Photocaged Morpholino Oligomers for the Light-Regulation of
Gene Function in Zebrafish and Xenopus Embryos
Alexander Deiters,*,†R. Aaron Garner,‡,§Hrvoje Lusic,†,#Jeane M. Govan,†
Mike Dush,‡Nanette M. Nascone-Yoder,‡and Jeffrey A. Yoder*,‡
Department of Chemistry, North Carolina State UniVersity, Raleigh,
North Carolina 27695, United States, Department of Molecular Biomedical Sciences, College of
Veterinary Medicine, North Carolina State UniVersity, Raleigh, North Carolina 27606, United States,
and The Center for ComparatiVe Medicine and Translational Research, North Carolina State
UniVersity, Raleigh, North Carolina 27606, United States
Received June 20, 2010; E-mail: firstname.lastname@example.org; email@example.com
Abstract: Morpholino oligonucleotides, or morpholinos, have emerged as powerful antisense reagents for
evaluating gene function in both in vitro and in vivo contexts. However, the constitutive activity of these
reagents limits their utility for applications that require spatiotemporal control, such as tissue-specific gene
disruptions in embryos. Here we report a novel and efficient synthetic route for incorporating photocaged
monomeric building blocks directly into morpholino oligomers and demonstrate the utility of these caged
morpholinos in the light-activated control of gene function in both cell culture and living embryos. We
demonstrate that a caged morpholino that targets enhanced green fluorescent protein (EGFP) disrupts
EGFP production only after exposure to UV light in both transfected cells and living zebrafish (Danio rerio)
and Xenopus frog embryos. Finally, we show that a caged morpholino targeting chordin, a zebrafish gene
that yields a distinct phenotype when functionally disrupted by conventional morpholinos, elicits a chordin
phenotype in a UV-dependent manner. Our results suggest that photocaged morpholinos are readily
synthesized and highly efficacious tools for light-activated spatiotemporal control of gene expression in
Morpholino oligonucleotides were first described in 1997, as
novel RNase H-resistant antisense reagents applicable to cell
culture studies,1,2and since have been used in ViVo to block
translation,3modify mRNA splicing,4and block microRNA
function.5Morpholinos also have become important for inves-
tigating gene function during embryonic development in species
as diverse as zebrafish, medaka, carp, chicken, Xenopus, sea
lamprey, sea urchin, and Drosophila.3,4,6-9However, these
reagents must be injected directly into the cells of an early
embryo and are constitutively active. Thus, morpholinos that
produce early embryonic lethality eliminate the possibility of
assessing gene function at later stages of development. More-
over, because the morpholino is distributed to the progeny of
injected cells, spatial control of gene silencing cannot be
obtained with standard antisense agents.
One strategy to eliminate the early lethality phenotypes and
to achieve spatiotemporal control over gene silencing is through
the application of photoresponsive morpholinos which are
inactive until exposed to a short dose of non-damaging
ultraviolet (UV) light.10,11This strategy is especially relevant
to studies employing transparent zebrafish embryos: the trans-
parent nature of the zebrafish embryo permits small regions of
the embryo and even single cells to be UV-irradiated in ViVo.
A first generation of light-activated antisense agents for zebrafish
embryos has been reported.12,13Indeed, it was recently shown
that a morpholino can be linked to a short, complementary
blocking morpholino via a photosensitive linker: once irradiated
within a zebrafish embryo, the link between the morpholinos is
broken, and the small number of base pairs is insufficient to
maintain a morpholino duplex, releasing the antisense mor-
pholino, which can then base-pair with its target mRNA.10
†Department of Chemistry and The Center for Comparative Medicine
and Translational Research.
‡Department of Molecular Biomedical Sciences and The Center for
Comparative Medicine and Translational Research.
§Present address: Department of Chemistry and Chemical Biology,
Harvard University, Cambridge, MA 02138.
#Present address: Department of Chemistry, Boston University, Boston,
(1) Summerton, J.; Stein, D.; Huang, S. B.; Matthews, P.; Weller, D.;
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(10) Shestopalov, I. A.; Sinha, S.; Chen, J. K. Nat. Chem. Biol. 2007, 3,
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(12) Blidner, R. A.; Svoboda, K. R.; Hammer, R. P.; Monroe, W. T. Mol.
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Published on Web 10/20/2010
10.1021/ja1053863 2010 American Chemical Society
15644 9 J. AM. CHEM. SOC. 2010, 132, 15644–15650
Although effective, this strategy requires the synthesis of two
morpholinos, the synthesis of the photosensitive linker, and the
chemical connection of all three through covalent bond forma-
tion followed by purification.14The length of the linker and
the sequence and length of the inhibiting morpholino need to
be carefully designed. Moreover, two morpholinos are released
after irradiation, which leads to a greater risk of off-target
An alternative strategy has been developed that utilizes a
RNA-based inhibitor strand that is fully complementary to the
morpholino and contains a photocleavable linkage: once irradi-
ated within a zebrafish embryo, the link joining two short RNA
oligomers is broken, and, as with the preceding strategy, the
small number of RNA base pairs is insufficient for maintaining
a duplex with the morpholino.11However, this approach requires
a 5-10-fold molar excess of the inhibitor strand to saturate the
morpholino, and since the injection of high amounts of nucleic
acids into an embryo can be lethal, this strategy is not feasible
for morpholinos that are only effective at high concentration.
