or nucleases. We demonstrate that one of these
groups, cyclo-dodecyl DMNPE or CD-DMNPE, results in a
modified dsRNA that allows gene expression to be switched
from full expression to native-like knockdown using light. Using
this modified RNA, we were able to construct distinct patterns
of gene expression in monolayers, including gradients.
1
) 0.64; H NMR (400 MHz, DMSO-d6, E or Z isomers, ratio of
isomer 1/isomer 2 ) 1:3): isomer 1 [δ 7.93 (d, J ) 8.8 Hz, 1H),
7.66 (s, 1H), 6.89 (s, 1H), 5.43 (s, 2H), 4.63 (s, 2H), 3.92 (m, 4H),
2.06 (s, 3H), 1.46-1.58 (m, 4H), 1.15-1.46 (m, 16H), 0.91-0.96
(m, 2H)]; isomer 2 [7.88 (d, J ) 8.8 Hz, 1H), 7.39 (s, 1H), 6.99 (s,
1H), 6.41 (s, 2H), 4.59 (s, 2H), 3.90 (m, 4H), 2.55 (s, 3H),
1.46-1.58 (m, 4H), 1.15-1.46 (m, 16H), 0.91-0.96 (m, 2H)]; 13
C
NMR (100 MHz, DMSO-d6): only isomer 2 peaks visible δ 197.5,
166.1, 151.5, 145.3, 127.1, 115.4, 111.0, 99.2, 66.9, 43.8, 29.8,
27.6, 23.4, 23.2, 22.7, 22.4, 21.5; UV/vis (DMSO): λmax (ελ): 263
nm (7323 M-1 cm-1), 346 nm (4470 M-1 cm-1); reversed phase
HPLC-MS (exact conditions as used for compound 3): retention
time (min) 13.32, 13.90; ESI-MS (m/z): [M]+ calcd for
C23H36N4O5, 449.3; found, 449.3.
Experimental Section
N-Cyclododecyl-2-(4-(1-diazoethyl)-2-methoxy-5-nitrophenoxy)-
acetamide (Compound 5). Compound 4 (10 mg, 22.3 µmol) was
dissolved in 500 µL of dry dimethyl sulfoxide and protected from
light. Manganese(IV) oxide (10 mg, 115 µmol) was added to 75
µL of this solution and shaken gently for 45 min, while protected
from light. The characteristically red-orange mixture was centri-
fuged using a microcentrifuge and the supernatant was filtered
through Celite 545 supported by glass wool in a glass pipet. This
pad was washed with 58.8 µL of dimethyl sulfoxide to give a final
diazo concentration of 25 mM. Compound 5 was freshly prepared
each time to cage dsRNA. As per the use of diazo-DMNPE,
compound 5 was not isolated or further characterized, beyond its
UV-visible spectrum. The UV-vis analysis showed peaks at 280,
346, and 450 nm.
Caging of dsRNA. Caging of dsRNA with diazo-DMNPE was
performed following our previously described methods.7 Caging
with compound 5 (diazo-CD-DMNPE) was done similarly with the
following modifications: To 250 µL of 50 µM dsRNA in tris-
acetate-EDTA buffer, freshly prepared compound 5 in DMSO (50
µL, 1.25 µmol) was added and gently shaken for 12 h, protected
from light. The addition of freshly prepared compound 5 in DMSO
(50 µL, 1.25 µmol) was repeated every 12 h for four times. Finally,
a double amount of compound 5 in DMSO (100 µL, 2.50 µmol)
was added and allowed to react for 24 h, protected from light.
