Combinatorial control of gene function with wavelength-selective caged morpholinos

Article abstract of DOI:10.1016/bs.mie.2019.04.007

Caged morpholino oligonucleotides (cMOs) are useful research tools in developmental biology because they allow spatiotemporal control of gene expression in whole organisms. While cMOs are usually triggered by light of a single wavelength, the introduction of spectrally distinct chromophores can enable combinatorial regulation of multiple genes. This chapter describes the general principles and methods of wavelength-selective cMO design and synthesis from commercially available reagents. Synthetic protocols for the linkers and the two-step cMO assembly are described in detail, as well as the microinjection and photoactivation techniques. Following these protocols, spectrally separated cyclic cMOs for multiple genes of interest can be prepared, enabling their inhibition in zebrafish embryos and other animal models.

Full text of DOI:10.1016/bs.mie.2019.04.007

ARTICLE IN PRESS
Combinatorial control of gene
function with wavelength-
selective caged morpholinos
Sankha Pattanayaka, Luis Angel Vázquez-Maldonadob,
Alexander Deitersb,*, James K. Chena,c,
*
aDepartment of Chemical and Systems Biology, Stanford University School of Medicine, Stanford,
CA, United States
bDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA, United States
cDepartment of Developmental Biology, Stanford University School of Medicine, Stanford, CA, United States
*Corresponding authors: e-mail address: deiters@pitt.edu; jameschen@stanford.edu
Contents
1. Introduction
2
4
4
5
6
2. Design and synthesis of cyclic cMOs
2.1 Design of cyclic cMOs
2.2 Design of wavelength-selective cMOs
2.3 Methods
3. Microinjection of cMOs into zebrafish embryos
4. Photoactivation of cMOs
5. Conclusion
Acknowledgements
References
16
16
18
19
19
Abstract
Caged morpholino oligonucleotides (cMOs) are useful research tools in developmental
biology because they allow spatiotemporal control of gene expression in whole organ-
isms. While cMOs are usually triggered by light of a single wavelength, the introduction
of spectrally distinct chromophores can enable combinatorial regulation of multiple
genes. This chapter describes the general principles and methods of wavelength-
selective cMO design and synthesis from commercially available reagents. Synthetic
protocols for the linkers and the two-step cMO assembly are described in detail, as well
as the microinjection and photoactivation techniques. Following these protocols,
spectrally separated cyclic cMOs for multiple genes of interest can be prepared,
enabling their inhibition in zebrafish embryos and other animal models.
#
Methods in Enzymology
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2019.04.007
2019 Elsevier Inc.
All rights reserved.
1

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Sankha Pattanayak et al.
1. Introduction
Embryonic development is controlled by a complex network of
genetic programs that are precisely regulated to create tissues and organs
(Perrimon, Pitsouli, & Shilo, 2012). One of the major objectives of modern
developmental biology is to understand how these genes act in space and
time to coordinate embryonic patterning. In recent years, genetic technol-
ogies such as antisense oligonucleotides, RNA interference, and genome-
editing tools have enabled new ways to dissect embryonic pathways at
the transcriptional or translational level (Housden et al., 2017). In particular,
morpholino antisense oligonucleotides (MOs) have been widely used
to inhibit gene expression in a variety of model systems including zebrafish,
Xenopus, and chick embryos (Ekker & Larson, 2001; Nasevicius & Ekker,
2000). MOs are chemically modified DNA derivatives that are composed
of nucleobase-appended morpholine rings connected with neutral pho-
sphorodiamidate linkages (Fig. 1A) (Bhadra, Pattanayak, & Sinha, 2015;
Summerton & Weller, 1997). These oligonucleotides are resistant to cellular
nucleases due to their unnatural backbones and persist in vivo for up to 4 days
(Bill, Petzold, Clark, Schimmenti, & Ekker, 2009). MOs are typically
25-base sequences, designed to block translation start sites or RNA splice
junctions, and the resulting loss-of-function phenotypes can be rapidly
A
B
Hairpin cMO
MO
Duplex cMO
MO
O
Base
O
Base
X
O
Inhibitor Linker
Inhibitor
Inhibitor
O
O
N
P
O
Nucleobase-caged cMO
MO
Cyclic cMO
O
P
O
N
O
O
Base
O
Base
MO
O
O
N
O
X
O
O
O
DNA: X = H
RNA: X = OH
NO2
NO2
NO2
Fig. 1 Morpholinos and their caged versions. (A) DNA/RNA and MO chemical structures.
(B) cMO design strategies showing hairpin, duplex, nucleobase-caged, and cyclic cMOs.
The photolabile moiety has been indicated as diamond shapes. Adapted with permission
from Yamazoe, S., Shestopalov, I. A., Provost, E., Leach, S. D., & Chen, J. K. (2012). Cyclic
caged morpholinos: Conformationally gated probes of embryonic gene function.
Angewandte Chemie, International Edition 51(28), 6908–6911.