Furthermore, after UV irradiation, this strategy releases two
RNA oligomers into an embryo, which may induce off-target
Here, we present a different, substantially more direct
approach to the generation of light-activatable morpholinos
through the incorporation of a photocaged monomer during
oligomer polymerization. The direct installation of light-
removable (caging) groups on specific morpholino bases ef-
ficiently blocks their ability to hybridize to their target mRNA
until the caging group is removed by irradiation with UV light
of 365 nm. The effectiveness of this caging strategy is fully
demonstrated by the UV-dependent activity of these reagents
in cell culture and live embryos.
General Information. Chromatography was carried out on
Merck silica gel, 60 Å. NMR spectra were obtained on a Varian
Oxford 300 MHz spectrometer. Absorbance spectra were obtained
on an HP UV/vis spectrometer 8453. HPLC and HRMS were
obtained using an Agilent Technologies 6210 LC-TOF ESI
morpholin-2-yl]methyl Acetate (2). Ac2O (232 mg, 215 µL, 2.28
mmol) was added dropwise over 5 min to a solution of compound
1 (1 g, 2.07 mmol; gift from Gene Tools, LLC) and DMAP (spatula
tip) in pyridine (10 mL) under a N2atmosphere at 0 °C. The reaction
was allowed to stir for 12 h at room temperature. The pyridine
was evaporated under vacuum, and the residue was purified by silica
gel chromatography using hexanes:EtOAc (1:1) containing 1%
TEA, affording 2 as a white foam in 95% yield (1.03 g, 1.97 mmol).
1H NMR (300 MHz, CDCl3): δ ) 1.38-1.48 (m, 2 H), 1.84 (s, 3
H), 2.04 (s, 3 H), 3.09-3.14 (m, 1 H), 3.35-3.39 (m, 1 H),
4.03-4.06 (m, 2 H), 4.38-4.41 (m, 1 H), 6.14-6.18 (m, 1 H),
6.99 (s, 1 H), 7.17-7.49 (m, 15 H).13C NMR (75 MHz, CDCl3):
δ ) 12.7, 21.0, 38.4, 49.3, 52.1, 64.7, 74.9, 76.3, 80.6, 110.9, 126.8,
128.2, 129.4, 135.6, 150.2, 164.0, 171.0. HRMS: m/z calcd for
C31H31N3O5[M + H]+, 526.23367; found, 526.27330.
methyl Acetate (4). NPOM-Cl 3 (609 mg, 2.35 mmol)29in DMF
(1 mL) was added dropwise over 5 min to a solution of compound
2 (1 g, 1.96 mmol) and Cs2CO3(1.92 g, 5.88 mmol) in DMF (10
mL) under a N2atmosphere at 0 °C. The flask was wrapped in
aluminum foil, and the reaction was allowed to stir for 12 h at
room temperature. The reaction was taken up in EtOAc (100 mL)
and washed with NaHCO3, water, and brine (100 mL each). The
organic layer was dried over Na2SO4and filtered, and the volatiles
were evaporated. The product was purified by silica gel chroma-
tography using hexanes:EtOAc (2:1) containing 1% TEA, affording
4 as a yellow foam in 78% yield (1.14 g, 1.52 mmol).1H NMR
(300 MHz, CDCl3): δ ) 1.38-1.48 (m, 2 H), 1.52 (d, J ) 7.2 Hz,
3 H), 1.78 (s, 3 H), 2.06 (s, 3 H), 3.12 (d, J ) 11.7 Hz, 1 H),
3.35-3.39 (m, 1 H), 4.03-4.05 (m, 2 H), 4.38-4.41 (m,
1 H), 5.12-5.38 (m, 3 H), 5.95-6.13 (m, 3 H), 6.82-6.88 (m, 1
H), 7.15-7.49 (m, 17 H).13C NMR (75 MHz, CDCl3): δ ) 13.4,
21.2, 24.0, 49.5, 52.6, 64.8, 70.0, 73.2, 75.0, 77.1, 81.2, 103.1,
105.0, 105.3, 106.7, 110.1, 126.7, 128.1, 129.3, 134.2, 137.9, 142.3,
146.9, 150.3, 152.2, 163.0, 170.8. HRMS: m/z calcd for
C41H40N4O10 [M + H]+, 749.28174; found, 749.28162. UV/vis
(CH2Cl2): λmax(?) ) 239 (20 000), 271 (10 000), 344 nm (4400).
