Light-responsive capture and release of DNA in a ternary supramolecular complex

Article abstract of DOI:10.1002/anie.201103707

The wavelength determines whether DNA is captured in a light-responsive ternary supramolecular complex or released (see scheme). The reversible binding of DNA is triggered by a photoisomerization, which switches the complex from a multivalent to a monoval

Full text of DOI:10.1002/anie.201103707

DOI: 10.1002/anie.201103707
Photoresponsive Systems
Light-Responsive Capture and Release of DNA in a Ternary
Supramolecular Complex**
Siva Krishna Mohan Nalluri, Jens Voskuhl, Jelle B. Bultema, Egbert J. Boekema, and
Bart Jan Ravoo*
One of the main advances in supramolecular chemistry has
been the progress from the general principles of molecular
recognition and self-assembly to applications in medicine and
material sciences. The correction of genetic defects and
disorders by gene therapy is an important challenge in
modern medicine. Cationic amphiphiles,[1] polymers,[2] and
dendrimers[3] have been investigated for their propensity to
transfect DNA and RNA in the form of nanoscale “lipo-
plexes” and “polyplexes”. Several approaches have been
proposed to develop transfection agents which also include a
release mechanism, such as release triggered by a change in
pH,[4] the reduction of disulfides,[5] and the light-induced
dissociation of covalent bonds.[6]
The light-induced release of DNA or RNA from a carrier
system is a most elegant way to combine efficiency, mild
conditions, and the absence of any additives. Photolabile
esters can easily be dissociated upon irradiation at 350 nm.[6,7]
On the other hand, also noncovalent complexes can be light-
sensitive. Azobenzenes constitute a well-known class of light-
responsive compounds that can be reversibly isomerized from
trans to cis form by irradiation at 350 nm and from cis to trans
form by irradiation at 455 nm. The inclusion of azobenzene as
a guest into a cyclodextrin host is light-responsive: the rodlike
and apolar trans isomer forms a stable inclusion complex with
a-cyclodextrin (a-CD) as well as with b-cyclodextrin (b-CD),
while the bent and polar cis isomer does not fit in either CD.
The light-controlled molecular recognition of CDs with
azobenzenes has been used to develop light-responsive
hydrogels,[8] molecular shuttles,[9] micelles and vesicles,[10]
surfaces,[11] and drug-delivery vehicles.[12]
binding mode to a low-affinity, monovalent binding mode.
The ternary system is based on bilayer vesicles of amphiphilic
CDs and the molecular recognition of guest molecules at the
surface of such host vesicles.[13] The inclusion of functional
guests at the surface of the CD vesicles offers a straightfor-
ward alternative to the cumbersome synthesis of CD poly-
mers and cationic amphiphilic CDs that have shown potential
in gene delivery.[14]
We have recently reported a binary supramolecular
system in which the photoisomerization of a divalent azo-
benzene linker can be used as a trigger to induce as well as
reverse the molecular recognition and adhesion of CD
vesicles in two directions: adhesion upon irradiation with
visible light (455 nm) and dissociation upon irradiation with
UV light (350 nm).[15] Herein, we describe the light-respon-
sive formation and dissociation of a ternary complex of
vesicles composed of amphiphilic CD 1, azobenzene–sper-
mine conjugate 2, and DNA.
Herein, we report the self-assembly of a ternary supra-
molecular system based on a light-responsive azobenzene–
cyclodextrin complex that can reversibly capture and release
DNA. The key innovation of this noncovalent complex is a
photoinduced transition from a high-affinity, multivalent
Azobenzene–spermine conjugate 2 binds to CD vesicles
through inclusion of the hydrophobic trans-azobenzene group
and to negatively charged DNA through an electrostatic
interaction of the positively charged spermine unit. The
surface of the CD vesicles decorated with conjugate trans-2
displays multiple protonated spermine groups and thus
exhibits high-affinity multivalent DNA binding. A ternary
complex (“supramolecular lipoplex”) is formed between CD
vesicles, conjugate trans-2, and DNA through multiple non-
covalent interactions. Upon UV irradiation, trans-2 isomer-
izes to cis-2, and conjugate cis-2 detaches from the vesicle
surface. As a consequence, the multivalent display of
spermine groups is disrupted and the resulting low-affinity
monovalent spermine units readily dissociate from the DNA.