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3
generated in a non-Mendelian manner (Corey & Abrams, 2001). While it
may be required to alter the MO length for a specific application, shorter
sequences can have diminished efficacy and longer sequences can have
decreased specificity. When MOs are microinjected into early-stage
embryos (typically before four-cell stage in zebrafish) they uniformly distrib-
ute throughout the organism and constitutively inhibit protein synthesis.
While MOs are routinely used to interrogate gene functions in zebrafish
and other organisms, their constitutive activity renders these reagents inad-
equate for studying genes that have pleiotropic functions. Since gene expres-
sion is inhibited immediately after microinjection, conventional MOs do
not allow any spatiotemporal control. Moreover, silencing essential genes
in early development can cause embryonic lethality, making them challeng-
ing to study (Timme-Laragy, Karchner, & Hahn, 2012). To overcome these
limitations, we and others have recently developed caged morpholinos
(cMOs) that remain inactive in vivo until activated by light of the appropriate
wavelength. The first generation of cMOs were composed of an RNA-
targeting MO, tethered with a short complementary MO inhibitor through
a photocleavable linker, thereby forming a hairpin structure (Fig. 1B). The
hairpin MOs contained dimethoxynitrobenzyl (DMNB)- or bro-
mohydroxyquinoline (BHQ)-based linkers, which can be cleaved with
ultraviolet (365nm) or two-photon irradiation, respectively (Ouyang
et al., 2009; Shestopalov, Sinha, & Chen, 2007). Several other MO-caging
strategies also have been developed. MOs have been caged by duplexing the
antisense oligonucleotide with an inhibitory strand (Fig. 1B) that contains an
internal photolabile nitrobenzyl-based linker (Tallafuss et al., 2012;
Tomasini, Schuler, Zebala, & Mayer, 2009). Alternatively, cMOs were gen-
erated by incorporating multiple nucleobase-caged morpholino monomers
during solid-phase synthesis (Fig. 1B) (Deiters et al., 2010; Liu & Deiters,
2014). The most recent strategy to generate cMOs utilizes restricting linear
morpholinos into a closed circular configuration, thereby preventing target
mRNA hybridizations (Fig. 1B) (Yamazoe, Shestopalov, Provost, Leach, &
Chen, 2012). The cyclic cMOs provide several advantages over previous
hairpin, duplex, and nucleobase-caged versions because they are modular
in linker design, can be synthesized and purified easily, do not use auxiliary
nucleotides, and utilize a single photolabile group. The first generation of
cyclic cMOs utilized DMNB-based linkers that are readily cleaved by
ultraviolet light (Yamazoe et al., 2012).
The application of cMOs in optically transparent model organisms like
zebrafish has provided new insights into gene functions during embryonic

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Sankha Pattanayak et al.
development. For example, we have used hairpin cMOs to optically inhibit
the expression of mesodermal genes in zebrafish, including no tail-a (tbxta),
heart of glass (heg), and floating head (flh) (Ouyang et al., 2009). The
nucleobase-caged cMOs have been used inhibit zebrafish chordin expression
(Deiters et al., 2010). Similarly, the cyclic cMOs have been used to perturb
genes like tbxta, t-box 16/spadetail (tbx16), pancreas transcription factor 1 alpha
(ptf1a), ets variant gene 2 (etv2), and focal adhesion kinase (fak) (Gutzman et al.,
2018; Yamazoe et al., 2012). We also have integrated cMOs with photo-
activatable lineage tracers, fluorescence-activated cell sorting and mRNA
profiling to dynamically elucidate Tbxta- and Tbx16-regulated transcriptomes
at different stages of mesoderm development (Payumo, McQuade, Walker,
Yamazoe, & Chen, 2016; Shestopalov, Pitt, & Chen, 2012).
In these examples, cMOs were used to inhibit individual genes since
their reliance on ultraviolet light-sensitive caging groups precludes sequen-
tial control of multiple genes. However, such a capability is highly desirable
since cMOs can then be used to decipher the combinatorial activities of two
or more genes in a more complex biological network. To achieve this goal,
we recently reported the development of spectrally separated cMOs that can
be activated by illumination with distinct wavelengths of light. In this chap-
ter, we describe the detailed methods for cyclic cMO preparation based on
our published article (Yamazoe, Liu, McQuade, Deiters, & Chen, 2014).
2. Design and synthesis of cyclic cMOs
2.1 Design of cyclic cMOs
As described in Section 1, the general strategy to design cMOs is to restrict
their hybridization with the target mRNA until being activated by light.
The basis of cyclic cMOs is the concept that oligonucleotide duplex forma-
tion has limited tolerance to local curvature of the hybridizing strands
(Ramstein & Lavery, 1988). Our first generation of cyclic cMOs were pre-
pared by connecting the two ends of a linear MO 1 using a DMNB-based
photolabile linker 2 (Fig. 2) (Yamazoe et al., 2012). The bifunctional linker
was synthesized with an amine-reactive N-hydroxysuccinimide (NHS) ester
functionality and a thiol-reactive chloroacetamide group. The 25-base MO
was designed to have corresponding 50-amine and 30-alkyl disulfide end-
modifications. To prepare the cyclic cMO, the linear MO was first
completely converted to the linker-MO conjugate through the NHS
ester-amine reaction using an excess amount of the linker, while keeping
the thiol-functionality protected as the disulfide group. Subsequent disulfide

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Combinatorial control of gene function
5
O
O
O
O
NO2
N
O
5’
3’
a
H
N
N
+
H2N
SSR
Cl
N
O
O
H
O
O
O
1
2
MO
DMNB linker
O
O
O
NO2
O
b
H
N
N
Cl
N
O
O
SSR
H
O
O
3
4
Fig. 2 Synthesis of cyclic cMOs. The DMNB linker is shown as an example. (a) 0.1M
Na2B4O7, pH 8.5, DMSO, 69–95%; (b) tris(2-carboxyethyl)phosphine, 0.1M Tris-HCl
buffer, pH 8.4, 84–92%. Adapted with permission from Yamazoe, S., Shestopalov, I. A.,
Provost, E., Leach, S. D., & Chen, J. K. (2012). Cyclic caged morpholinos: Conformationally
gated probes of embryonic gene function. Angewandte Chemie, International Edition
51(28), 6908–6911.
reduction generates the free thiol that reacts spontaneously with the
chloroacetamide to give the desired macrocycle in high yields (Fig. 2).
We tested the strategy by targeting Tbxta, the zebrafish ortholog of mam-
malian Brachyury (Yamazoe et al., 2012). Tbxta deficiency is characterized by
loss of notochord and posterior mesoderm, abnormal medial floor plate and
somitemispatterning.WhenthecyclictbxtacMOswereinjectedintozebrafish
zygotes at a dose of 1ng/embryo and cultured in the dark, 75% of the embryos
exhibited no developmental defects at 24h post fertilization (hpf ). However,
when the cyclic cMO was uncaged through illumination with ultraviolet light
at 3.5 hpf, 90% of the embryos showed complete tbxta-mutant phenotype.
2.2 Design of wavelength-selective cMOs
Since the design of cMOs is modular, they can be synthesized with a variety
of linkers and chromophores. To prepare spectrally differentiated cMOs, we
chose 2-nitrobenzyl (NB) and [7-(diethylamino)coumarin-4-yl]methyl
(DEACM) chromophore-based linkers (Fig. 3A) because of their photo-
deprotection efficiencies and minimal spectral overlap. Swapping the
DMNB moiety with the NB chromophores blue-shifts the absorption max-
imum and makes it less susceptible to photolysis at wavelengths >400nm. In
contrast, the DEACM chromophore efficiently photolyzes at wavelengths
over 400nm, thus would allow us to orthogonally control the cleavage of
these two linkers (Fig. 3B) (Yamazoe et al., 2014).

