Light-activated gene expression from site-specific caged DNA with a biotinylated photolabile protection group

Article abstract of DOI:10.1039/b922502a

A new method for site-specific caging of plasmid DNA was developed to enable light-activation of gene expression in living cells by exposure to a low dose of light.

Full text of DOI:10.1039/b922502a

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Light-activated gene expression from site-specific caged DNA
with a biotinylated photolabile protection groupw
Satoshi Yamaguchi,a Yanjie Chen,a Soh Nakajima,a Toshiaki Furutad and
Teruyuki Nagamune*abc
Received (in Cambridge, UK) 27th October 2009, Accepted 21st December 2009
First published as an Advance Article on the web 20th January 2010
DOI: 10.1039/b922502a
A new method for site-specific caging of plasmid DNA was
developed to enable light-activation of gene expression in living
cells by exposure to a low dose of light.
was performed by overlap extension PCR using a 50-amino-
modified DNA primer (Fig. 1a).3 Second, the amine group
incorporated within the PCR product was reacted with the
amine-reactive group of the biotinylated caging agent 1
(Fig. 1b). Third, streptavidin was bound to the biotin group
of the site-specifically caged plasmid DNA, as a steric regulator
to block transcription factors binding to the promoter
sequence. In this study, the enhanced green fluorescent protein
(EGFP)-expression plasmid, pEGFP-N1, was selected as
a model plasmid to analyze gene expression in living cells.
Site-specific caged plasmid DNAs were transfected into HeLa
cells by lipofection and EGFP expression was then activated
by UV light in living cells.
Spatiotemporal regulation of gene expression within living cells
and organisms provides exciting possibilities for elucidating
complex biological processes.1 In particular, light-activated
‘caged’ nucleic acids are promising chemical tools for the
spatiotemporal control of gene expression.2 Plasmid DNAs
encoding genes are generally selected for target gene regulation
on the basis of their stability and ease of use. However, only a
few research groups have reported caged plasmid DNAs.2b,c
This is mainly because the gene expression of conventional
caged plasmids could not be effectively induced by a low dose of
light.2b In the conventional caging method, a random reaction
between photolabile protecting groups and the phosphate back-
bone of DNA is employed. This caging reaction produces a
number of different plasmid structures, ranging from plasmids
with no bound caging groups to plasmids with a large number
of caging groups. In addition, some caging groups are located
on sites that are unrelated to the expression of the target gene.
To block gene expression perfectly, a high average number of
caging groups per plasmid is needed. Therefore, activation
of gene expression required a high dose of light. However,
irradiation with a high dose of light caused a decrease of
gene expression and cell death because of phototoxicity.2b,d
Accordingly, blocking gene expression with a small number of
caging groups is required to enable light-activation with a low
dose of light. Here, we report the design, synthesis, and photo-
induced activation of a novel, caged plasmid DNA that is
site-specifically labelled with one biotinylated photolabile group
within the promoter region.
The primers used in the overlap extension PCR are shown in
Fig. 1a. The forward primer was covalently modified with a
hexylamine moiety at the 50 end. Each primer was extended
by PCR using a DNA polymerase. According to previous
reports,3 the product of overlap extension PCR is a circular
molecule, containing two staggered nicks. In our design, the
calculated Tm value of the 51 bp sequence between the two
staggered nicks is above 65 1C. Therefore, under physiological
conditions, the PCR product probably annealed to form a
circular double-stranded molecule. In addition, the hexyl-
amine moiety was located in the nick, 3 bp upstream of the
TATA box of the cytomegalovirus (CMV) promoter.
Biotinylated caging agent 1 was designed based on a 6-bromo-
7-hydroxycoumarin-4-ylmethyl (Bhc) group. Bhc is a recently
developed photolabile protection group that has a photo-
sensitivity superior to that of classical 2-nitrobenzyl (NB)-based
Site-specific caging of expression plasmids was performed
by a three-step procedure. First, site-specific amino-modification
a Department of Chemistry and Biotechnology, School of Engineering,
The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, 113-8656,
Tokyo, Japan. E-mail: nagamune@bioeng.t.u-tokyo.ac.jp;
Fax: +81 (0)3 5841 8657; Tel: +81 (0)3 5841 7328
b Department of Bioengineering, School of Engineering,
The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, 113-8656,
Tokyo, Japan
c Center for NanoBio Integration (CNBI), The University of Tokyo,
7-3-1, Hongo, Bunkyo-ku, 113-8656, Tokyo, Japan
d Department of Biomolecular Science, Toho University,
2-2-1, Miyama, Funabashi, Chiba 274-8510, Japan
w Electronic supplementary information (ESI) available: Full experi-
mental procedures, schematic illustration of the enzymatic digestion
and the splitting of non-caged plasmid, and electrophoretic mobility
shift analysis data of non-caged plasmid. See DOI: 10.1039/b922502a
Fig. 1 Structure of overlap primers used in the overlap PCR (a) and
schematic illustration of the synthesis and photochemistry of site-
specific caged DNA (b).
