In vivo light-driven DNA binding and cellular uptake of nucleic acid stains

Article abstract of DOI:10.1021/cb300100r

Chemical derivatization of nucleic stains such as ethidium bromide or DAPI with tailored, photoresponsive caging groups, allows for on demand spatiotemporal control of their in vivo nucleic acid binding, as well as for improving their cellular uptake. This effect was particularly noteworthy for a nitro-veratryloxycarbonyl-caged derivative of ethidium bromide that, in contrast with the parent stain, is effectively internalized into living cells. The activation strategy works in light-accessible, therapeutically relevant settings, such as human retinas, and can even be applied for the release of active compounds in the eyes of living mice.

Full text of DOI:10.1021/cb300100r

Articles
pubs.acs.org/acschemicalbiology
In Vivo Light-Driven DNA Binding and Cellular Uptake of Nucleic Acid
Stains
Mateo I. Sanchez,† Jose Martínez-Costas,‡ Francisco Gonzalez,§,∥ María A. Bermudez,§
́
́
M. Eugenio Vazquez,*,† and Jose L. Mascarenas*,†
́
́
̃
†Departamento de Química Organ
Química Bioloxica e Materiais Moleculares, Unidad Asociada al CSIC, Universidad de Santiago de Compostela, 15782 Santiago de
́ ́
ica and Departamento de Bioquímica y Biología Molecular, Centro Singular de Investigacion en
‡
́
Compostela, Spain
§Departamento de Fisiología, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
∥Servicio de Oftalmología, Complejo Hospitalario Universitario de Santiago de Compostela, 15706 Santiago de Compostela, Spain.
S
* Supporting Information
ABSTRACT: Chemical derivatization of nucleic stains such as ethidium bromide or DAPI with tailored, photoresponsive caging
groups, allows for “on demand” spatiotemporal control of their in vivo nucleic acid binding, as well as for improving their cellular
uptake. This effect was particularly noteworthy for a nitro-veratryloxycarbonyl-caged derivative of ethidium bromide that, in
contrast with the parent stain, is effectively internalized into living cells. The activation strategy works in light-accessible,
therapeutically relevant settings, such as human retinas, and can even be applied for the release of active compounds in the eyes
of living mice.
he development of nucleic-acid-targeted drugs continues
to be a major research endeavor at the interface between
T
chemistry and biomedicine. A large variety of DNA-binding
agents have been developed, and many of them have found
application as drugs or as DNA stains.1−5 Unfortunately, many
of these molecules present selectivity and toxicity problems that
seriously restrict their therapeutic potential. In this context,
derivatizing these molecules with photolabile appendages that
confer advantageous physicochemical features while providing
for external control of their activity might open important
therapeutic avenues.6−8 Needless to say, the use of light-
activated compounds in photodynamic therapy might greatly
benefit from current technical advances that make it possible to
irradiate almost any tissue in the human body.9
Figure 1. Structures of DAPI (1), its caged derivative (©1), and
ethidium bromide (2).
DAPI (4′,6-diamidino-2-phenylindole, 1; Figure 1) and
ethidium bromide (EtBr, 3,8-diamino-5-ethyl-6-phenyl-phenan-
thridinium bromide, 2) are among the best known nucleic acid
stains10−14 and have found wide and important applications in
molecular and cell biology. Ethidium bromide binds nucleic
acids by intercalation and has been extensively used for
detecting double-stranded DNA (dsDNA), mainly in gel
electrophoresis assays. However, it cannot be used for direct
staining of cellular nucleic acids, as it is poorly internalized. In
contrast, the blue-fluorescent DAPI can be used to stain cell
nuclei and, unlike the non-specific EtBr, prefers to bind A/T-
rich DNA sequences, inserting into their narrow minor groove.