dione (5). Compound 4 (1 g, 1.33 mmol) and K2CO3(552 mg, 4.00
mmol) were dissolved in MeOH (10 mL) at room temperature. The
reaction was stirred for 3 h, after which it was filtered, and the
solvent was evaporated under vacuum. The residue was purified
by silica gel chromatography using hexanes:EtOAc (1:1) containing
1% TEA, affording 5 as a yellow foam in 93% yield (874 mg,
1.24 mmol).1H NMR (300 MHz, CDCl3): δ ) 1.38-1.45 (m, 2
H), 1.48-1.52 (m, 3 H), 1.76-1.78 (d, J ) 4.5 Hz, 3 H), 1.91-1.95
(m, 0.5 H), 1.99-2.07 (m, 0.5 H), 3.08 (d, J ) 11.7 Hz, 1 H), 3.28
(d, J ) 12.0 Hz, 1 H), 3.51-3.62 (m, 2 H), 4.22-4.38 (m, 1 H),
5.15-5.39 (m, 3 H), 5.97-6.14 (m, 3 H), 6.85 (d, J ) 9.6 Hz, 1
H), 7.15-7.49 (m, 17 H).13C NMR (75 MHz, CDCl3): δ ) 13.2,
23.9, 49.0, 51.9, 64.0, 70.1, 73.1, 73.6, 77.9, 81.1, 103.0, 105.0,
105.3, 106.0, 110.2, 126.7, 128.2, 129.4, 134.2, 138.0, 142.3, 146.9,
150.5, 152.1, 163.1. HRMS: m/z calcd for C39H38N4O9[M + H]+,
707.27118; found, 707.27130. UV/vis (CH2Cl2): λmax(?) ) 240
(20 000), 267 (10 000), 344 nm (3600).
Morpholinos. The caged compound 5 was activated and
incorporated into EGFP and chordin morpholino oligomers (se-
quences indicated in Scheme 2) by Gene Tools, LLC15-18
(Philomath, OR). Morpholino stocks were resuspended in water,
aliquoted, and stored at -20 °C.
Morpholino Melting Temperature Determination. The melting
temperature (Tm) of each morpholino hybridized to its RNA
complement target was determined as described previously,19with
the following modifications. The morpholino and RNA complement
(0.5 µM) were incubated in 0.15 M NaCl, 0.05 M NaH2PO4, pH
7.2. The samples were protected from light or irradiated at 365 nm
with an UV transilluminator (3 mW/cm2) for 10 min, heated to
100 °C for 5 min, and then cooled to 25 °C at a rate of 2 °C/min
using a Cary 100 Bio UV/vis spectrometer with a temperature
controller (Varian). The absorbance was recorded at 260 nm every
1 °C. The Tmwas determined by the maximum of the first derivative
of the absorbance vs temperature plot. Standard deviations were
calculated from three individual experiments.
Cell Culture. COS-7 cells were grown to 50-70% confluency
at 37 °C and 5% CO2 in complete culture media (DMEM
supplemented with 10% FBS, 50 IU/mL penicillin, and 50 µg/mL
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W. H.; Olson, A. J.; Chen, J. K. J. Am. Chem. Soc. 2009, 131, 13255–
(15) Summerton, J. E.; Weller, D. D. Uncharged morpholino-based
polymers having achiral intersubunit linkages. U.S. Patent 5,034,506,
July 23, 1991.
(16) Summerton, J. E.; Weller, D. D. Uncharged morpholino-based
polymers having phosphorous containing chiral intersubunit linkages.
U.S. Patent 5,185,444, February 9, 1993.
(17) Summerton, J. E.; Weller, D. D.; Stirchak, E. P. Alpha-morpholino
ribonucleoside derivatives and polymers thereof. U.S. Patent 5,378,841,
January 3, 1995.
(18) Summerton, J. E.; Weller, D. D. Morpholino-subunit combinatorial
library and method. U.S. Patent 5,698,685, December 16, 1997.
(19) Stein, D.; Foster, E.; Huang, S. B.; Weller, D.; Summerton, J. Antisense
Nucleic Acid Drug DeV. 1997, 7, 151–157.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 44, 2010
Photochemical Gene Regulation
streptomycin) and split into aliquots containing 106cells, which
were pelleted and resuspended in 100 µL of Cell Line Nucleofector
Solution “R” (Amaxa Inc., Gaithersburg, MD) supplemented with
0.10 µg of pEGFP-N1 (Clontech Laboratories, Inc., Mountain View,
CA), 0.42 µg of pDsRed2-N1 (Clontech Laboratories, Inc.), and
150 µM morpholino (EGFP-MO0or EGFP-MO4). Cell suspensions
were then transfected with a Nucleofector I using program “W-
01”. Solutions were incubated at 25 °C for 10 min prior to the
addition of 500 µL of complete culture medium. A fraction of each
cell suspension (100 µL) was then transferred into a single well of
a six-well plate containing 2.9 mL of complete culture medium.
Plates with transfected cells were incubated at 37 °C, 5% CO2for
1 h prior to UV exposure. UV exposure was performed for 2 min
using a DAPI fluorescence filter (340-380 nm excitation) on a
Leica DM5000B compound microscope.20Fluorescence from
transfected cells was imaged 24 h after UV exposure with a Zeiss
SteREO Lumar.V12 microscope. Fluorescence images were taken
at identical exposure times for control (no morpholino), no UV,
and UV-exposed cells in the same experiment.