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Sankha Pattanayak et al.
O
O
NEt2
A
O
NO2
O
O
O
O
O
H
H
N
N
O
N
Cl
N
O
N
Cl
N
O
O
N
O
O
H
H
O
O
O
O
5
6
DEACM linker
NB linker
B
470 nm
365 nm
Fig. 3 Sequential and wavelength-activation of cyclic cMOs. (A) Chemical structures
of the NB linker and the DEACM linker. (B) Concept of sequential activation of cMOs,
prepared by using these two linkers. Adapted with permission from Yamazoe, S.,
Liu, Q., McQuade, L. E., Deiters, A., & Chen, J. K. (2014). Sequential gene silencing using
wavelength-selective caged morpholino oligonucleotides. Angewandte Chemie, International
Edition 53(38), 10114–10118.
The cyclic cMOs were prepared by using procedures analogous to our
first generation of DMNB-containing linkers (Fig. 2) (Yamazoe et al.,
2012). We have systematically evaluated the validity of this approach
in zebrafish embryos using the tbxta cMO as a test system (Yamazoe
et al., 2014). We found that when the embryos were illuminated with
365-nm light at 3.5 hpf, 86% of NB cMO-injected embryos exhibited
strong tbxta mutant phenotypes, whereas DEACM cMO-injected
embryos exhibited only variable defects. Conversely, upon 470-nm light
illumination, 95% of DEACM cMO-injected embryos showed complete
loss of tbxta functions, while 91% of NB cMO-injected embryos devel-
oped normally.
We further tested the applicability of the approach in two additional reg-
ulators of zebrafish mesoderm development, the Tbx16 and Flh (Yamazoe
et al., 2014). Our results showed that NB and DEACM cyclic cMOs could
be used in combination to sequentially silence genes.
2.3 Methods
2.3.1 Synthesis of the photocleavable linkers
2.3.1.1 Materials and instruments
1. All reactions were carried out in flame-dried glassware and stirred mag-
netically under a nitrogen atmosphere, unless stated otherwise.

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2. Reactions were monitored by thin layer chromatography (TLC), using
glass-backed silica gel 60F254 (Merck or Sorbent Technologies, 250-μm
thickness), and a compact ultraviolet lamp.
3. Solvents: tetrahydrofuran (THF), dioxane, and toluene were distilled
from sodium/benzophenone ketyl prior to use; dichloromethane
(DCM), dimethylformamide (DMF), acetonitrile, and methanol were
˚
distilled from calcium hydride and stored over 4A molecular sieves.
4. Reagents obtained from commercial sources were stored under nitrogen
and used directly without further purification.
5. Yields refer to chromatographically and spectroscopically pure
compounds.
6. Flash chromatography was performed with silica gel (EM Science silica
˚
˚
gel 60A, 70–230 mesh; Sorbtech silica gel 60A, 230–400 mesh) as a
stationary phase.
1
7. The H NMR and 13C NMR spectra were recorded on a 300 or
400MHz Varian NMR spectrometer, and chemical shifts are in δ
units (ppm).
8. Electrospray ionization (ESI) mass spectra were obtained using a Micro-
mass ZQ single quadrupole liquid chromatography-mass spectrometer
(LC-MS) or a Micromass Q-TOF hybrid quadrupole LC-MS.
2.3.1.2 Preparation of the DEACM Linker 6
The DEACM linker 6 can be synthesized from commercially-available
7-(diethylamino)-4-methyl-2H-chromen-2-one (7) in 10 steps (Fig. 4).
Briefly, coumarin 7 was oxidized with selenium dioxide and subsequently
allylated to produce the alcohol 9, which was protected with a tert-
butyldimethylsilyl (TBS) group to provide the alkene 10. The alkene 10
was subjected to hydroboration to give the alcohol 11. The chloroacetamide
group was then installed in three steps to obtain 12, followed by the removal
of the silyl group with tetra-n-butylammonium fluoride (TBAF), delivering
the secondary alcohol 13. Finally, the hydroxyl group of 13 was activated by
disuccinimidyl carbonate (DSC) to yield the linker 6. A detailed protocol for
the synthesis of 6 can be found below.
2.3.1.2.1 7-(Diethylamino)-2-oxo-2H-chromene-4-carbaldehyde (2)
1. Add selenium dioxide (3.32g, 30.0mmol) to commercially-available
7-(diethylamino)-4-methyl-2H-chromen-2-one (7, 4.64g, 20.0mmol)
dissolved in anhydrous dioxane (120mL) in a round-bottom flask.
2. Attach a Liebig condenser to the round-bottom flask.