Hence, the ternary supramolecular complex disassembles and
DNA is effectively released upon UV irradiation. Further-
more, upon visible-light irradiation, the conjugate cis-2
[*] S. K. M. Nalluri, J. Voskuhl, Prof. Dr. B. J. Ravoo
Organic Chemistry Institute and Graduate School of Chemistry
Westfꢀlische Wilhelms-Universitꢀt Mꢁnster
Corrensstrasse 40, 48149 Mꢁnster (Germany)
Dr. J. B. Bultema, Prof. Dr. E. J. Boekema
Department of Biophysical Chemistry, Groningen Biomolecular
Sciences and Biotechnology Institute, University of Groningen
Nijenborgh 7, 9747 AG, Groningen (The Netherlands)
[**] We thank the Graduate School of Chemistry in Mꢁnster (fellowship
to SKMN) and the Deutsche Forschungsgemeinschaft (grant Ra
1732/1-2) for financial support. We acknowledge COST Action
CM0703 “Systems Chemistry”.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201103707.
Angew. Chem. Int. Ed. 2011, 50, 9747 –9751
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Communications
Figure 1. The light-responsive formation and dissociation of a ternary complex of vesicles of composed of CD (a-CD 1a or b-CD 1b),
azobenzene–spermine conjugate trans-2, and DNA (50-mer single-stranded DNA (ssDNA) or salmon testes double-stranded DNA (dsDNA)
consisting of 2000 base pairs).
isomerizes back to trans-2 and the ternary complex is formed
again. The light-responsive ternary complex is illustrated in
Figure 1.
Amphiphilic a-CD 1a and b-CD 1b were synthesized as
reported previously.[13b,c] Unilamellar CD bilayer vesicles with
a diameter of 100–150 nm were prepared in buffer at pH 7.2
by extrusion.[13] Conjugate 2 was synthesized as reported in
the Supporting Information. The reversible photoisomeriza-
tion of conjugate 2 is shown in Figures S1 and S2 in the
Supporting Information. The interaction of conjugate trans-2
with a-CD and b-CD was measured by using isothermal
titration calorimetry (ITC). It was found that trans-2 forms
inclusion complexes with a-CD (Ka ꢀ 6 ꢀ 103 mÀ1) as well as
with b-CD (Ka ꢀ 4 ꢀ 103 mÀ1). The ITC data are summarized in
Table S1 in the Supporting Information.
The ternary supramolecular system of a-CD 1a, conjugate
trans-2, and single-stranded DNA (ssDNA) was investigated
in particular detail. The optical density (OD600) of a solution
of vesicles of a-CD 1a at a concentration of 30 mm is less than
0.05. When conjugate trans-2 is added to vesicles of a-CD 1a,
there is no change in either the OD600 or the average vesicle
diameter (Figure 2Aand 3A). However, the z-potential of
the vesicles increases from roughly À26 mV in the absence of
guest[13c] to about +11 mV upon addition of trans-2 (Fig-
ure 3C), indicating that the surface of the CD vesicles
displays a high density of positively charged spermine
groups. When 50-mer ssDNA is added to the binary mixture
of vesicles of a-CD 1a and conjugate trans-2, the OD600
Figure 2. Formation of a ternary complex of vesicles of a-CD 1a,
conjugate trans-2, and 50-mer ssDNA. A) Time-dependent measure-
ment of OD600. B) Maximum OD600 versus concentration of trans-2
and P/N charge ratio. Concentrations: [1a]=30 mm and
[ssDNA]=1.6 mm in aqueous buffer (2 mm HEPES and 10 mm EDTA at
increases from approximately 0.05 to 0.9 within 30 min
(Figure 2A). According to dynamic light-scattering (DLS)
measurements, the average particle size increases to more
than 1000 nm (Figure 3A). These observations indicate that
rapid aggregation of vesicles occurs in the ternary complex of
vesicles of 1a, conjugate trans-2, and ssDNA. In every other
combination (i.e. absence of vesicles of 1a, absence of
conjugate 2, and/or absence of ssDNA), no aggregation was
observed (see Figure S4 in the Supporting Information).
