Cyclic caged morpholinos: Conformationally gated probes of embryonic gene function

Article abstract of DOI:10.1002/anie.201201690

Feeling a bit cagey: Morpholino-based antisense reagents have been caged through oligonucleotide cyclization (see scheme), enabling photocontrol of gene expression in zebrafish embryos and larvae. Using these reagents, the timing of exocrine cell fate commitment in the developing pancreas has been examined. Copyright

Full text of DOI:10.1002/anie.201201690

Angewandte
Chemie
DOI: 10.1002/anie.201201690
Gene Silencing
Cyclic Caged Morpholinos: Conformationally Gated Probes of
Embryonic Gene Function**
Sayumi Yamazoe, Ilya A. Shestopalov, Elayne Provost, Steven D. Leach, and James K. Chen*
Deciphering the molecular mechanisms that underlie
embryogenesis requires an ability to alter gene function
with spatial and temporal precision. Synthetic reagents can be
valuable tools in this discovery process, especially in model
organisms currently intractable to targeted genomic changes.
In particular, morpholino-based antisense oligonucleotides
(MOs) have been widely used to inhibit gene expression in
metazoans that develop ex utero (Figure 1a),[1–4] and caged
versions of these reagents (cMOs) can enable conditional
gene silencing, by using targeted illumination.[5–9] Composed
of morpholino-based nucleosides and a phosphorodiamidate
backbone, these nuclease-resistant probes are typically
injected into zygotes as 25-base oligomers, after which they
hybridize to complementary RNAs and disrupt splicing or
translation. While cMOs can provide important new insights
into embryonic gene function, their utility has been hindered
by their empirical design, synthetic complexity, in vivo
instability, reliance on complementary inhibitors, and/or use
of multiple caging groups. Herein, we describe next-gener-
ation cMOs that overcome these limitations. We demonstrate
that cyclic cMOs are effective reverse-genetic tools in zebra-
fish embryos, and we use these reagents to examine the timing
of exocrine fate commitment in the developing pancreas.
We previously developed hairpin cMOs that can be
activated with either ultraviolet (UV; 360 nm) or two-
photon irradiation (Figure 1b).[5,10] The RNA-targeting MO
is tethered to a shorter, complementary MO inhibitor through
a dimethoxynitrobenzyl (DMNB)- or bromohydroxyquino-
line (BHQ)-based linker, thereby forming a hairpin structure
that is resistant to RNA hybridization. Photocleavage of the
Figure 1. MOs and their caged derivatives. a) Comparison of DNA/
RNA and MO chemical formulas. b) Hairpin caged MO. c) MO/caged
linker separates the two oligonucleotides, allowing the 25-
base MO to interact with its RNA target. Hairpin cMOs have
been used to conditionally inactivate the expression of several
zebrafish genes, including no tail-a (ntla), floating head (flh),
oligonucleotide duplex. d) MO with caged bases. e) Cyclic caged MO.
RNA-targeting MOs are shown in black, inhibitory oligonucleotides in
gray, and caging groups in white.
heart of glass (heg), and ETS1-related protein (etsrp).[5,10] We
have also combined ntla cMOs, with caged fluorophores,
fluorescence-activated cell sorting, and microarray technolo-
gies to dynamically profile the Ntla-dependent transcrip-
tome.[11] Despite these successes, hairpin cMOs have limita-
tions. Their design involves careful optimization of the
thermodyamics of inhibitor binding, their purification
requires high-performance liquid chromatography (HPLC),
and the inhibitory oligonucleotide can increase MO toxicity.
Other oligonucleotide caging strategies have been devel-
oped, each with its own advantages and disadvantages. For
example, chimeras of peptide nucleic acid (PNA) and 2’-O-
methyl (2’-OMe) RNA that form hairpins have been used to
conditionally inhibit gene function,[6] although the photo-
released 2’-OMe RNAs may be toxic to embryos.[7] Duplexes
composed of the targeting MO and two complementary,
[*] Dr. S. Yamazoe, Dr. I. A. Shestopalov, Prof. Dr. J. K. Chen
Departments of Chemical and Systems Biology and Developmental
Biology, Stanford University School of Medicine
269 Campus Drive, CCSR 3155, Stanford, CA 94305 (USA)
E-mail: jameschen@stanford.edu
Homepage: http://chen.stanford.edu
Dr. E. Provost, Prof. Dr. S. D. Leach
Departments of Surgery, Oncology, and Cell Biology, McKusick-
Nathans Institute of Genetic Medicine, Johns Hopkins University
733 N. Broadway, BRB 471, Baltimore, MD 21205 (USA)
[**] We thank A. Puri and I. Hinkson for their assistance. This work was
supported by the NIH (R01 GM087292 and DP1 OD003792 to
J.K.C.; R01 DK061215 to S.D.L.).