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Sankha Pattanayak et al.
RO
O
RO
OH
a
d–e
b
Et2N
O
O
Et2N
O
O
Et2N
O
O
Et2N
O
O
7
8
9, R = H
11
c
10, R = TBS
O
O
N
O
O
O
H
N
H
RO
O
N
O
N
Cl
O
N
Cl
j
O
f–h
H
H
O
O
Et2N
O
O
Et2N
O
O
12, R = TBS
13, R = H
6
i
Fig. 4 Synthesis of the DEACM linker. (a) SeO2, dioxane, reflux, 42%; (b) allyltributyltin,
ZnCl2, CH3CN/H2O, 83%; (c) TBSCl, imidazole, DMF, 97%; (d) BH3-Me2S, THF, 0°C;
(e) NaOH, H2O2, 0°C—RT, 49% over two steps; (f ) CDI, DCM; (g) ethylenediamine,
DCM; (h) 2-chloroacetyl chloride, DIPEA, DCM, 0°C—RT, 65% over three steps;
(i) TBAF, THF, 0°C, 44%; (j) DSC, DMAP, DCM, 82%. Adapted with permission from
Yamazoe, S., Liu, Q., McQuade, L. E., Deiters, A., & Chen, J. K. (2014). Sequential gene
silencing using wavelength-selective caged morpholino oligonucleotides. Angewandte
Chemie, International Edition 53(38), 10114–10118.
3. Reflux the mixture at 60°C using an oil bath, while stirring overnight.
4. Let the reaction mixture cool to room temperature, then filter through
Celite (25g) and concentrate the filtrate under reduced pressure.
5. Purify by silica gel chromatography eluting with hexanes/ethyl acetate
(4:1) to afford 7-(diethylamino)-2-oxo-2H-chromene-4-carbaldehyde
1
(8) as a red solid (2.06g, 42%). Characterization properties: H NMR
(300MHz, CDCl3): δ¼1.12–1.20 (t, J ¼7.2Hz, 6H), 3.33–3.40
(q, J ¼7.2Hz, 4H), 6.36 (s, 1H), 6.42–6.43 (d, J ¼2.7Hz, 1H),
6.53–6.57 (dd, Ja ¼9.0Hz, Jb ¼2.7Hz, 1H), 8.19–8.22 (d, J ¼2.7Hz,
1H), 10.95 (s, 1H). 13C NMR (400MHz, CDCl3): δ¼12.4, 44.7, 97.4,
103.6, 109.4, 117.1, 126.9, 143.7, 150.9, 157.3, 161.7, 192.5. HRMS-
ESI: m/z calculated for C14H15NO3 [M+H]+: 246.1130; observed:
246.1130.
2.3.1.2.2 7-(Diethylamino)-4-(1-hydroxybut-3-en-1-yl)-2H-chromen-
2-one (9)
6. Stiramixtureof7-(diethylamino)-2-oxo-2H-chromene-4-carbaldehyde
(8, 500mg, 2.0mmol), allyltri-n-butyltin (0.99mL, 3.0mmol), and zinc
chloride (409mg, 3.0mmol), dissolved in a 4:1 mixture of acetonitrile
and water (10mL) at room temperature overnight.

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7. Concentrate the mixture under reduced pressure.
8. Add water (10mL) to the residue and extract with dichloromethane
(DCM, 3ꢀ10mL).
9. Wash the combined organic layers with brine (10mL).
10. Dry the organic layer over anhydrous sodium sulfate (10g), and filter.
11. Concentrate the filtrate under reduced pressure.
12. Purify the crude product by silica gel chromatography eluting with
hexanes/ethyl acetate (1:1) to afford 7-(diethylamino)-4-(1-hydro-
xybut-3-en-1-yl)-2H-chromen-2-one (9) as a green solid (477mg,
1
83%). Characterization properties: H NMR (400MHz, CDCl3):
δ¼1.14–1.18 (t, J ¼7.2Hz, 6H), 2.38–2.45 (m, 1H), 2.57–2.63
(m, 1H), 3.33–3.38 (q, J ¼7.2Hz, 4H), 4.97–5.00 (dd, Ja ¼8.0Hz,
Jb ¼4.0Hz, 1H), 5.10–5.17 (m, 2H), 5.81–5.90 (m, 1H), 6.22 (s, 1H),
6.40–6.41 (d, J ¼2.4Hz, 1H), 6.52–6.55 (dd, Ja ¼9.2Hz, Jb ¼2.4Hz,
1H), 7.36–7.38 (d, J ¼9.2Hz, 1H). 13C NMR (400MHz, CDCl3):
δ¼12.5, 41.6, 44.7, 68.7, 97.8, 105.1, 106.2, 108.6, 118.8, 124.9,
133.6, 150.3, 156.4, 158.2, 163.0. HRMS-ESI: m/z calculated for
C17H21NO3 [M+H]+: 288.1660; observed: 288.1602.
2.3.1.2.3 4-(1-((tert-Butyldimethylsilyl)oxy)but-3-en-1-yl)-7-
(diethylamino)-2H-chromen-2-one (10)
13. To a solution of 7-(diethylamino)-4-(1-hydroxybut-3-en-1-yl)-
2H-chromen-2-one (9, 862mg, 3.0mmol) in dimethylformamide
(DMF, 3.4mL), add imidazole (612mg, 9.0mmol) and tert-
butyldimethylsilyl chloride (904mg, 6.0mmol).
14. Stir the solution at room temperature overnight.
15. Quench the reaction with saturated aqueous sodium bicarbonate
(15mL).
16. Extract the resulting aqueous phase with ethyl acetate (3ꢀ10mL).
17. Wash the combined organic layers with water (15mL) and then brine
(10mL).
18. Dry the organic layer over anhydrous sodium sulfate (5g), and filter.
19. Concentrate the filtrate under reduced pressure.
20. Purify the crude product by silica gel chromatography eluting with
hexanes/ethyl acetate (4:1) to afford 4-(1-((tert-butyldimethylsilyl)-
oxy)but-3-en-1-yl)-7-(diethylamino)-2H-chromen-2-one (10) as a
1
yellow solid (1.16g, 97%). Characterization properties: H NMR
(400MHz, CDCl3): δ¼0.03 (s, 6H), 0.86 (s, 9H), 1.13–1.16
(t, J ¼7.2Hz, 6H), 2.38–2.48 (m, 2H), 3.32–3.37 (q, J ¼7.2Hz, 4H),