We have recently demonstrated that caging of the amidinium
moieties of DAPI and other bisbenzamidinium DNA binders
with suitable photoresponsive protecting groups suppresses
their DNA binding.15 Importantly, irradiation with UV light
Received: December 6, 2011
Accepted: May 2, 2012
© XXXX American Chemical Society
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removes the caging groups and regenerates the parent active
compounds, thus allowing the control of the in vitro DNA
binding activity of these molecules by external stimuli (UV
light). We were then challenged to demonstrate that such
conditional activation strategy could also be used in living cells
and tissues, a process that should be easily monitored by the
increased fluorescence of these molecules upon staining of the
cell nuclei. In addition, we were also intrigued by the possibility
of extending the caging strategy to other DNA binders lacking
amidinium groups, such as ethidium bromide (EtBr).
Herein we demonstrate that both DAPI (1) and EtBr (2) can
be efficiently caged using photolabile groups and that their
nucleic acid binding ability can be restored in living cells and in
therapeutically relevant tissues such as retinas, in a spatially and
temporarily controlled manner. We also show that it is even
possible to release the stains in the eyes of living mice by using
an external irradiation source.
Figure 2. (Left) Fluorescence microscopy of Vero cells incubated with
5.0 μM ©1 showing the fluorescence changes inside the cells (t = 0,
brightfield and fluorescence; t = 4−16 min, fluorescence); arrows
highlight the brighter areas. (Right) Schematic representation of the
proposed activation dynamics of caged DAPI, which is, at least in part,
stored in the cell membrane. Initial background fluorescence at t = 0
most probably results from non-specific interaction between uncaged
DAPI and the hydrophobic polymeric Mowiol 4−88 matrix used as
coverslip mounting solution.
RESULTS AND DISCUSSION
■
As expected from the relatively low pKa of the amidines and
their propensity to act as leaving groups, the caging groups of
DAPI derivative ©115 can be readily removed in the presence
of dsDNAs using a standard gel transilluminator lamp as
irradiation source (λ = 300−375 nm, Supplementary Figure
S1). For the in vivo assays, we incubated independent samples
of Vero cells16 with 5 μM ©1 for 30 min and monitored their
evolution by fluorescence microscopy. Initial irradiation
experiments were carried out using the transilluminator, but
the UV light source from the microscope could also effectively
photolyze ©1. This allowed an easy monitoring of the changes
in fluorescence emission of the dye inside the cells in real time.
Remarkably, the fluorescence emission is initially relatively
weak and diffuse, but apparently more intense on the perimeter
(membrane) of the cells. Over time it becomes progressively
concentrated in the cell nuclei, while growing fainter at the cell
edges. Indeed, after 20 min under the microscope, the edges
become barely distinguishable and only the nuclei are observed
(Supplementary Video S1). Control experiments with lip-
osomes confirm that, as expected, ©1 is basically non-emissive,
and addition of DAPI to a liposome solution results in a
marked increase in its emission intensity (Supporting
Information, page S12). Therefore it can be safely stated that
the fluorescence observed in the membrane is due to uncaged
DAPI.
sought to extend this strategy to EtBr (2), which binds DNA by
intercalation and lacks the amidinium groups of DAPI. Despite
its wide use as an in vitro DNA stain, EtBr has very limited value
for in vivo experiments due to its poor cell membrane
permeability. We envisioned that masking the amino groups
of EtBr with the non-polar Nvoc caging group might not only
interfere with its DNA binding but also improve the
internalization properties of the stain by increasing its
hydrophobic character. Therefore the bis-Nvoc phenanthridyl
derivative ©2 was prepared by treatment of the commercial
EtBr with nitro-veratryloxycarbonyl (Nvoc) chloride under
basic conditions (Scheme 1).
Scheme 1. Synthesis of Nvoc-Caged Ethidium Bromide ©2
In order to separate the effects of the uncaging and cellular
redistribution, Vero cells were incubated with ©1 in PBS for 30
min and irradiated for approximately 3 min in the trans-
illuminator (enough to uncage most of the stain). Comparison
of micrographs obtained immediately after the irradiation and
after 5 min of incubation confirms that the uncaged dye
requires some time to accumulate and stain the cell nuclei
(Supporting Information, page S17). All of these results are
consistent with the presence of the hydrophobic caged DAPI
(©1) in the cytoplasmic membrane and probably also in other
cell locations. Upon irradiation, photolysis of ©1 releases the
fluorogenic DAPI, which is highly emissive in the hydrophobic
phospholipid matrix. However, the uncaged DAPI is very
hydrophilic (ACD/Laboratories LogD (pH 7.4) = −1.03)17,18
and is therefore driven out of the membrane and freely diffuses
toward the cell nuclei, finally forming a stable, fluorescent DNA
complex (Figure 2).