Zebrafish. All experiments involving live zebrafish (Danio rerio)
were performed in accordance with relevant institutional and
national guidelines and regulations and were approved by the North
Carolina State University Institutional Animal Care and Use
Committee. Zebrafish embryos were collected by natural mating,
visualized with a Nikon SMZ800 microscope equipped with a 400
nm long-pass filter (GG400, Chroma Technology Corp., Rocking-
ham, VT), and microinjected at the 1-4-cell stage with morpholinos
and/or synthetic mRNAs using a pressure microinjector (PV830
picopump, World Precision Instruments, Sarasota, FL) at 60 psi
and a mechanical micromanipulator (World Precision Instruments)
with pulled glass capillary needles (World Precision Instruments).
Embryos were photographed with a Leica DM5000B microscope
equipped with a Retiga 1300 digital CCD camera and SimplePCI
For EGFP morpholino studies, zebrafish embryos were micro-
injected with in Vitro transcribed capped synthetic EGFP mRNA
(1.5 fmol, 0.40 ng), synthetic mCherry mRNA (0.7 fmol, 0.16 ng),
and EGFP-MO4(30 fmol, 0.54 ng). All injections were made into
the cytoplasm during the 1-cell stage, and some embryos were
exposed to 365 nm UV light (2 min, 25 W hand-held UV lamp,
2.1 mW/cm2) immediately following microinjection.21Embryos
were maintained in egg water22with 0.5 ppm methylene blue at
28 °C for 24 h before being photographed. A 1:20 molar ratio of
EGFP mRNA to EGFP-MO4allowed for the most efficient
photoregulation of EGFP-MO4activity. Fluorescence images were
taken at identical exposure times for all embryos in the same
For chordin studies, approximately 1 nL of a 500 µM chordin-
MO4, 0.15% phenol red solution was microinjected into the yolk
of 1-4-cell stage zebrafish embryos. Embryos were either protected
from light or exposed to 365 nm UV light (25 W hand-held UV
lamp, 2.1 mW/cm2) for 2 min at various time points after injection.
Embryos were maintained in egg water22with 0.5 ppm methylene
blue at 28 °C for 24-28 h before being scored and photographed.
Xenopus. All experiments involving live Xenopus laeVis were
performed in accordance with relevant institutional and national
guidelines and regulations and were approved by the North Carolina
State University Institutional Animal Care and Use Committee.
Xenopus embryos were obtained by in Vitro fertilization as
previously described.23Fertilized embryos were dejellied in 2%
cysteine-HCl (pH 7.8-8.1) and cultured in 0.1X Marc’s Modified
Ringers (MMR).23Before injection, embryos were equilibrated in
a solution of 3% Ficoll-400 in 0.75X MMR. In each experiment,
all embryos were obtained from a single clutch (i.e., eggs were
from the same adult female frog). A mixture of in Vitro transcribed
capped synthetic EGFP mRNA (1.6 fmol, 0.42 ng), synthetic
mCherry mRNA (7.0 fmol, 1.6 ng), and EGFP-MO4(46.8 fmol,
0.84 ng) was injected in a total volume of 0.6 nL in one animal
pole blastomere at the 8-16-cell stage using a pressure microin-
jector (World Precision Instruments) at 60 psi and a mechanical
micromanipulator (World Precision Instruments) with pulled glass
capillary needles (borosilicate with rod, World Precision Instru-
ments). A 1:30 molar ratio of EGFP mRNA to EGFP-MO4allowed
for the most efficient photoregulation of EGFP-MO4activity.
Injected embryos were recovered in the dark in 1.5% Ficoll in 0.5X
MMR for 20-30 min at room temperature before being gradually
transferred to 0.1X MMR. Embryos were either protected from light
or exposed to UV light for 2 min at 25 °C at the 32-64-cell stage,
using a Zeiss Lumar fluorescent Stereomicroscope equipped with
a DAPI filter and a 1.2× objective at 120× magnification, focusing
on the animal pole. Control and UV-irradiated embryos were then
cultured at 16 °C until stage 24.24Photographs were taken using a
Zeiss Lumar fluorescent stereomicroscope with an AxioCam MRc
camera and Axiovision image capture software. Fluorescence
images were taken at identical exposure times for all embryos in
the same experiment.
Results and Discussion
Development of NPOM-Caged Morpholinos. We hypoth-
esized that the incorporation of a photosensitive group directly
on a morpholino base would block the ability of a morpholino
to base-pair with its target mRNA until the caging group is
removed by UV irradiation (Scheme 1). By installing the caging
group directly on the base of a phosphoramidite morpholino
building block, it can be included in the direct synthesis of a
morpholino and site-specifically incorporated into the oligomer,
providing an efficient strategy for synthesizing caged morpholi-
nos. This strategy is based upon earlier successes in using a
novel 6-nitropiperonyloxymethyl caging group (NPOM) to
disrupt Watson-Crick base-pairing in DNA:DNA and DNA:
RNA duplexes.25-28This caging group is particularly effective
at caging nitrogen heterocycles and has the advantage of
disrupting hydrogen-bonding between nucleotides while provid-
ing the stability needed for use in conventional DNA or
(20) UV exposure of 1 min was not as effective at reducing EGFP
expression in transfected cells.