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Sankha Pattanayak et al.
4.85–4.88 (m, 1H), 4.97–5.02 (m, 2H), 6.13 (s, 1H), 6.13–6.14 (s, 1H)
6.45–6.46 (d, J ¼2.8Hz, 1H), 6.52–6.55 (dd, Ja ¼9.2Hz, Jb ¼2.8Hz,
1H), 7.43–7.45 (d, J ¼9.2Hz, 1H). 13C NMR (400MHz, CDCl3):
δ¼ꢁ5.1, ꢁ4.8, ꢁ3.6, 12.4, 18.0, 25.7, 42.9, 44.6, 71.0, 97.8, 105.7,
105.9, 108.4, 117.8, 125.2, 133.8, 150.2, 156.5, 158.3, 162.7.
HRMS-ESI: m/z calculated for C23H35NO3Si [M+H]+: 402.2464;
observed: 402.2468.
2.3.1.2.4 4-(1-((tert-Butyldimethylsilyl)oxy)-4-hydroxybutyl)-7-
(diethylamino)-2H-chromen-2-one (11)
21. Dissolve
4-(1-((tert-butyldimethylsilyl)oxy)but-3-en-1-yl)-7-
(diethylamino)-2H-chromen-2-one (10, 80mg, 0.20mmol) in tetra-
hydrofuran (THF, 700μL).
22. Cool down to 0°C.
23. Add a 2M solution of borane–dimethyl sulfide complex in THF
(500μL, 1.0mmol) dropwise over 5min.
24. Stir at 0°C for 3h.
25. Still at this temperature, slowly add a 3M aqueous solution of sodium
hydroxide (500μL) and 30% hydrogen peroxide in water (400μL).
26. Allow the mixture to warm up to room temperature for 2h while
stirring.
27. Extract the reaction mixture with ethyl acetate (3ꢀ2mL).
28. Wash the combined organic layers with water (2mL) and brine (2mL).
29. Dry the organic layer over anhydrous sodium sulfate (5g), and filter.
30. Concentrate the filtrate under reduced pressure.
31. Purify the crude product by silica gel chromatography eluting with
hexanes/ethyl acetate (1:1) to afford 4-(1-((tert-butyldimethylsilyl)-
oxy)-4-hydroxybutyl)-7-(diethylamino)-2H-chromen-2-one (11) as
1
a yellow solid (41mg, 49%). Characterization properties: H NMR
(400MHz, CDCl3): δ¼ꢁ0.04 (s, 3H) 0.08 (s, 3H), 0.91 (s, 9H),
1.18–1.21 (t, J ¼7.2Hz, 6H), 1.62–1.90 (m, 5H), 3.37–3.42
(q, J ¼7.2Hz, 3H), 3.62–3.67 (m, 2H), 4.91–4.93 (dd, Ja ¼6.4 Hz,
Jb ¼4.0Hz, 1H), 6.17 (s, 1H), 6.49–6.50 (d, J ¼2.8 Hz, 1H),
6.54–6.57 (dd, Ja ¼9.2 Hz, Jb ¼2.8Hz, 1H), 7.47–7.49 (d, J ¼9.2Hz,
1H). 13C NMR (400MHz, CDCl3): δ¼ꢁ5.0, ꢁ4.6, 12.6, 18.3, 25.9,
28.5, 34.5, 44.8, 62.7, 71.0, 97.9, 105.9, 106.1, 108.5, 125.4, 150.3,
156.6, 158.7, 162.7. HRMS-ESI: m/z calculated for C23H37NO4Si
[M + H]+: 420.2570; observed: 408.4193.

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2.3.1.2.5 4-((tert-Butyldimethylsilyl)oxy)-4-(7-diethylamino-2-oxo-
2H-chromen-4-yl)butyl (2-(2-chloroacetamido)ethyl)carbamate (12)
32. To a solution of 4-(1-((tert-butyldimethylsilyl)oxy)-4-hydroxybutyl)-
7-(diethylamino)-2H-chromen-2-one (11, 47mg, 0.11mmol) in
DCM (3mL), add 1,10-cabonyldiimidazole (45mg, 0.28mmol).
33. Stir at room temperature for 3h.
34. Dilute the reaction mixture by adding more DCM (2mL).
35. Wash the solution with water (5mL) and brine (5mL).
36. Dry the organic layer over anhydrous sodium sulfate (2g), and filter.
37. Concentrate the filtrate under reduced pressure.
1
38. Confirm the crude product by H NMR and use in the next step
without further purification.
39. Dissolve the activated alcohol in DCM (2.5mL).
40. Add ethylenediamine (16μL, 0.25mmol).
41. Stir at room temperature for 2h.
42. Remove the solvent and excess ethylenediamine under reduced
pressure.
43. Confirm the crude product by 1H NMR and use in the next step with-
out further purification.
44. Dissolve the amine residue in DCM (2.0mL).
45. Add N,N-diisopropylethyl amine (8μL, 0.05mmol) and stir at room
temperature for 10min.
46. Cool down to 0°C.
47. Add 2-chloroacetyl chloride (16μL, 0.20mmol) dropwise.
48. Stir at 0°C for 10min.
49. Quench the reaction with saturated aqueous sodium bicarbonate
(2mL).
50. Extract the aqueous layer with DCM (3ꢀ2mL).
51. Wash the combined organic layers with brine (2mL).
52. Dry the organic layer over anhydrous sodium sulfate (3g), and filter.
53. Concentrate the filtrate under reduced pressure.
54. Purify the crude product by silica gel chromatography eluting with hex-
anes/ethyl acetate (1:1) to afford 4-((tert-Butyldimethylsilyl)oxy)-4-
(7-diethylamino-2-oxo-2H-chromen-4-yl)butyl (2-(2-chloroacetamido)
ethyl)carbamate (12) as a yellow foam (42mg, 65%). Characterization
1
properties: H NMR (400MHz, CDCl3): δ¼ꢁ0.04 (s, 3H) 0.08
(s, 3H), 0.91 (s, 9H), 1.18–1.22 (t, J ¼7.2 Hz, 6H), 1.71–1.80 (m, 4H),
3.35–3.43 (m, 8H), 4.03 (s, 2H), 4.05–4.06 (m, 2H), 4.89 (m, 1H), 5.04