As expected, ©2 did not show any fluorescence in the
presence of a short dsDNA oligonucleotide, and competition
experiments with uncaged EtBr confirmed that ©2 is unable to
bind DNA. Irradiation of a solution of ©2 in Tris-HCl buffer,
100 mM NaCl, pH 7.5, with UV light (standard trans-
illuminator lamp, λ = 300−375 nm) promoted the cleavage of
the photolabile groups, thus regenerating the active EtBr
intercalator (2). Importantly, this can be achieved in the
presence of DNA, leading to a large increase (60-fold) in the
emission intensity at 600 nm, which is consistent with the
intercalation of the released EtBr into the DNA (Figure 3).
Once we demonstrated the possibility of in vivo UV-
controlled DNA interaction with a minor groove binder, we
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mainly localized in the nucleoli, as demonstrated by
colocalization experiments with DAPI and mitotracker dyes
(for further information see the Supporting Information, pages
S14 and S16).
These results suggest that, in contrast to the parent
phenanthridinium 2, the designed photoresponsive derivative
©2 seems to efficiently cross cell membranes, accumulating
inside the cell in a latent form, until irradiation with UV light
releases the active intercalator, which binds and stains nucleolar
nucleic acids. A control experiment at increasing irradiation
times revealed a progressive increase in the fluorescence
emission from the cells, reaching a maximum after approx-
imately 10 min of irradiation (Figure 4, top). It is probable that
some monocaged EtBr derivatives are also present, but cannot
be monitored by fluorescence imaging. The use of a more
potent illumination source should warrant a more rapid and
efficient generation of the active binder.
Figure 3. Fluorescence emission spectra of a 0.8 μM solution of ©2 in
Tris-HCl buffer at increasing irradiation times, in the presence of a
hairpin ds-oligonucleotide. No further increase in fluorescence was
observed after approximately 15 min of irradiation.
In addition to temporal activation, the caging strategy should
also allow for the spatial control of the interaction. To
demonstrate that, we prepared a cell monolayer on a 100 mm
tissue culture plate (Figure 5A) and irradiated it through a
HPLC monitoring of the photolysis reaction showed that the
process generates Nvoc-monoprotected temporary intermedi-
ates, but both caging groups are fully removed after 18−20 min
of irradiation (Supporting Information, page S10). Control
experiments with a mixture of specifically synthesized C3 and
C8 Nvoc-monoprotected EtBr showed that these compounds
are not fluorescent in the presence of DNA, despite retaining
some DNA binding affinity (Supporting Information, pages
S9−S10). Therefore, uncaging of both groups is required for
good DNA binding and for DNA-dependent fluorescent
staining.
Figure 5. (A) cell monolayer in the culture plate before irradiation;
(B) same culture after irradiation through a cardboard cutout.
Once the photocontrolled DNA binding of ethidium was
confirmed, we studied whether the presence of the hydro-
phobic appendage could improve the cell transport properties
of the dye. Therefore, independent samples of Vero cells were
treated in parallel with 12.5 μM caged ©2 and 20.0 μM EtBr
(2) and examined by fluorescent microscopy after 30 min
(excitation filter 530−550 nm/emission filter 590 nm). As
shown in Figure 4A/C, in both cases the cells were essentially
non-fluorescent, and only a very faint emission could be
detected in the case of EtBr. However, UV irradiation of the
cells incubated with ©2 (10 min) resulted in a very significant
increase in the emission intensity, a result that must be
correlated with photorelease of ethidium bromide from the
inactive derivative ©2 (Figure 4B). Remarkably, staining is
cardboard cutout of our university acronym, USC. After 15 min
of irradiation through the mask, we looked at the culture
through a gel imaging system, observing the expected cellular
photopatterning only for the cell populations that were
irradiated (Figure 5B). Therefore, derivative ©2 is a light-
responsive, transport-efficient version of ethidium bromide and
should represent a significant addition to the as yet short
arsenal of nucleic acid stains that are effective in vivo.