Supporting information for this article (experimental details) is
available on the WWW under http://dx.doi.org/10.1002/anie.
201201690.
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tandem oligonucleotides connected by a nitrobenzyl-based
linker have also been reported (Figure 1c).[7,9] While these
duplexes are easier to prepare than hairpin reagents, they
release two potentially cytotoxic oligonucleotides upon
photocleavage and can be less stable in vivo. Finally, MOs
containing four nitropiperonyloxymethyl-caged thymine
bases have been synthesized, bypassing the need for an
inhibitory oligomer (Figure 1d).[8] Activating these reagents,
however, requires the photolysis of multiple caging groups
per oligonucleotide, which can be difficult to achieve without
damaging levels of UV irradiation.
To overcome these limitations, we sought to develop
cMOs that are easier to synthesize, rely on a single caging
group, and do not involve a complementary inhibitor.
Because oligonucleotide duplexes have limited tolerance for
bending,[12] we hypothesized that cyclized MOs with a photo-
cleavable linker could be used for conditional gene silencing
(Figure 1e). A recent report that circular DNA oligonucleo-
tides bind inefficiently to their complementary RNAs in vitro
lends further support to this concept.[13]
a linear ntla MO recreates the ntla mutant phenotype at
a dose of 1 ng per embryo,[5,10] we injected an equivalent
amount of the photocleavable cyclic ntla cMO into zygotes
and irradiated a subset of the embryos with UV light for ten
seconds at 3 h post fertilization (hpf).[15] The embryos were
cultured until 24 hpf, at which point their phenotypes were
scored according to four morphological classes (Figure 2a).[10]
The cyclic ntla cMO was found to be comparable to the
hairpin cMO reagent in terms of efficacy, if not slightly better,
with approximately 75% (n = 24) of the cyclic cMO-injected
embryos exhibiting no developmental defects in the absence
of UV light and 90% (n = 20) showing a complete ntla mutant
phenotype following UV irradiation (Figure 2b). Western
blot analysis of 10-hpf zebrafish confirmed the UV light-
dependent loss of Ntla protein in cyclic ntla cMO-injected
embryos (Figure 2c). The cyclic ntla cMO could also be used
to knockdown ntla function in a spatially restricted manner;
photoactivation of the reagent within the embryonic shield at
6 hpf converted notochord progenitors into medial floor plate
cells, as has been reported with hairpin ntla cMOs (Fig-
ure 2d).[5,11]
We tested this new caging strategy by targeting ntla, the
zebrafish orthologue of mammalian Brachyury.[14–16] Loss-of-
function phenotypes for this T-box transcription factor
include ablation of the notochord and posterior mesoderm,
ectopic medial floor plate cells, and somite mispatterning. We
synthesized a DMNB-functionalized linker with an N-
hydroxysuccinimide ester and a chloroacetamide group 7
(Scheme 1) and reacted it with a 25-base ntla MO 8
(Supporting Information, Table S1) containing 5’-amine and
3’-alkyl disulfide modifications. Reduction of the linear MO–
linker intermediate 9 yielded the free thiol, which sponta-
neously reacted with the chloroacetamide to give the desired
macrocycle 10. Liquid chromatography–mass spectrometry
(LC–MS) analysis indicated that the macrocyclization reac-
tion went to completion, and the final product was purified by
gel filtration. The total yield for the 25-base cyclic ntla cMO
synthesis starting with the linker and the targeting MO was
80%, approximately a tenfold improvement over our hairpin
cMO protocol.[10]
In principle, the basal activity of cyclic cMOs should
decrease with macrocycle size, as bending within the smaller
MO macrocycles will be more acute. To test this, we
synthesized nonphotocleavable cyclic MOs corresponding to
21-, 23-, and 25-base oligonucleotides that target the ntla
sequence (Table S1 and Scheme S1). The cyclic MOs or the
corresponding linear MOs were then mixed with equimolar
amounts of 25-base complementary RNA (Table S1), and
melting curves were recorded (Table 1). Although these
in vitro assays cannot recreate the complexity of MO/RNA
interactions in vivo, the thermodynamic insights they provide
can be informative.[10] As expected, the cyclic MOs exhibited
reduced affinities for RNA compared to the linear MOs, and
greater differences in Tm and DG values were recorded as MO
length decreased.