(21) In trial experiments with chordin-MO4-injected zebrafish embryos, a
2 min UV exposure was found to be the minimum required to elicit
a severe chordin phenotype in 80-100% of the embryos.
(22) Westerfield, M. The Zebrafish Book. Guide for the Laboratory Use of
Zebrafish (Danio rerio); University of Oregon Press: Eugene, OR,
(23) Sive, H. L.; Grainger, R. M.; Harland, R. M. Early DeVelopment of
Xenopus LaeVis; Cold Spring Harbor Laboratory Press: Cold Spring
Harbor, NY, 1998.
(24) Nieuwkoop, P. D.; Faber, J. External and internal stage criteria in the
development of Xenopus laevis. In Normal Table of Xenopus laeVis
(Daudin); Nieuwkoop, P. D., Faber, J., Eds.; North-Holland Publishing
Co.: Amsterdam, 1956; pp 162-168.
(25) Lusic, H.; Lively, M. O.; Deiters, A. Mol. Biosyst. 2008, 4, 508–511.
(26) Lusic, H.; Young, D. D.; Lively, M. O.; Deiters, A. Org. Lett. 2007,
(27) Young, D. D.; Edwards, W. F.; Lusic, H.; Lively, M. O.; Deiters, A.
Chem. Commun. 2008, 462–464.
(28) Young, D. Y.; Lusic, H.; Lively, M. O.; Yoder, J. A.; Deiters, A.
ChemBioChem 2008, 9, 2937–2940.
Scheme 1. Photochemical Gene Regulation with Caged
aSquares represent the 6-nitropiperonyloxymethyl (NPOM) caging group.
15646J. AM. CHEM. SOC. 9 VOL. 132, NO. 44, 2010
Deiters et al.
morpholino syntheses.26While stable under physiological condi-
tions, oligonucleotides harboring these caging groups are
returned to nearly full activity following a brief irradiation with
365 nm UV light.25–28We adopted this caging approach for
the synthesis of the caged morpholino monomer 5 (Scheme 2).
Compound 1 (gift from Gene Tools, LLC) was acetylated in
95% yield to deliver 2. The NPOM caging group was then
installed on 2 using the chloromethyl ether 3 (synthesized in
three steps29) and Cs2CO3 in DMF at room temperature,
providing 4 in 78% yield. The free 5′-hydroxyl group in 5 was
obtained through saponification of the acetyl ester with K2CO3
in methanol (93% yield). Using standard polymerization
chemistry,16,30the caged morpholino monomer 5 was then
incorporated into morpholino sequences shown to block the
translation of EGFP31and chordin3(Scheme 2). The morpholino
sequence selected to target EGFP expression (EGFP-MO0) has
been shown to effectively knock down EGFP expression both
in cell culture and in ViVo.31The morpholino sequence selected
to target the expression of endogenous zebrafish chordin
(chordin-MO0) routinely induces a specific morphological
phenotype when injected into zebrafish embryos.3,32,33Four
caged monomers were included in each morpholino, since a
common control for morpholino experiments is to include a
nonactive morpholino with four mismatched bases,1,3,34and we
have shown that three to four NPOM caging groups can
functionally disrupt DNA:DNA and phosphorothioate DNA:
RNA duplexes.27,28A morpholino nomenclature was adopted
in which a superscript numeral represents the number of caging
groups within a morpholino (e.g., EGFP-MO4includes four
caging groups, and EGFP-MO0includes zero caging groups).
NPOM Caging Groups Disrupt Morpholino:RNA Duplexes.
In order to evaluate the effectiveness of NPOM caging groups
to disrupt morpholinos’ base-pairing, we determined the Tmfor
morpholino:RNA duplexes before and after 365 nm UV
exposure. We evaluated EGFP-MO0, chordin-MO0, EGFP-
MO4, and chordin-MO4. As indicated in Table 1 (and Supporting
Information, Figure S1), the presence of four NPOM caging
groups on EGFP-MO4resulted in a ∼30 °C decrease in its Tm
as compared to the noncaged EGFP-MO0. This observation is
consistent with a 27 °C decrease in Tmwhen four mismatches
are included in a 25mer morpholino:RNA duplex.1Exposure
of EGFP-MO4to UV resulted in increasing its Tmto be virtually
the same as that of EGFP-MO0. Similar experiments demon-
strate that the presence of four caging groups on chordin-MO4
decreases the Tmof its morpholino:RNA duplex by ∼10 °C and
that exposure of chordin-MO4to UV results in increasing its
Tmto be nearly identical to that of chordin-MO0. This smaller
change in Tmis likely because the caging groups are clustered
near the ends of the chordin-MO4, leaving more contiguous
noncaged internal bases that might weakly base-pair with its
target RNA. However, the number of contiguous hybridizing
bases in chordin-MO4is fewer than the reported number
required for a morpholino to block gene function.35In addition,
gel shift experiments clearly demonstrate that the NPOM caging
groups on both EGFP-MO4and chordin-MO4effectively disrupt
the stable formation of morpholino:RNA duplexes (Supporting
Information, Figures S2 and S3); morpholino:RNA duplexes
are restored with as little as a 2 min exposure to UV (Supporting
Information, Figure S4). It must be noted that changes in UV
source, morpholino concentration, and biological context may
alter the efficiency of decaging.