Furthermore, the z-potential of the ternary complex is
approximately + 0.4 mV (Figure 3C). The near-neutral z-
potential of the ternary complex indicates that positively
charged spermine-decorated vesicles and negatively charged
DNA aggregate upon charge neutralization. Indeed the rate
and extent of formation of the ternary complex is dependent
pH 7.2).
on the surface coverage of positively charged spermine
moieties at the vesicle surface. If less conjugate trans-2 is
added to the vesicles of a-CD 1a, the surface density of trans-
2 will be lower and hence the vesicles interact more slowly
with ssDNA and it takes longer before the aggregation
reaches a maximum OD600 (Figure 2A). The maximum
OD600 was plotted against the concentration of trans-2 and
the overall positive (P)/negative (N) charge ratio in the
ternary system. The P/N ratio was calculated by assuming that
there are 3 positive charges per trans-2 and 50 negative
charges for each 50-mer ssDNA. As shown in Figure 2B,
aggregation has a threshold P/N ratio close to 1.0.
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at 350 nm for 40 min decreases both the OD600 from roughly
0.52 to 0.12 and the average particle size from more than
1000 nm to about 200 nm (Figure 3A,B). The z-potential of
the remaining particles decreases from approximately
+0.4 mV to À20.6 mV (Figure 3C). UV irradiation induces
the photoisomerization of trans-2 to cis-2 (see Figures S1 and
S2) and the OD600, DLS and z-potential measurements
consistently indicate that the ternary system dissociates into
its components (CD vesicles, cis-2, and ssDNA) upon UV
irradiation. As shown in Figure 3B, the dissociation of the
ternary complex occurs within the first 20 min of UV
irradiation. The negative surface potential of the ternary
system after UV irradiation can be ascribed to the fact that
the positively charged spermine groups of cis-2 dissociate
from the surface of the vesicles, since only trans-azobenzenes
form inclusion complexes with a-CD. As a consequence, the
spermine groups are no longer displayed in a high-affinity
multivalent arrangement, and they readily dissociate from the
DNA since monovalent binding is insignificant at these
concentrations.[16] The negatively charged vesicles do not bind
to negatively charged ssDNA because of electrostatic repul-
sion. We conclude that ssDNA is released from the vesicle
surface upon UV irradiation of the ternary system.
Figure 3. Light-responsive formation of a ternary complex of vesicles
of a-CD 1a (30 mm), conjugate trans-2 (30 mm), and 50-mer ssDNA
(0.7 mm). A) Light-responsive size distribution according to DLS.
B) Time-dependent increase and decrease of OD600 under alternate
irradiation with UV light (350 nm) and visible light (455 nm). The inset
shows the reversible light-responsive formation of the ternary complex
induced by conjugate 2. C) z-Potential. All measurements were per-
formed in aqueous buffer (2 mm HEPES and 10 mm EDTA at pH 7.2).
Figure 4. Cryo-TEM images. A) Vesicles of a-CD 1a. B,C) Ternary com-
plex of vesicles of 1a, conjugate trans-2, and dsDNA. D) Vesicles of b-
CD 1b. E,F) Ternary complex of vesicles of 1b, conjugate trans-2, and
dsDNA. DNA strands are marked with black arrows.
Upon subsequent irradiation of the ternary system of
vesicles of a-CD 1a, conjugate cis-2, and ssDNA with visible
light at 455 nm for 40 min (to convert cis-2 to trans-2), both
the OD600 increases from approximately 0.14 to 0.53 and the
average particle size increases from roughly 200 nm to more
than 1000 nm (Figure 3A,B). The z-potential increases from
about À20.6 mV to À4.4 mV (Figure 3C). The OD600, DLS,
and z-potential measurements consistently indicate that the
ternary complex of vesicles of 1a, conjugate trans-2, and
ssDNA reassembles upon visible-light irradiation. In other
words, the light-induced formation and dissociation of the
ternary complex is reversible and thus allows the light-
responsive capture and release of low-molecular-weight
DNA. The reversibility of the light-induced formation of
the ternary complex is quantitative over two cycles provided
The critical role of charge neutralization was confirmed by
the fact that the addition of excess spermine·4HCl (50 mm)
leads to an immediate dissociation of the ternary complex:
excess spermine screens the electrostatic interaction and in
addition may act as a competitive monovalent binder to DNA
(see Figure S5 in the Supporting Information). Also the
addition of enzyme DNase I (30 UmlÀ1) leads to slow
dissociation of the ternary complex (see Figure S5 in the
Supporting Information). This observation can be explained
by the fact that the ternary complex dissociates upon
enzymatic hydrolysis of the captured DNA.