We next tested photoactivatable versions of these cyclic
MOs and their linear counterparts in zebrafish embryos. As
before, we UV-irradiated a subset of the cMO-injected
embryos at 3 hpf and evaluated their morphological pheno-
types at 24 hpf and Ntla protein levels at 10 hpf. Although the
We next investigated the efficacy of the cyclic ntla cMO in
zebrafish embryos. Because we had previously shown that the
Scheme 1. Cyclic ntla cMO synthesis. a) allyltrimethylsilane, TiCl4, CH2Cl2, 98%; b) O3, MeOH; c) NaBH4, MeOH, 74% over 2 steps; d) tosyl
chloride, pyridine, 63%; e) methylamine, tetrahydrofuran (THF), 87%; f) methyladipoyl chloride, N,N-diisopropylethylamine (DIPEA), CH2Cl2,
57%; g) 1,1’-carbonyldiimidazole, CH2Cl2; h) ethylenediamine, CH2Cl2; i) 2-chloroacetyl chloride, Et3N, CH2Cl2, 54% over 3 steps; j) LiOH, THF,
H2O; k) N,N’-disuccinimidyl carbonate, pyridine, CH3CN, 68% over 2 steps; l) 5’-amine, 3’-alkyl disulfide ntla MO (21-, 23-, or 25-base), 0.1m
Na2B4O7 pH 8.5, dimethylsulfoxide (DMSO), 69-95%; m) tris(2-carboxyethyl)phosphine (TCEP) resin, 0.1m Tris-HCl buffer, pH 8.4, 84–92%.
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cyclic ntla cMOs exhibited less basal activity as they
decreased in size, they also had an equivalent loss of efficacy
upon photocleavage (Figure 2b–c). Linear versions of the 21-
and 23-base reagents were similarly less potent, indicating
that this activity reduction is due to diminished MO/RNA
interactions. Thus, for this method, the 25-base cyclic cMO
provides the best dynamic range of caged and uncaged
activities.
To test the generality of cyclic cMOs, we synthesized a 25-
base cMO targeting pancreas transcription factor 1 alpha
(ptf1a; Table S1), which is required for exocrine cell differ-
entiation in the developing pancreas.[17–19] Ptf1a is selectively
expressed in pancreatic exocrine cells, and their formation
can therefore be directly visualized in transgenic zebrafish
that express enhanced green fluorescent protein (eGFP)
under the control of ptf1a promoter-derived cis-regulatory
elements (Tg(ptf1a:eGFP) zebrafish).[20] We injected
Tg(ptf1a:eGFP) zygotes with the cyclic ptf1a cMO at a dose
of 2 ng per embryo and UV irradiated a subset of the embryos
at 3 hpf for ten seconds. Another set of Tg(ptf1a:eGFP)
zygotes was injected with an equivalent amount of a hairpin
ptf1a cMO that was designed according to previously
established thermodynamic parameters.[10] The intensity of
eGFP fluorescence within the pancreatic field at three days
post fertilization (dpf) was then scored for each experimental
condition (Figure 3a).
The hairpin ptf1a cMO partially impeded pancreatic
development even without photoactivation, possibly because
of linker degradation or the ability of the hairpin to
interconvert between stem-loop and linear forms during the
three-day period (Figure 3b). In contrast, the majority of
Tg(ptf1a:eGFP) zygotes injected with the cyclic ptf1a cMO
developed normally in the absence of UV light (76%, n = 21)
but failed to form an exocrine pancreas upon UV irradiation
(95%, n = 21; Figure 3b). Endocrine pancreas development
was not affected, as gauged by whole-mount in situ hybrid-
ization with an antisense probe for insulin (Figure S1). These
findings demonstrate the general efficacy of cyclic cMOs and
suggest that they might be better probes of later embryo-
logical processes than hairpin-based cMOs.