Spatial Control of EGFP-MO4in Transfected Cells. EGFP is
an easily visualized protein and thus a highly useful target for
evaluating antisense agents.3,11,31To assess the photosensitivity
of EGFP-MO4in cells, COS-7 cells (a monkey kidney cell line)
were co-transfected with a plasmid encoding EGFP, a plasmid
encoding a control red fluorescent reporter (DsRed2), and either
EGFP-MO0or EGFP-MO4. A small region of the cell culture
plate was then briefly irradiated with UV light. The EGFP-
MO0effectively blocks EGFP translation, whereas EGFP-MO4
blocks EGFP translation only in the region which had been
exposed to UV light (Figure 1). This loss of EGFP is not the
result of UV quenching, since the cells were exposed to UV
light immediately after transfection, prior to EGFP production.
Neither EGFP-MO0nor uncaged EGFP-MO4resulted in a
complete knock-down of EGFP fluorescence in transfected cells;
this is likely the result of the high levels of EGFP mRNA
generated from the expression plasmid and/or relatively inef-
(29) Lusic, H.; Deiters, A. Synthesis 2006, 8, 2147–2150.
(30) Fox, C. M.; Reeves, M. D.; Weller, D. D. Method of synthesis of
morpholino oligomers. International patent application WO/2009/
064471, May 22, 2009.
(31) Schnapp, E.; Tanaka, E. M. DeV. Dyn. 2005, 232, 162–170.
(32) Fisher, S.; Amacher, S. L.; Halpern, M. E. DeVelopment 1997, 124,
(33) Hammerschmidt, M.; Pelegri, F.; Mullins, M. C.; Kane, D. A.; van
Eeden, F. J.; Granato, M.; Brand, M.; Furutani-Seiki, M.; Haffter, P.;
Heisenberg, C. P.; Jiang, Y. J.; Kelsh, R. N.; Odenthal, J.; Warga,
R. M.; Nusslein-Volhard, C. DeVelopment 1996, 123, 95–102.
(34) Bill, B. R.; Petzold, A. M.; Clark, K. J.; Schimmenti, L. A.; Ekker,
S. C. Zebrafish 2009, 6, 69–77.
(35) Summerton, J. E. Curr. Top. Med. Chem. 2007, 7, 651–660.
Scheme 2. Synthesis of the Caged Morpholino Monomer 5 and
Synthesized Morpholino Oligomer Sequencesa
aT* denotes the caged monomer 5. The NPOM caging group is shown
Table 1. Morpholino Melting Temperatures (Tm)a
82.4 ( 1.0
53.8 ( 1.1
84.5 ( 1.0
80.1 ( 0.6
70.8 ( 0.5
79.9 ( 1.1
aExperimentally determined melting temperatures (Tm) of duplexes of
noncaged and caged morpholinos with their targeted RNA sequences.
The distribution of the four caging groups has a substantial influence on
the melting temperature of the morpholino:RNA duplexes, as discovered
earlier for DNA:DNA duplexes.27When the caging groups are evenly
distributed along the length of the morpholino (e.g., EGFP-MO4), a
greater change in Tm is observed. Standard deviations were determined
from three independent experiments.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 44, 2010
Photochemical Gene Regulation
ficient transfection of morpholinos. Nevertheless, this experiment
demonstrates that, in agreement with previous observations
regarding disruption of oligonucleotide duplex formation25–28
and our Tmand gel-shift experiments, the installation of four
NPOM caging groups on a morpholino oligomer effectively
inhibits antisense activity in cultured mammalian cells and that
this activity can be readily restored using UV irradiation, even
in a spatially controlled fashion.
EGFP-MO4Regulation of EGFP Expression in Zebrafish
Embryos Is UV-Dependent. We then assessed whether the caged
EGFP-MO4is effective in regulating gene expression in ViVo.
We utilized the zebrafish embryo, a developmental model
organism with transparent embryos in which morpholinos are
routinely employed.3,34Embryos were co-injected at the 1-4-
cell stage with in Vitro transcribed, synthetic mRNA encoding
EGFP, EGFP-MO4and synthetic mRNA encoding the red
fluorescent protein mCherry (as a control). Figure 2 demonstrates
that, in the absence of UV irradiation, injected embryos express
both EGFP and mCherry and that UV exposure specifically
disrupts EGFP expression. Importantly, zebrafish embryos
exposed to UV light display a normal phenotype (also see Figure
4F, below).36As expected, embryos co-injected with EGFP
mRNA and EGFP-MO0display little or no EGFP expression,
regardless of UV exposure (data not shown). In addition, this
loss of EGFP expression is not the result of UV quenching, as
the embryos are exposed to UV light immediately after injection,
well prior to EGFP protein production. These results demonstrate
that NPOM-caged morpholinos can be used to achieve effective
light-controlled regulation of gene expression in live embryos.