The above results encouraged us to evaluate the performance
of both caged DAPI (©1) and caged ethidium (©2) in a more
stringent, therapeutically relevant setting. Therefore we
incubated mice eye retinas with 5.0 μM ©1 and 12.5 μM ©2
(PBS buffer) and analyzed the resulting cellular staining before
and after irradiation. Before irradiation, no staining was visible,
whereas after irradiation both ©1 and ©2 were effectively
uncaged in the retina, and the cell nuclei became fluorescent
(Figure 6a,b; see also Supplementary Video S2). Importantly,
the same results were reproduced with human retinas (Figure
6d,e), confirming that these compounds can be activated in the
complex cellular environment of human tissues.
In a step forward, we also explored the possibility of in vivo
activation of these molecules by direct external irradiation of
the eyes in living mice. Therefore, mice were treated with caged
DAPI (©1) using a femoral vein injection, and a transpupillar
irradiation of one of their eyes was made. The mice were
sacrificed, and the retinas were extracted and flat mounted for
study. As expected, only the retina of the irradiated eyes
showed a clear fluorescence emission, which was mostly
concentrated in the vascular walls (Figure 6c), possibly because
the blood-retinal barrier prevented most of the molecules from
entering the retinal cells. This is a known phenomenon that
occurs in healthy retinas; however, if the retinas present some
Figure 4. (Top) Fluorescence microscopy of Vero cells (12.5 μM ©2)
at increasing irradiation times. (Bottom) 12.5 μM ©2, after 30 min
incubation:( A) before irradiation and uncaging; (B) after uncaging by
UV irradiation for 15 min; (C) control experiment with 20.0 μM of
ethidium bromide 2 after 30 min incubation.
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METHODS
■
General. All reagents were acquired from commercial sources.
Reactions were followed by analytical RP-HPLC with an Agilent 1100
series LC/MS using an Eclipse XDB-C18(4.6 mm × 150 mm, 5 μm)
analytical column. Standard conditions for analytical RP-HPLC
consisted of a linear gradient from 5% to 95% of solvent B for 30
min at a flow rate of 1 mL/min (solvent A: water with 0.1% TFA,
solvent B: acetonitrile with 0.1% TFA). Compounds were detected by
UV absorption at 220, 270, 304, and 330 nm. Final products were
purified on a Buchi Sepacore preparative system consisting of a C-615
̈
pump manager with two C-605 pump modules for binary solvent
gradients, a C-660 fraction collector, and a C-635 UV Photometer.
Purification was made using reverse phase of water/acetonitrile 0.1%
TFA, using a prepacked preparative cartridge (150 mm × 40 mm)
Figure 6. Fluorescence microscopy of mouse and human retinas. (a)
Mouse retina incubated with 5.0 μM of ©1, after ∼5 min irradiation.
(b) Mouse retina incubated with 12.5 μM ©2, after ∼10 min
irradiation. (c) Retina extracted from a mouse previously injected with
©1 in the right femoral vein (100 μL, 5.0 μM) and transpupillary
irradiated for ∼10 min before sacrificing the animal; the nuclei of
aligned cells forming a vessel image can be clearly observed. (d)
Human retina incubated with 5.0 μM ©1, after ∼5 min irradiation. (e)
Human retina incubated with 12.5 μM of 2, after ∼10 min irradiation.
(f) Human retina incubated with 12.5 μM of ©2 before irradiation.
Control, non-irradiation experiments were carried in all cases, and
none of them showed traces of fluorescence. Images in panels a and b
were acquired with 1 s exposure time, and images in panels c−f with
400 ms. All images with ISO 400 sensitivity.
with reverse-phase RP18 silica gel (Buchi order no. 54863). The
̈
fractions containing the products were freeze-dried, and their identity
was confirmed by ESI-MS(+). NMR spectra were recorded using
Varian Mercury 300 or Bruker DPX 250 spectrophotometers and
processed using the MestreNova v6.1.1-6384 suite (Mestrelab
Research). 1H NMR spectra were processed applying Global Spectrum
Deconvolution (GSD).