ꢀc
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photolabile groups.4a Because of its high sensitivity to long-
wavelength UV light, Bhc has been previously employed for
preparing caged analogues of various biologically active
molecules.4 In addition, it was recently reported that the
7-hydroxyl moiety of the Bhc group can be alkylated without
decreasing the overall photo-sensitivity to light.4d Based on
this finding, we decided to attach a biotin moiety at the
7-position of Bhc (Fig. 1b). According to previous reports,4a,d,e
the biotinylated caging agent was activated at the 4-position
with 4-nitrophenyl carbonate ester as an amine-reactive group.
As shown in Fig. 1b, biotinylated caging agent 1 can be
covalently attached to PCR products via a carbamate bond.
The synthesis of 1 was accomplished as shown in Scheme 1
(see ESIw for detailed procedures). The starting compound 2
was prepared in three steps, as previously reported.4e The Boc
protecting group of 2 was removed in dichloromethane
(DCM) containing 10% trifluoroacetic acid (TFA). The amine
group was then biotinylated using biotin-N-hydroxysuccinimidyl
ester and triethylamine (TEA) in DMF to yield compound 3
(56% over two steps). Next, the biotinylated caging agent 1
was obtained by reaction of 3 with 4-nitrophenyl chloro-
formate and TEA in CH2Cl2.
probably denatured during electrophoresis. Considering the
sites of the two nicks (3 bp upstream and 41 bp downstream of
the TATA box), the two DNA fragments were identified as the
108 bp fragment between the upstream nick and the SmaI site
and the 261 bp fragment between the SnaBI site and the
downstream nick (Fig. S1, ESIw). If this hypothesis is correct,
only the shorter fragment has a hexylamine moiety and can
react with biotinyl caging agent 1. In fact, as the concentration
of 1 increased, the band of the shorter fragment became
thinner (Fig. 2a, position A), while the band of the longer
fragment remained almost the same (Fig. 2a, position B), and
coincidentally, a new, blurred band appeared (Fig. 2a, position
C). In the Alexa Fluor 488-fluorescent image of the same gel, a
blurred band was similarly observed at the same position as
the new band in the EtBr-fluorescent image (Fig. 2b). Thus,
this new band at position C is derived from conjugates of
caged DNA and fluorescent-labelled streptavidin (Fig. 2b).
When intact pEGFP-N1 containing no hexylamine moiety
was incubated with caging agent 1 and analyzed as described
above, the band of digested DNA did not shift as the
concentration of 1 increased (Fig. S2, ESIw). These results
indicate that PCR products were site-specifically modified
with a biotinylated caging group through chemical reaction
between the hexylamine moiety of the PCR products and the
biotinylated caging agent 1. Furthermore, it was also con-
firmed that streptavidin can bind to the biotinylated caging
group on the DNA, as designed.
The PCR product was incubated with biotinylated caging
agent 1 at various concentrations of 1 from 0 to 10 mM in a
sodium carbonate buffer. After removal of excess caging agent
1 by column chromatography and gel filtration, site-specific
caging of PCR products was investigated by restriction
enzyme digestion and electrophoretic mobility shift analysis.5
Site-specific caged DNA was digested with restriction enzymes
(SnaBI and SmaI) at two sites: 213 bp upstream (SnaBI site)
and 98 bp downstream (SmaI site) of the TATA box. The
resulting DNA fragments, containing a caged fragment,
were incubated with fluorescent (Alexa Fluor 488)-labelled
streptavidin for 1 h at room temperature, followed by electro-
phoresis in 1% agarose gel. By imaging the EtBr-fluorescence
of the electrophoresed gel, contrary to our expectation, two
DNA fragments of approximately 100 bp and 250 bp were
detected in the non-caged DNA sample (i.e. amino-modified
PCR products with 0 mM caging agent 1) (Fig. 2a). Considering
the restriction sites, it was expected that a DNA fragment of
318 bp would be obtained by digestion with the restriction
enzymes (Fig. S1, ESIw). This result could be explained by the
expected DNA fragment being split into two shorter DNA
fragments because the DNA duplex between the two staggered
nicks, which were introduced in overlap extension PCR,
Next, we investigated the photo-cleavage of site-specific
caged DNA in vitro. The site-specific caged DNA solutions
were irradiated with UV light for various periods of time. The
photo-irradiated samples were analyzed as described above.
As the dose of light increased, the band of caged DNA
fragment-streptavidin conjugate became thinner (Fig. 2c and d,
position C), and simultaneously, the band of the non-caged
DNA fragment thickened (Fig. 2c, position A). Thus, the
Fig. 2 Electrophoretic mobility shift analysis of the caging reaction
and the photo-cleavage reaction. (a), (b) Site-specific caged plasmid
DNA, prepared at various concentrations of caging agent 1 (0–10 mM),
was digested with two restriction enzymes (SmaI and SnaBI) and
incubated with fluorescent (Alexa Fluor 488)-labeled streptavidin,
followed by electrophoresis in 1% agarose gel. (c), (d) Site-specific
caged plasmid DNA prepared at the caging agent concentration of
10 mM was irradiated by ultraviolet light (365 nm, 0–1.9 J cmꢁ2) and
analyzed as in (a), (b). After electrophoresis, the fluorescent images of
gels were obtained by scanning the fluorescence of EtBr (a), (c) and
Alexa Fluor 488 (b), (d).