EGFP-MO4Regulation of EGFP Expression in Xenopus Em-
bryos Is UV Dependent. To test the broader applicability of this
approach for species beyond zebrafish, the caged EGFP
morpholino was also investigated in Xenopus frog embryos.
Although not as optically transparent, caged compounds have
successfully been used in Xenopus.37,38One advantage of
Xenopus is the ability to target specific tissues by injecting
individual cells of the early embryo to limit the distribution of
injected reagents.39Xenopus embryos were co-injected in a
single cell at the 8-16-cell stage with synthetic EGFP mRNA,
EGFP-MO4, and synthetic mCherry mRNA. All injected
embryos were phenotypically normal, and Figure 3 clearly
demonstrates that, although mCherry expression was unaffected,
EGFP-MO4blocks EGFP production only after UV exposure.
As in zebrafish, this loss of EGFP expression cannot be the
result of UV quenching, as the embryos are exposed to UV
light immediately after injection, which is prior to EGFP
(36) Dong, Q.; Svoboda, K.; Tiersch, T. R.; Monroe, W. T. J. Photochem.
Photobiol. B 2007, 88, 137–146.
(37) Cambridge, S. B.; Geissler, D.; Calegari, F.; Anastassiadis, K.; Hasan,
M. T.; Stewart, A. F.; Huttner, W. B.; Hagen, V.; Bonhoeffer, T. Nat.
Methods 2009, 6, 527–531.
(38) Minden, J.; Namba, R.; Mergliano, J.; Cambridge, S. Sci. STKE 2000,
(39) Moody, S. A.; Kline, M. J. Anat. Embryol. (Berl.) 1990, 182, 347–
Figure 1. Spatial control of EGFP-MO4activity in mammalian cell culture.
In a 96-well plate, COS-7 cells were co-transfected with plasmids encoding
EGFP (pEGFP-N1) and DsRed2 (pDsRed2-N1) (A,B) with no morpholino,
(C,D) with EGFP-MO0, or (E,F) with EGFP-MO4. Cells transfected with
EGFP-MO4were briefly irradiated in a small circle at the center of the
well. Expression levels of EGFP and DsRed were evaluated 24 h after
transfection and irradiation, and representative images are shown on the
right and left, respectively. (G,H) A magnified view of (E,F) is shown.
Figure 2. Light-activation of EGFP-MO4in zebrafish embryos. Zebrafish
embryos at the 1-4-cell stage were microinjected with synthetic mRNA
encoding EGFP, EGFP-MO4(1:20 molar ratio), and synthetic mRNA
encoding mCherry and were (A,B) protected from light or (C,D) im-
mediately exposed to UV light. Expression levels of EGFP and mCherry
were evaluated 24 h after injection and irradiation, and representative images
of zebrafish tails are shown on the right and left, respectively. The
fluorescence observed in (D) is not derived from EGFP but is autofluores-
ence of the yolk sac (ys), as previously observed.37Photos of entire embryos
are shown in insets.
Figure 3. Light-activation of EGFP-MO4in Xenopus embryos. Xenopus
embryos were microinjected with synthetic mRNA encoding EGFP, EGFP-
MO4(1:30 molar ratio), and synthetic mRNA encoding mCherry and (A-C)
protected from light or (D-F) immediately exposed to UV light. Expression
levels of EGFP (right) and mCherry (middle) were evaluated at embryonic
stage 24 (∼1 day after injection) and are shown in comparison to bright-
field images (left).
15648 J. AM. CHEM. SOC. 9 VOL. 132, NO. 44, 2010
Deiters et al.
production. In conclusion, the direct caging approach can be
used for the light-regulation of morpholino activity in cell
culture, zebrafish embryos, and Xenopus embryos.
chordin-MO4Regulation of the chordin Gene in Zebrafish
Embryos Is UV Dependent. The above results show that our
NPOM-caged EGFP morpholino enables light-regulated control
of EGFP expression in cell culture and in ViVo. In order to
determine if NPOM-caged morpholinos can effectively regulate
endogenous genes, we chose to target the zebrafish chordin gene.