Synthetic Procedure for 3,8-Bis({[(4,5-dimethoxy-2-
nitrobenzyl)oxy]carbonyl}amino)-5-methyl-6-phenylphenan-
thridinium (Nvoc2-ethidium, ©2. To a solution of commercially
available ethidium bromide (2) (100 mg, 0.253 mmol) in 25 mL of
DIEA/DMF (0.195 M) was added nitroveratryl chloride (209 mg,
0.759 mmol). The resulting mixture was stirred for 16 h under Ar and
in the dark. After concentration, the residue was purified by
preparative reverse-phase chromatography (Buchi Sepacore) (5 min
̈
isocratic 15% B, followed by linear gradient from 15% to 95% B during
30 min, and 10 min isocratic at 95% B). The combined fractions were
concentrated and freeze-dried to obtain the desired product (©2) as a
trifluoroacetic salt (77 mg, 0.086 mmol, 34%).
In Vitro Fluorescence Experiments with ©2. A fluorescence
cuvette containing a 0.82 μM solution of ©2 in Tris-HCl buffer 20
mM, 100 mM NaCl, pH 7.5, and 0.136 μM hairpin DNA
oligonucleotide AACGTT was irradiated with UV light, and
fluorescence emission spectra were recorded after different irradiation
times. Hairpin oligonucleotide: AACGTT: GGC AAGCTT CGC
TTTTT GCG AAGCTT GCC.
degree of damage the compounds should be able to enter the
cells.19 These results confirm that the designed compounds can
be selectively activated in the posterior segment of eyes of living
animals using an external irradiation source and suggest the eye
as a suitable initial target organ for future therapeutic
intervention using this type of light-activatable compounds.
Delivery of drugs in a minimally invasive, safe, and effective
manner in the eye is still a major therapeutic challenge.20 Given
the well-established use of lasers in ophthalmology, the
uncaging tactic should open new opportunities to treat specific
eye diseases that require the selective release of active drugs in
specific areas of the eye.21,22
In conclusion, we have demonstrated that the Nvoc caging
strategy can be applied to classic DNA intercalators such as
EtBr. Since these molecules are nucleic-acid-dependent
fluorescent probes, they provide an excellent opportunity for
real-time monitoring of the activation process in living cells. For
the Nvoc-DAPI (©1), we could observe the uncaging process
and cell redistribution of the released stain in real time.
Moreover, the caging strategy can be exploited not only for the
“on demand” spatiotemporal control of the nucleic acid binding
but also for fine-tuning the physicochemical properties of the
substrates. In the case of the Nvoc-caged derivative ©2, this
allows for a rapid cell internalization, overcoming the poor cell
transport ability of the parent ethidium bromide. Importantly,
we have also demonstrated that this strategy can be successfully
implemented in therapeutically relevant tissues such as the
retina, it even being possible to activate the dyes with external
irradiation in the eyes in living mice. To the best of our
knowledge, our results provide the first evidence on the use of
caged compounds in eyes and demonstrate that focalized
release of drugs can be made with a minimally invasive
technique, opening a new door for therapeutic and perhaps
diagnostic applications.
Cell Uptake Experiments. Vero cells were maintained in DMEM
(Dulbecco Modified Eagle Medium) containing 10% of FBS (fetal
bovine serum). The day before the cellular uptake experiments, cells
were seeded in 12-well plates containing glass coverslips (15 mm).
Cells were then washed 3 times in PBS and overlaid with 1 mL of fresh
PBS with no serum added. Compound ©1 was added in order to
obtain a final concentration of 5.0 μM, and in the case of ©2 12.5 μM.
Samples were incubated for 30 min at RT in the absence of light. For
video recording and snapshots of Figure 2, after incubation the
coverslips were mounted on glass slides with Mowiol 4−88 [100 mg
mL−1 in 100 mM Tris-HCl pH 8.5, 25% glycerol, and 0.1% DABCO
(as an antifading agent)]. Irradiation of selected samples was
performed with a standard gel UV transilluminator (8 W, λexc 300−
370 nm). Images were obtained with an Olympus DP-71 digital
camera mounted on an Olympus BX51 fluorescence microscope
(Olympus Corp.) equipped with a 360−370 nm excitation filter and
420 nm emission filter for the cells treated with ©1 and were further
processed (cropping, resizing and global contrast and brightness
adjustment) with Adobe Photoshop (Adobe Systems). In the case of
©2 we used a 530−550 nm excitation filter and a 590 nm emission
filter.