Purification of Caged dsRNA. As previously described.10
Mass Spectrometry of Caged dsRNA. As previously de-
scribed.10
tert-Butyl 2-(4-acetyl-2-methoxyphenoxy)acetate (Compound
1). Compound 1 was synthesized as described in the literature.13
2-(4-Acetyl-2-methoxy-5-nitrophenoxy)acetic Acid (Compound
2). Compound 2 was synthesized as described in the literature.13
2-(4-Acetyl-2-methoxy-5-nitrophenoxy)-N-cyclododecylaceta-
mide (Compound 3). Compound 2 (50.0 mg, 186 µmol), cy-
clododecylamine (68.1 mg, 372 µmol), and 1-hydroxybenzotrizole
hydrate (56.9 mg, 372 µmol) were dissolved in 900 µL of
dimethylformamide (DMF). 1-Ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride (59.6 mg, 311 µmol) was added to the
resulting solution and allowed to shake for 15 h. The product was
purified by partitioning the reaction mixture between ethyl acetate
(10 mL) and saturated sodium chloride (10 mL). The ethyl acetate
layer was washed twice with saturated sodium chloride solution
and the combined aqueous layers were washed once with ethyl
acetate. The combined organic layers were washed with saturated
sodium bicarbonate solution, 1 N HCl, and again with saturated
sodium bicarbonate solution. The organic layer was then dried with
magnesium sulfate and evaporated to give an off-white solid. Yield
1
63.3 mg (78.4%), TLC (EtOAc/MeOH, 75:25 v/v): Rf ) 0.60; H
NMR (400 MHz, DMSO-d6): δ 7.95 ppm (d, J ) 8.4 Hz, 1H),
7.54 (s, 1H), 7.26 (s, 1H), 4.67 (s, 2H), 3.95 (s, 3H), 3.88-3.95
(m, 1H), 2.52 (s, 3H), 1.50-1.64 (m, 4H), 1.15-1.41 (m, 18H);
13C NMR (100 MHz, DMSO-d6): δ 199.3, 165.8, 153.5, 147.8,
137.6, 132.1, 109.9, 108.7, 67.7, 56.6, 44.1, 30.0, 29.8, 23.3, 23.1,
22.8, 21.3; UV/vis (DMSO): λmax (ελ): 262 nm (8037 M-1 cm-1),
343 nm (4500 M-1 cm-1); MS (m/z): [M]+ calcd for C23H34N2O6,
435.2; found, 435.5; reversed phase HPLC-MS (flow rate 0.4
mL/min, runtime 30 min, injection volume 25 µL) solvent A
(0.1% formic acid in H2O), solvent B (0.1% formic acid in
acetonitrile (ACN)), gradient 50% B to 100% B over 10 min,
isocratic 100% B for 17 min, 100% B to 0% B over 3 min, C8
Hypersil column (5 µm, 100 × 4.6 mm, Varian): retention time
(min) 12.37; ESI-MS (m/z): [M]+ calcd for C23H34N2O6, 435.2;
found, 435.2.
Cell Culture and Irradiation. As previously described,10 with
the following modifications: HeLa cells were plated in a 96 well
format, at 70% confluency, and allowed to culture for 18-20 h.
Cells were then co-transfected with pEGFP-C1 (0.099 µg/well),
pDsRed2-N1 (0.132 µg/well) plasmids, uncaged or caged dsRNA
(0.20 pmol, 1.56 nM for 1× concentration and 0.98 pmol, 7.80
nM for 5× concentration), and lipofectamine (1.13 µL/well) in Opti-
MEM (120 µL/well) for 6 h. Cells were washed with Opti-MEM
(150 µL) and irradiated for 10 min in Opti-MEM (100 µL) using
a Blak-Ray lamp (Model XX-15 L, 30 W) placing the lamp 10 cm
above the 96-well plate. One half of the plate-lid was protected
from light using aluminum foil and the other half was exposed to
UV light filtered through a WG-320 long pass filter (Edmund
Optics). Cells were then cultured in antibiotic free Dulbecco’s
Modified Eagle Medium (DMEM; 200 µL) containing fetal bovine
serum (FBS; 10%) for 42 h and washed with phosphate buffered
(E/Z)-N-Cyclododecyl-2-(4-(1-hydrazonoethyl)-2-methoxy-5-ni-
trophenoxy)acetamide (Compound 4). Compound 3 (32 mg, 73.6
µmol) was dissolved in 2 mL of a 1:1 mixture of acetonitrile and
95% ethanol. Hydrazine monohydrate (71.2 µL, 1.47 mmol) and
glacial acetic acid (6.6 µL, 110.4 µmol) were added to the solution
(13) Holmes, C. P. J. Org. Chem. 1997, 62, 2370–2380.
9
J. AM. CHEM. SOC. VOL. 133, NO. 3, 2011 441