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Sankha Pattanayak et al.
(m, 1H), 6.17 (s, 1H), 6.50–6.51 (d, J ¼2.8Hz, 1H), 6.54–6.57 (dd,
Ja ¼8.8Hz, Jb ¼2.4Hz, 1H), 7.13 (br, 1H), 7.44–7.47 (d, J ¼8.8Hz,
1H). 13C NMR (400MHz, CDCl3): δ¼ꢁ5.0, ꢁ4.5, 12.6, 18.3, 24.9,
25.9, 29.8, 34.6, 40.5, 40.8, 42.6, 44.8, 64.9, 70.8, 97.9, 105.9, 106.1,
108.4, 125.3, 150.4, 156.7, 157.4, 158.4, 162.7, 166.9. HRMS-ESI:
m/z calculated for C28H44ClN3O6Si [M+H]+: 582.2766; observed:
582.2761.
2.3.1.2.6 4-(7-Diethylamino-2-oxo-2H-chromen-4-yl)-4-hydroxybutyl
(2-(2-chloroacetamido)ethyl) carbamate (13)
55. To a solution of 4-((tert-butyldimethylsilyl)oxy)-4-(7-diethylamino-2-
oxo-2H-chromen-4-yl)butyl (2-(2-chloroacetamido)ethyl)carbamate
(12, 38mg, 0.06mmol) in THF (1.5mL), add TBAF (1M in THF,
0.1mL, 0.1mmol) dropwise.
56. Stir at room temperature for 1h.
57. Concentrate under reduced pressure.
58. Purify the crude product by silica gel chromatography eluting with
chloroform/acetone (1:1) to afford 4-(7-diethylamino-2-oxo-2H-
chromen-4-yl)-4-hydroxybutyl (2-(2-chloroacetamido)ethyl) carba-
mate (13) as a yellow foam (13mg, 44%). Characterization properties:
1H NMR (400MHz, CDCl3): δ¼1.17–1.21 (t, J ¼7.2Hz, 6H),
1.80–1.90 (m, 4H), 3.34–3.45 (m, 9H), 4.03 (s, 2H), 4.10–4.13
(m, 2H), 5.01 (m, 1H), 5.44 (m, 1H), 6.25 (s, 1H), 6.45–6.46
(d, J ¼2.0Hz, 1H), 6.54–6.57 (dd, Ja ¼9.2Hz, Jb ¼2.4Hz, 1H), 7.21
(br, 1H), 7.37–7.39 (d, J ¼9.2Hz, 1H). 13C NMR (400MHz, CDCl3):
δ¼12.6, 25.3, 33.7, 40.6, 40.7, 42.7, 44.8, 64.9, 69.3, 97.9, 105.1, 106.2,
108.7, 125.1, 150.5, 156.5, 157.5, 158.9, 163.1, 167.2. HRMS-ESI: m/z
calculated for C22H30ClN3O6 [M+H]+: 468.1901; observed: 468.1870.
2.3.1.2.7 4-(7-Diethylamino-2-oxo-2H-chromen-4-yl)-4-((((2,5-
dioxopyrrolidin-1-yl)oxy)carbonyl) oxy)butyl (2-(2-chloroacetamido)
ethyl)carbamate (6)
59. Dissolve 4-(7-Diethylamino-2-oxo-2H-chromen-4-yl)-4-hydroxybutyl
(2-(2-chloroacetamido)ethyl) carbamate (13, 13mg, 0.03mmol) in
DCM (500μL).
60. Add a catalytic amount of 4-dimethylaminopyridine (10mol%, 0.3mg).
61. Add N,N0-disuccinimidyl carbonate (36mg, 0.14mmol).
62. Stir reaction mixture at room temperature overnight.
63. Concentrate under reduced pressure.

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64. Purify the crude product by silica gel chromatography eluting
with DCM/acetone (2:1) to afford 4-(7-diethylamino-2-oxo-2H-
chromen-4-yl)-4-((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl) oxy)butyl
(2-(2-chloroacetamido)ethyl)carbamate (6) as a yellow solid (15mg,
82%). Characterization properties: 1H NMR (400MHz, CDCl3):
δ¼1.19–1.22 (t, J ¼7.2Hz, 6H), 1.72–1.86 (m, 2H), 2.10–2.14
(m, 2H), 2.84 (s, 4H), 3.35–3.42 (m, 8H), 4.03 (s, 2H), 4.15–4.18
(t, J ¼7.2Hz, 2H), 5.43 (m, 1H), 5.96–5.99 (t, J ¼6.0Hz, 1H),
6.15 (s, 1H), 6.51–6.52 (d, J ¼2.8Hz, 1H), 6.59–6.63
(dd, Ja ¼9.2Hz, Jb ¼2.8Hz, 1H), 7.18 (br, 1H), 7.33–7.35
(d, J ¼9.2Hz, 1H). 13C NMR (400MHz, CDCl3): δ¼12.5, 14.3,
24.3, 25.5, 25.6, 31.9, 40.3, 42.6, 44.9, 64.5, 78.3, 98.2, 105.1,
109.4, 124.6, 150.9, 151.2, 151.6, 156.7, 157.4, 161.8, 167.3,
172.2. HRMS-ESI: m/z calculated for C27H33ClN4O10 [M+H]+:
609.1963; observed: 609.1990.
2.3.1.3 Preparation of the NB Linker 5
The photocleavable NB-linker can be synthesized from commercially avail-
able 1-(2-nitrophenyl)ethane-1,2-diol (14) in eight steps (Fig. 5), and
detailed procedures for the synthetic steps have been previously reported
(Yamazoe et al., 2012). Briefly, the primary hydroxyl of 14 was tosylated
and substituted with methylamine to obtain the amino alcohol 15. It was
then reacted with methyl adipoyl chloride to get the methyl ester 16.
O
HO
O2N
O
NO2
NO2
N
a–b
c
d–f
O
O
OH
N
H
OH
OH
14
15
16
O
O
O
O
H
H
N
Cl
N
O
O
Cl
O
O
O
N
N
N
N
N
g–h
H
H
O
O
O
O
O2N
O2N
17
5
Fig. 5 Synthesis of the NB linker. (a) TsCl, pyridine, 99%; (b) methylamine,THF, 91%;
(c) methyladipoyl chloride, DIPEA, CH2Cl2, 41%; (d) CDI,CH2Cl2; (e) ethylenediamine,
CH2Cl2; (f ) 2-chloroacetyl chloride, Et3N,CH2Cl2, 65% over three steps; (g) LiOH, THF,
H2O; (h) DSC, pyridine,CH3CN, 62% over two steps. Ts¼toluene-sulfonyl, DIPEA¼ N,N-
diisopropylethylamine. Adapted with permission from Yamazoe, S., Liu, Q., McQuade, L. E.,
Deiters, A., & Chen, J. K. (2014). Sequential gene silencing using wavelength-selective
caged morpholino oligonucleotides. Angewandte Chemie, International Edition 53(38),
10114–10118.