Experiments with Retinas and with Live Mice. Animal retinas
were obtained from 3-month-old wild type SV129 mice originally
acquired from Charles River Laboratories. The animals were terminally
anesthetized, the eyes were enucleated, the retinas were dissected from
the eyecup, and flat mounts were prepared with the ganglion cell layer
uppermost. Then the retinas were incubated with a solution of caged
ethidium (©2, 12.5 μM in PBS buffer) or caged DAPI (©1, 5.0 μM,
PBS buffer). The uncaging was performed with a standard gel UV
transilluminator for ©2 (λ = 300−375 nm). In the case of ©1, it was
D
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found that the light of the microscope was intense enough to cleave
the photolabile groups. The irradiated samples were observed in a
fluorescence microscope equipped with a 530−550 nm excitation filter
and a 590 nm emission filter for the cells treated with ©2 and a 360−
370 nm excitation filter and 420 nm emission filter for the cells treated
with ©1. The same procedure was followed in the case of a human
retina obtained from a patient who underwent ocular evisceration
because of a painful terminal glaucoma. Once the retina was removed
from the eye, it was placed in DMEM containing 10% of FBS, 1%
penicillin/streptomycin, and 1% glutamate and and cut into several
pieces. Each piece was placed on a slide fluorescent microscope, and
the incubation procedure described above was followed. Consent
permission was obtained from the patient, and the procedure was
authorized by the Ethical Committee for Clinical Research of the
Xunta de Galicia.
For in vivo experiments the animals were anesthetized by
intraperitoneal injection of 4% chloral hydrate (400 mg/kg body
weight), and a drop of tropicamide solution (10 mg mL−1) was
instilled on the eyes to produce mydriasis. The right femoral vein was
cannulated, and 0.1 mL of a solution of caged DAPI (©1, 5.0 μM in
PBS) was injected. Then, the right eye was transpupillary irradiated (λ
= 300 to 375 nm). Finally, the animals were sacrificed by cervical
dislocation, the eyes were enucleated, and the retinas mechanically
dissected out were mounted and viewed in the fluorescence
microscope. All experiments involving animals were conducted
according to the Bioethical Committee of our institution and adhered
to the ARVO statement for the Use of Animals in Ophthalmic and
Vision Research.
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ASSOCIATED CONTENT
* Supporting Information
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The rule of five revisited: applying LogD in place of LogP in drug-
likeness filters. Mol. Pharmaceutics 4, 556−560.
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Nat. Rev. Mol. Cell Biol. 8, 414−420.
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(20) Janoira, K. G., Gunda, S., Boddu, S. H., and Mitra, A. K. (2007)
Novel approaches to retinal drug delivery. Expert Opin. Drug Delivery
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(21) Thrimawithana, T. R, Young, S., Bunt, C. R., Green, C., and
Alany, R. G. (2011) Drug delivery to the posterior segment of the eye.
Drug Discovery Today 16, 270−277.
■
S
Synthesis, fluorescence spectroscopy, and further experiments
with cells. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: joseluis.mascarenas@usc.es; eugenio.vazquez@usc.es.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(22) Gomez-Ulla., F., Gonzalez, F., and Torreiro, M. G. (1998)
Diode laser photocoagulation in idiopaticpolypoidalvasculopathy.
Retina 18, 481−483.
We are thankful for support given by the Spanish grants
SAF2007-61015, SAF2010-20822-C02, CTQ2009-14431/
BQU, Consolider Ingenio 2010 CSD2007-00006, and the
Xunta de Galicia INCITE09 209 084PR, GRC2010/12,
PGIDIT08CSA-047209PR. M.I.S. thanks the Spanish Ministry
of Education for FPU Ph.D. fellowships.
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