Most interestingly, UV irradiation of the ternary complex
of vesicles of a-CD 1a, conjugate trans-2, and 50-mer ssDNA
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Communications
that the irradiation time is sufficient (40 min at 350 nm and
In summary, a light-responsive ternary complex is formed
by self-assembly of three components: vesicles of amphiphilic
CDs, azobenzene–spermine conjugates trans-2, and 50-mer
ssDNA. The photoisomerization of azobenzene leads to a
light-responsive capture and release of ssDNA. The key
innovation in this ternary system is a light-induced switch
from a high-affinity multivalent display to a low-affinity
monovalent binding mode. We envisage that the incorpora-
tion of selective target ligands at the surface of vesicles[13d,e]
along with the light-responsive conjugates could make these
systems versatile for the capture, delivery, and release of low-
molecular-weight DNA and RNA to target cells for gene
therapy.
40 min at 455 nm), the vesicle concentration is limited to
30 mm (so that the maximum OD600 is about 0.5), and the
concentration of ssDNA is approximately 2.0 mm.
In the same way, experiments were performed with
vesicles of b-CD 1b (instead of a-CD 1a). The inclusion
complexes of trans-2 with a-CD and b-CD are of comparable
stability (see Table S1 in the Supporting Information), and the
addition of conjugate trans-2 and 50-mer ssDNA to vesicles of
b-CD 1b leads to the formation of a light-responsive ternary
supramolecular complex (see Figure S6 in the Supporting
Information). Also in this case, electrostatic complexation has
a threshold P/N ratio close to 1.0. Light-induced formation
and dissociation of the ternary complex is quantitatively
reversible and thus allows the light-responsive capture and
release of DNA.
Received: May 31, 2011
Revised: July 28, 2011
Published online: September 5, 2011
Also the interaction of CD vesicles (a-CD 1a as well as b-
CD 1b), conjugate trans-2, and double-stranded (ds) DNA
consisting of 2000 base pairs (instead of 50-mer ssDNA)
resulted in the formation of ternary complexes (see Figur-
es S7 and S8 in the Supporting Information). The average
particle size increases to more than 1000 nm. Complex
formation has a threshold P/N ratio close to 1.0. The P/N
ratio was estimated by assuming that there are 3 positive
charges per trans-2 and 4000 negative charges per dsDNA.
The z-potential of the ternary complex is close to neutral. The
formation of the complex was confirmed by using cryo-TEM
(Figure 4). It is evident from Figure 4A,D that CD vesicles
are spherical and unilamellar in the absence of guest.
However, after the sequential addition of trans-2 and
dsDNA, large aggregates of (multilamellar) vesicles and
dsDNA are observed due to multivalent noncovalent inter-
actions with conjugate trans-2. The vesicles form extensive
areas of close contact surrounded by long strands of dsDNA
(Figure 4B,C,E,F). It appears that DNA functions as an
electrostatic glue that induces the tight adhesion, flattening,
and stacking of CD vesicles, resulting in dense, multilamellar
complexes. Electrostatic complexation may result in signifi-
cant dehydration of the bilayer surfaces.
Unlike the light-responsive ternary system described for
ssDNA, UV irradiation of the ternary complex of CD vesicles
(a-CD 1a or b-CD 1b), conjugate trans-2, and dsDNA does
not have any significant effect on either the maximum OD600
(which remains roughly 0.40) or the average particle size
(which remains more than 1000 nm). UV irradiation of the
ternary complex decreases the z-potential from about
À0.42 mV to À15.4 mV. The decrease of z-potential can be
ascribed to the partial release of the positively charged
spermine unit of cis-2 from the vesicle surface. However, the
combination of OD600, DLS, and z-potential measurements
indicate that even after UV irradiation, a stable ternary
complex persists. We propose that the ternary complex of
vesicles, trans-2, and dsDNA is more robust owing to
increased multivalency and increased kinetic stability as a
consequence of the length of the dsDNA.[16] In other words,
the formation of the ternary complex is not reversible and
cannot be used to capture and release high-molecular weight-
DNA.
Keywords: azobenzenes · cyclodextrins · DNA ·
molecular recognition · vesicles
.
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It can be estimated that in a charge-neutral complex, ssDNAwill
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