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Sankha Pattanayak et al.
The secondary alcohol of 16 was subsequently activated with 1,1-
carbonyldiimidazole and converted to a primary amine with the treatment
of ethylenediamine. The chloroacetamide moiety was then installed by
capping this amine with 2-chloroacetyl chloride to yield 17. Finally,
base-catalyzed hydrolysis and activation with N,N0-disuccinimidyl carbon-
ate provided the fully functionalized NB linker 5.
2.3.2 Synthesis and purification of cMOs
2.3.2.1 Materials and instruments
1. Custom MO oligomers with 50-primary amine and 30-disulfide amide
modifications were purchased from Gene Tools, LLC.
2. Reaction buffers: 0.1M Na2B4O7 (pH 8.5), 0.1M Tris-HCl buffer
(pH 8.4).
3. A vortex mixer, a Speedvac, a sonicator, and a benchtop centrifuge.
4. Illustra NAP-5 columns (GE Healthcare, Catalog number—17-0853-01).
5. Pierce™ Filter Spin Cups (ThermoFisher, Catalog number—69700).
6. Pierce™ Immobilized TCEP Disulfide Reducing Gel (ThermoFisher,
Catalog number—77712).
7. SulfoLink™ Coupling Resin (ThermoFisher, Catalog number—20401).
8. A Nanodrop™ UV-vis spectrophotometer.
2.3.2.2 General procedure for the cMO preparation
1. Dissolve the lyophilized, 25-base MO oligomer with 50-primary amine
and 30-disulfide amide modifications (50.0nmol) in 0.1M Na2B4O7
(pH 8.5, 100 μL) buffer in an Eppendorf tube. Sonicate to dissolve if
required.
2. Add a solution of the photocleavable linker 5 or 6 (172nmol) in 15 μL
DMSO to the above MO solution.
3. Cover with aluminum foil and shake overnight using a vortex mixer.
4. Purify the linker-MO conjugate from the unreacted linker and salts
using a NAP-5 column. To do so, equilibrate the NAP-5 column three
times with 5mL water each time. Dilute the reaction mixture to 500 μL
with water and load onto the column. Elute the MO with water and
collect 500 μL fractions in Eppendorf tubes. Check fractions to have
absorbance at 260nm and combine. Generally, the first 1mL contains
the MO.
5. Concentrate to 200 μL using a Speedvac.
6. It’s important to eliminate all the unreacted linker impurities to avoid
side reaction in the subsequent step. Since Step 4 may not remove the

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linker completely, acidify with 2 μL AcOH to protonate any hydro-
lyzed linker. Then wash the aqueous layer with CHCl3 (3ꢀ200 μL)
and EtOAc (3ꢀ200 μL) to remove the linker. Care should be taken
not to remove the opaque interface of the solvents.
7. Neutralize with 10% NH4OH and confirm the pHꢂ8.0.
8. Speedvac to dryness and dissolve in 200 μL water. To calculate the yield
using a Nanodrop spectrophotometer, dilute 1 μL of the MO solution
in 9 μL 0.1N HCl and measure the absorption at 260nm, following
MO-manufacturer’s instructions. Typical amide-coupling yields for
the MO-linker intermediates are 71–78%.
9. Use mass spectrometry to confirm the product identity and quality.
Dissolve 0.5ng of the MO-conjugate in 10 μL HPLC grade water
and record the mass spec on an ESI LC-MS instrument. The multi-
charged peaks can be deconvoluted using MaxEnt 1™ software
(Waters Corporation).
10. Remove water using a Speedvac and dissolve in 0.1M Tris-HCl buffer
(pH 8.4, 100 μL).
11. Wash the TCEP reducing gel (100 μL of the 50% slurry) with the same
Tris-HCl buffer (3ꢀ300 μL) using a Pierce™ Filter Spin Cup.
12. Add the MO-linker intermediate solution to the TCEP-resin and shake
overnight in dark.
13. Centrifuge at 13000g for 30s to collect the filtrate.
14. Wash the gel slurry with 0.1M Tris-HCl buffer (pH 8.4, 3ꢀ100 μL),
collect by centrifugation and combine with the filtrate.
15. Concentrate to 200 μL using a Speedvac.
16. Wash Sulfolink Coupling Resin (200 μL of the 50% slurry) with 0.1M
Tris-HCl buffer (pH 8.4, 400 μL) in a Pierce™ Filter Spin Cup.
17. Add the MO solution to the resin and shake for 15min and then left
upright without shaking for 30min.
18. Centrifuge at 13000 g for 30s to collect the filtrate.
19. Wash the gel slurry with 0.1M Tris–HCl buffer (pH 8.4, 3x100 μL),
collect by centrifugation and combine with the filtrate.
20. Remove the salts from the cyclic cMO using a NAP-5 column follow-
ing procedures described above.
21. Speedvac to dryness, dissolve in 200 μL water, quantify and calculate
the yield using a Nanodrop spectrophotometer. Typical thiol-halogen
reaction yields are 39–71%.
22. Use mass spectrometry to confirm the final cyclic cMO identity and
quality as described before.