To conclude these studies, we used the cyclic cMO
technology to examine the temporal requirements of ptf1a
function for exocrine pancreas formation. We linearized the
cyclic ptf1a cMO in zebrafish with UV irradiation conditions
optimized for various developmental stages (3–52 hpf; Fig-
ure S2). We then assessed exocrine tissue development at
3 dpf by whole-mount in situ hybridization with antisense
trypsin (Figure 4) and ptf1a (Figure S1) probes. The tran-
scription levels of these exocrine markers followed similar
trends, with a substantial decrease in transcript levels upon
UV irradiation up to 44 hpf. Linearization of the ptf1a cMO at
48 and 52 hpf, however, had only minor effects on their
expression. Assuming that cyclic ptf1a cMO efficacy is not lost
during these studies (MOs can persist in zebrafish for up to
Figure 2. Comparison of cyclic and hairpin ntla cMOs. a) Classification
of ntla loss-of-function phenotypes. 24-hpf embryos are shown, ante-
rior to the left and dorsal at the top. Scale bar=200 mm. b) Phenotypic
distributions for embryos injected with the indicated oligonucleotides
and either cultured in the dark or irradiated with UV light at 3 hpf.
Between 13 and 24 embryos were analyzed for each condition. c) Ntla
protein levels in 10-hpf embryos subjected to the indicated conditions.
Data are the average of at least four experiments (6 embryos per
condition) Æstandard error of the mean (s.e.m.), normalized with
respect to b-actin. d) Phenotypes observed in embryos injected with
caged fluorescein-conjugated dextran (cFD) Æthe cyclic ntla cMO and
irradiated within the shield at 6 hpf. Trunk regions of 36-hpf embryos
are shown as overlays of differential interference contrast (DIC) and
fluorescence micrographs (anterior left, dorsal up). Scale
bars=50 mm.
Table 1: Thermodynamic parameters of MO/RNA duplexes.
MO oligomer[a]
Tm [8C] linear[b]
DG [kcalmolÀ1] linear[b]
Tm [8C] cyclic[b]
DG [kcalmolÀ1] cyclic[b]
DTm [8C][c]
DDG [kcalmolÀ1 [c]
]
25-base
23-base
21-base
78.3Æ0.9
77.0Æ2.0
74.2Æ1.3
À22.1Æ2.3
À20.4Æ2.0
À19.7Æ1.1
75.2Æ1.2
68.7Æ1.3
60.2Æ1.3
À18.5Æ0.7
À15.9Æ1.0
À14.0Æ0.4
3.1Æ0.7
8.3Æ0.9
14.0Æ1.1
3.6Æ0.9
4.5Æ0.9
5.7Æ0.7
[a] See Table S1 for sequence information. [b] Average values of 3–12 experiments Æstandard deviation (s.d.). [c] Absolute differences between linear
MO/RNA and cyclic MO/RNA duplexes Æstandard error (s.e.).
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from commercially available oligonucleotides and linker 7,
and they can be purified without HPLC. They also bypass the
need for inhibitory oligomers and multiple caging groups. Our
results further suggest that these cyclic cMOs might be better
than current cMO technologies for studying organogenesis
and other late-stage developmental processes. These attri-
butes should facilitate the implementation of cMOs by the
developmental biology community and enhance future efforts
to decipher embryonic gene function.
Received: March 1, 2012
Revised: April 24, 2012
Published online: && &&, &&&&
Keywords: antisense oligonucleotides · developmental biology ·
.
gene expression · oligonucleotides · zebrafish
Figure 3. Comparison of cyclic and hairpin ptf1a cMOs. a) Classifica-
tion of ptf1a loss-of-function phenotypes according to pancreas-spe-
cific eGFP expression in Tg(ptf1a:eGFP) zebrafish. Fluorescence micro-
graphs of 3-dpf larvae are shown, anterior to the right and dorsal at
the top. Scale bar=100 mm. Ctrl=control without oligonucleotide
injected. b) Phenotypic distributions for embryos injected with the
indicated reagents and either cultured in the dark or UV irradiated at
3 hpf. Between 21 and 31 embryos were analyzed for each condition.
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Figure 4. Temporal analysis of ptf1a-dependent exocrine pancreas
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Angew. Chem. Int. Ed. 2012, 51, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Gene Silencing
Feeling a bit cagey: Morpholino-based
antisense reagents have been caged
through oligonucleotide cyclization (see
scheme), enabling photocontrol of gene
expression in zebrafish embryos and
larvae. Using these reagents, the timing
of exocrine cell fate commitment in the
developing pancreas has been examined.
S. Yamazoe, I. A. Shestopalov, E. Provost,
S. D. Leach, J. K. Chen*
&&&& — &&&&
Cyclic Caged Morpholinos:
Conformationally Gated Probes of
Embryonic Gene Function
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!