Scheme 1 Synthesis of the biotinylated caging agent 1.
ꢀc
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the poly(A) sequence in pEGFP. As a result, transferred
caged plasmid DNA both with and without streptavidin
did not change the level of EGFP expression (98 ꢂ 6.2%
and 97 ꢂ 6.7% of non-caged plasmid DNA, respectively,
p 4 0.4; t-test on 4 degrees of freedom). This result indicated
that caging of plasmid DNA at the site related to transcription
is required to suppress gene expression. Additionally, this
result confirmed that binding with streptavidin did not change
the transfection efficiency of caged plasmid DNA. Thus, the
decrease in gene expression from plasmid DNA caged near the
TATA box was probably due to streptavidin competitively
blocking transcription factor binding to the TATA box.
To investigate the light-activation of site-specific caged
plasmid DNA in living cells, the transfected cells were exposed
to various doses of UV light. With exposure to UV light,
the EGFP expression from the caged plasmid DNA with
streptavidin increased to above 83% of positive control
(Fig. 3). The maximum expression level was 92% at a light-
dose of 0.48 J cmꢁ2. A simple calculation suggested that
almost three quarters of the suppressed gene expression was
activated by light-irradiation under these conditions. In a
previous report of a caged pGFP randomly modified with
NB-based photolabile groups,2b at the maximum, only one
third of the suppressed gene expression was recovered by light-
doses of 0.25–5.6 J cmꢁ2. Thus, the present site-specific caged
plasmid DNA was more effectively activated by light.
Fig. 3 Effect of light on the EGFP expression in HeLa cells transfected
with site-specific caged plasmid DNA. Non-caged and site-specific
caged plasmid DNA with or without streptavidin were transferred into
HeLa cells by lipofection for 2 h. After transfection, some groups of
HeLa cell cultures were individually exposed to various doses of 365 nm
UV light (0–0.72 J cmꢁ2). After incubation for 24 h, each HeLa cell
culture was analyzed by flow cytometry. The percentages of EGFP
expressing cells were normalized to that of cells transfected with non-
caged plasmid DNA. Each data point represents the mean ꢂ S.E.
(n 4 5). *p o 0.01 and **p o 0.001 versus caged plasmid DNA with
streptavidin irradiated with 0 J cmꢁ2 of UV light (t-test).
in vitro photo-cleavage of the biotinylated caging group
(‘uncaging’) by UV irradiation was confirmed.
In summary, we have developed a new method for site-
specific caging of plasmid DNA with biotinylated caging
agent 1. The suppressed gene expression of site-specific caged
plasmid DNA was activated by a low dose of light more
effectively than that of conventional randomly-caged plasmid
DNA. In principle, the present site-specific caging method
may be applicable to any plasmid DNA coding gene, shRNAs
and other functional RNAs, such as RNA aptamers. Thus,
this site-specific caging method is a promising technology for
light-controlled gene expression within living cells.
Gene expression of site-specific caged plasmid DNA was
investigated in living cells. The EGFP-expression level of cells
transfected with non-caged plasmid DNA was 47 ꢂ 4.1%
(n = 9, mean ꢂ S.E.). The percentage of EGFP-positive cells
in each sample was normalized with that of this positive
control sample (Fig. 3). Without streptavidin, the expression
from caged plasmid DNA was slightly decreased compared
with the positive control (88 ꢂ 12%, in Fig. 3). On the
other hand, streptavidin bound to the caged plasmid DNA
decreased the expression level to 65 ꢂ 8.3% (Fig. 3, caged
plasmid DNA with streptavidin irradiated with 0 J cmꢁ2 of
UV light). This decrease due to the combination with streptavidin
was statistically significant (p o 0.001; t-test on 16 degrees of
freedom, Fig. 3). These results indicate that site-specific caged
plasmid DNA in combination with streptavidin can suppress
gene expression as predicted.
Notes and references
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To investigate how caged plasmid DNA with streptavidin
suppressed EGFP expression, we first visualized streptavidin
using fluorescent (R-phycoerythrin)-labelled streptavidin and
confocal laser scanning microscopy. When R-phycoerythrin-
labelled streptavidin bound to caged plasmid DNA was
transferred into cells, several red-fluorescent aggregates were
observed in transfected cells (Fig. S3,ESIw). On the other
hand, when R-phycoerythrin-labelled streptavidin was trans-
ferred with no plasmid DNA or non-caged plasmid DNA, no
red-fluorescent aggregate was detected in cells (Fig. S3, ESIw).
These results strongly suggested that streptavidin was introduced
into living cells with caged plasmid DNA as expected. Next, as
a control experiment, we investigated the gene expression
of the plasmid DNA caged at a site that is not related
to transcription: a site more than 900 bp downstream of
5 L. M. Hellman and M. G. Fried, Nat. Protoc., 2007, 2, 1849–1861.
ꢀc
This journal is The Royal Society of Chemistry 2010
2246 | Chem. Commun., 2010, 46, 2244–2246