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Sankha Pattanayak et al.
3. Microinjection of cMOs into zebrafish embryos
Microinjection is an effective and common way to introduce reagents
into zebrafish embryos. The MOs or cMOs were microinjected either into
the animal cell or the yolk, following previously described standard proce-
dures (Ekker, 2004). Injection solutions were prepared in cell-culture-grade
water containing 100mM KCl and 0.1% (w/v) phenol red. Prior to injec-
tion, the solutions were heated to 100C for 30s to dissociate any MO aggre-
gates, and were centrifuged (13000g, 1min) to remove undissolved particles.
One- to four-cell stage zebrafish embryos were arrayed in an agarose tem-
plate and submerged in E3 medium for microinjection. Typically, 1–2nL
solutions were injected either into the animal cell or the yolk prior to the
four-cell stage, and the embryos were subsequently cultured in E3 medium
at 28.5°C.
Accurate microinjection dosing of cMOs is essential to achieve their
optimum dynamic ranges. We recommend performing a careful titration
with the 25-base linear MO to find the minimum required dose, as described
for the tbxta hairpin cMO in our published paper (Ouyang et al., 2009). The
cMO injection solutions should be stored in air-tight O-ring-capped micro-
centrifuge tubes to avoid concentration changes due to evaporation. We also
recommend careful quantification of the injection volume by the mineral oil
droplet method (Nu€sslein-Volhard & Dahm, 2002). Care should be taken to
minimize exposure to ambient light or light from the microinjection stereo-
scope, especially while working with the DEACM linker. To minimize
photobleaching, the stereoscope light in the bottom can be covered with
UV or blue absorbing plastic such as 3M™ Sun Control window film
(3M USA Ltd. Catalog number—PR50).
4. Photoactivation of cMOs
The cMOs can be uncaged with the irradiation of appropriate
wavelengths of light. The light should also have enough intensity to effec-
tively complete the photochemical reaction within seconds with minimal
photodamage. As described earlier, to uncage NB cMOs we illuminate with
365-nm light, which in our hands only partially uncage DEACM cMOs. In
contrast, 470-nm light illumination is used to uncage DEACM cMOs. Our
set-up for the light irradiation is shown in Fig. 6. For global uncaging,

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Combinatorial control of gene function
17
A
B
C
Fig. 6 Photoactivation of cMOs. (A) Irradiation set-up for zebrafish embryos using an
agarose template in a Leica DM4500B upright fluorescence microscope equipped with
a 20ꢀ, 0.5 NA water-immersion objective. (B) Global irradiation of an embryo controlled
by Metamorph™ software. Full illumination of animal cells is shown. (C) Local irradiation
of an embryo using similar set-up. Localized illumination of animal cells is shown. The
illumination light beam has been indicated by the arrow-head.
zebrafish embryos were arranged in an agarose microinjection template
(560 μmꢀ960 μm) that contain evenly spaced individual wells (Fig. 6A
and B). The illumination was performed with a mercury short arc lamp
(Osram HBO™ 103W) using a Leica DM4500B upright compound
epifluorescence microscope equipped with a 20ꢀ water-immersion objec-
tive (Leica 506147, HCX APO L 20ꢀ/0.5 NA). To activate NB-cMOs,
individual embryos were irradiated with 365-nm light for 10s using a
narrow-band DAPI filter cube (Ex: 365nm, 10-nm bandpass; Chroma).
Similarly, DEACM-cMOs were activated with 470-nm light using a GFP
filter cube (Ex: 470nm, 40-nm bandpass; Leica) for 30s. The light intensities
were measured using a digital energy meter (Thor Labs PM100D) and
microscope slide sensor (Thor Labs S170C). Both light beams create a spot
with 2-mm diameter and the light intensities were as follows: 365-nm filter,
41mW/cm2; 470nm filter, 64mW/cm2.
In the microinjection template, we recommend orienting the
chorionated embryos such that the animal cells face upward and remain
directly in the path of the light beam. While unlikely, in mis-oriented
embryos, the light (especially UV) may not pass the top cell layers/yolk
to reach the bottom cells, resulting inefficient uncaging. To avoid exposure
to scattered light from another irradiation, the embryos can be placed in
alternate wells.
The above set-up can also be used for localized activation of cMOs by
reducing the size of the illumination field diaphragm (Fig. 6C). To further
trace the uncaged cells, a photoactivatable fluorescent tracer such as caged
fluorescein dextran can be co-injected with cMOs, and the targeted cells
can be isolated with fluorescence activated cell sorting for further analysis
(Payumo et al., 2016; Shestopalov et al., 2012).

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Sankha Pattanayak et al.
5. Conclusion
The protocols described in this chapter for wavelength-selective cyclic
cMO design and synthesis can likely be extended to other genes and organ-
isms. cMOs can complement genome-editing technologies like TALEN
(transcription activator-like effector nucleases) and CRISPR (clustered reg-
ularly interspaced short palindromic repeats) that have been successfully used
in zebrafish (Ata, Clark, & Ekker, 2016). However, these genetic technol-
ogies often require raising multiple animal generation to obtain homozygous
mutants, and are subject to Mendelian phenotypic distributions. Current
genome editing technologies also have limited capability in spatiotemporal
control. In comparison, cyclic cMOs can be synthesized and implemented
within days and can have 100% phenotypic penetration. These synthetic
reagents are thus valuable research tools, allowing rapid loss-of-function
studies with precise spatiotemporal control. Moreover, the wavelength-
selective cMOs discussed in this chapter permit combinatorial regulation
of multiple genes, extending the applicability of these tools in studying more
complex biological network.
While our studies demonstrate that multiple and spectrally distinct syn-
thetic reagents can have useful applications for in vivo studies, further advances
could expand their experimental scope. Current cMO technologies suffer
from narrow dynamic ranges, and the simultaneous use of multiple cMOs
could be associated with significant basal activities. For example, while the
tbxta and tbx16 cMOs are effective individually, their combined dark activities
precluded their combinatorial use in studying medial floor plate development
in zebrafish (Payumo, Walker, McQuade, Yamazoe, & Chen, 2015). This is
likely because these two genes are regulated reciprocally (Griffin, Amacher,
Kimmel, & Kimelman, 1998), and their mild basal activities have synergistic
effects on the mesoderm development. New cMO designs may be necessary
to overcome this limitation. Additionally, as described in Section 2.2, over-
lapping spectral features of the NB and DEACM groups can have some influ-
ence on the non-orthogonal activation of the two cMOs, and more red-
shifted chromophores maybe necessary to uncover the full potential of this
approach. As novel photolabile chromophores continue to be developed
(Gandioso et al., 2017; Gorka, Nani, Zhu, Mackem, & Schnermann,
2014; Hansen, Velema, Lerch, Szymanski, & Feringa, 2015; Jean-Gerard,
Vasseur, Scherninski, & Andrioletti, 2018), we anticipate that wavelength-
selective optochemical tools will increasingly provide insights into complex
biological processes that cannot be readily achieved through other means.

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19
Acknowledgements
We gratefully acknowledge support from NIH Grants R01 GM087292 (J.K.C.), R35
GM127030 (J.K.C.), and R01 GM112728 (A.D.).
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