We selected a well-characterized morpholino sequence (chordin-
MO0) which, when injected into 1-4-cell zebrafish embyros,
induces a shrunken head and a ventralized tail by 24-28 h post
fertilization (hpf), characteristic of the chordin phenotype.3,32,33
We routinely observe >90% of chordin-MO0-injected embryos
displaying these phenotypes (data not shown). To evaluate the
light-dependent efficacy of a NPOM-caged chordin morpholino
(chordin-MO4), it was injected into 1-4-cell stage zebrafish
embryos which were then either protected from light or
immediately subjected to a brief exposure to UV. Embryos were
allowed to develop until 24-28 hpf, at which time their
phenotype was assessed, and the severity of the chordin
phenotype was scored as normal, mild, moderate, or severe
(Figure 4A-D). The moderate and severe phenotypes are
deformities known to be induced by disruption of the chordin
gene,3,32,33and the mild phenotype is characterized by a slight
blunting in the animal’s tail. We suggest that this latter
phenotype is caused by a small amount of partial decaging of
chordin-MO4through handling under ambient light or by the
stretch of contiguous noncaged bases within chordin-MO4
weakly associating with its target mRNA. Note that the mild
phenotype is not caused merely by the presence of a NPOM-
caged morpholino, as this phenotype is not observed when
embryos are injected with EGFP-MO4(data not shown). One
hundred percent of control embryos that are not injected with
a morpholino displayed a normal phenotype at 24-28 hpf,
independent of UV exposure, confirming that irradiation does
not induce any observable developmental defect (Figure 4E,F;
no UV, n ) 26; +UV, n ) 32). The observation that 99% of
chordin-MO4-injected embryos not exposed to UV displayed
normal or mild phenotypes at 24-28 hpf confirms that the
NPOM-caged morpholinos are stable in the zebrafish embryo
(Figure 4G; n ) 74 embryos). In contrast, 90% of chordin-
MO4-injected embryos that are immediately exposed to UV light
displayed moderate and severe phenotypes (Figure 4H; n ) 88
embryos), suggesting an efficient removal of the NPOM caging
groups in ViVo after UV exposure, activation of the morpholino
antisense agent, and subsequent knock-down of chordin function.
In order to determine if NPOM-caged morpholinos can be
employed for temporal control of gene expression, zebrafish
embryos were injected with chordin-MO4and irradiated at
successively later stages of development (Figure 4I). Interest-
ingly, exposure of chordin-MO4-injected embryos to UV at any
time point prior to the developmental stage of gastrulation (∼10
hpf) reliably induced a severe chordin mutant phenotype, while
disruption of chordin during mid- to late-stage gastrulation
elicited milder anomalies. These results are highly consistent
with the known expression patterns and the timing of the
dorsalizing function of chordin in the zebrafish embryo.3These
results confirm that the NPOM-caged morpholino technology
is applicable to the light-regulation of different genes and allows
for the temporal investigation of gene function in live zebrafish
We have developed a caged morpholino monomer and
incorporated it in the synthesis of two different morpholino
antisense reagents. These caged antisense agents were inactive
in cell culture, zebrafish embryos, and Xenopus embryos until
irradiated with UV light of 365 nm. Using these reagents, we
demonstrated photochemical gene silencing in mammalian cells
and in live aquatic animals and observed the expected embryonic
phenotypes. No detectable toxic effects of the irradiation or
the caging groups were observed. The developed direct caging
approach can be easily applied to any morpholino oligomer,
Figure 4. Light-activation of chordin-MO4in zebrafish embryos. (A-H)
Zebrafish embryos were microinjected with chordin-MO4during the 1-4-
cell stage and either protected from light or immediately irradiated with
UV light, and their phenotype was assessed 24-28 h post fertilization (hpf).
Phenotypes include (A) normal, (B) mild, (C) moderate, and (D) severe.
(E,F) Uninjected control embryos display a normal phenotype regardless
of UV exposure. (G) Embryos injected with chordin-MO4and not exposed
to UV light of 365 nm display normal or mild phenotypes. (H) Embryos
injected with chordin-MO4and immediately exposed to UV light display
moderate and severe phenotypes. (I) Embryos were injected with chordin-
MO4as described above and divided into groups that were irradiated at
various time points after fertilization, and their phenotype was assessed at
24-28 hpf. Note that light-activation of chordin-MO4during or after the
developmental stage of gastrulation (∼10 hpf) fails to generate the severe
J. AM. CHEM. SOC. 9 VOL. 132, NO. 44, 2010
Photochemical Gene Regulation
since the straightforward design only requires insertion of Download full-text
caged building blocks in the oligomer synthesis. We are
currently developing the chemistry to synthesize caged
morpholino monomers for the other three bases, which will
provide greater flexibility in the design of caged morpholino
oligonucleotides. It is likely that this methodology will find
broad application in the cell and developmental biology
research community, since it is generally applicable to the
spatial and temporal regulation of gene function in multiple
Acknowledgment. We thank Gene Tools, LLC for synthesiz-
ing caged morpholino oligomers from our caged building block,
Syntrix Biosystems, Inc. for fruitful discussions, and R. Read
Tull for assistance with Xenopus microinjections. Financial
support from the National Institutes of Health (R01GM079114,
R01DK085300, and R43GM088964) is acknowledged. A.D. is
the recipient of a Beckman Young Investigator Award and a
Cottrell Scholar Award.
Supporting Information Available: Melting curves for caged
morpholinos and morpholino gel shift assay results. This
material is available free of charge via the Internet at http://
15650J. AM. CHEM. SOC. 9 VOL. 132, NO. 44, 2010
Deiters et al.