Functionalization of Fatty Acid Vesicles through Newly Synthesized Bolaamphiphile-DNA Conjugates

Article abstract of DOI:10.1021/bc500289u

The surface functionalization of fatty acid vesicles will allow their use as nanoreactors for complex chemistry. In this report, the tethering of several DNA conjugates to decanoic acid vesicles for molecular recognition and synthetic purposes was explored. Due to the highly dynamic nature of these structures, only one novel bola-amphiphile DNA conjugate could interact efficiently with or spontaneously pierce into the vesicle bilayers without jeopardizing their self-assembly or stability. This molecule was synthesized via a Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (click reaction), and consists of a single hydrocarbon chain of 20 carbons having on one end a triazole group linked to the 5′-phosphate of the nucleic acid and on the other side a hydroxyl-group. Its insertion was so effective that a fluorescent label on the DNA complementary to the conjugate could be used to visualize fatty acid structures. (Chemical Equation Presented).

Full text of DOI:10.1021/bc500289u

Article
pubs.acs.org/bc
Functionalization of Fatty Acid Vesicles through Newly Synthesized
Bolaamphiphile−DNA Conjugates
†
†
†
‡
‡,§
Michael C. Wamberg, Rafał Wieczorek, Søren Bo Brier, Jan Willem de Vries, Minseok Kwak,
‡
,†
Andreas Herrmann, and Pierre-Alain Monnard*
†Center for Fundamental Living Technology (FLinT), Department of Physics, Chemistry and Pharmacy, University of Southern
Denmark, Campusvej 55, DK-5230 Odense M, Denmark
‡
Department of Polymer Chemistry, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG
Groningen, The Netherlands
§
Department of Chemistry, Pukyong National University, Busan 608-737, South Korea
*
S Supporting Information
ABSTRACT: The surface functionalization of fatty acid vesicles
will allow their use as nanoreactors for complex chemistry. In
this report, the tethering of several DNA conjugates to decanoic
acid vesicles for molecular recognition and synthetic purposes
was explored. Due to the highly dynamic nature of these
structures, only one novel bola-amphiphile DNA conjugate could
interact efficiently with or spontaneously pierce into the vesicle
bilayers without jeopardizing their self-assembly or stability. This
molecule was synthesized via a Cu(I)-catalyzed [3 + 2] azide−
alkyne cycloaddition (click reaction), and consists of a single
hydrocarbon chain of 20 carbons having on one end a triazole
group linked to the 5′-phosphate of the nucleic acid and on the
other side a hydroxyl-group. Its insertion was so effective that a
fluorescent label on the DNA complementary to the conjugate could be used to visualize fatty acid structures.
INTRODUCTION
the vesicle surface can improve their functionality, for example,
by allowing the assembly of a complex catalyst or by tethering
two nanoreactors together.
Fatty acid vesicles are stabilized by hydrogen bonds between
protonated and ionized carboxylic acid headgroups, and are
■
The functionalization of self-assembled soft matter structures
with molecules that are able to undergo specific recognition is
being intensively pursued to develop applications in medicine,
material science, and synthetic biology.
between supramolecular assemblies and functional molecular
agents can be realized either by means of physical association
14,27
1
−9
Tight interactions
stable around a pH close to the pK of the acid, e.g., from pH
a
28
7.0 to 7.2 for decanoic acid (DA). They are also characterized
by higher critical vesicle concentrations (CVC) and higher
exchange rates of molecules between assemblies and solution
compared to similar phospholipid vesicles. Both parameters
(
noncovalent interactions) or by chemical derivatization of the
molecular agents with an amphiphilic moiety, such as
1
0
11
12
phospholipids, block copolymers1, and cholesterol. The
2−15
22
latter approach is usually preferred
as it leads to specific
increase with decreasing hydrocarbon chain length. These
properties lead to a higher sensitivity of fatty acid vesicles to
fluctuations in pH, ionic strength, and temperature, as well as
the presence of solutes in the suspension, all of which can
induce transient vesicle permeabilization or in extreme cases
complete structure disruption. The more dynamic bilayer
membranes, however, allow for morphological studies of the
vesicle alterations resulting from uptake of additional
and durable interactions. However, most of the examples in
biosensing or medicine are dealing with phospholipid lip-
osomes, e.g., composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine (POPC).
Our interest is to achieve functionalization of fatty acid
nanostructures with polyelectrolytes (nucleic acids, peptides or
sugars). Fatty acids are simple single-chain amphiphiles with an
acid headgroup and are capable of forming vesicular
23
1
6,17
amphiphile molecules or the in situ production of new
structures.
These vesicles can be used in synthetic
19
1
8,19
amphiphiles, as during photocatalysis.
chemistry (as nanoreactors) or in medical applications for
targeted drug delivery. Fatty acid vesicles have also been
studied as models for primitive membranes,
20
1
8,21−25
due to their
Received: July 1, 2014
simple chemical structure and their availability in meteoritic
organic compounds. In every application, functionalization of
Revised: August 18, 2014
26
©
XXXX American Chemical Society
A
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Figure 1. (a) Synthetic scheme of amphiphile fabrication employing azide units and a 5′-hexynyl substituted DNA 15-mer that are connected by
click chemistry. (b) Overview of bolaamphiphile structures and related derivatives studied in this project. DA structure added for comparison. Gray
rectangles denote regions of expected hydrophilic headgroup placement. Note that the transmembrane arrangement of oligo-1, 2, and 4 is
postulated; oligo-4 is still under investigation as it might adopt a folded conformation and reside in one monolayer only.
In earlier work, we have designed a self-replicating chemical
system, a protocell, which is based on the functionalization of
DA fatty acid membranes to carry out photochemical
previously derivatized molecules. Indeed, short nucleic acids are
relatively well solubilized in an aqueous medium and their
amphiphilic derivatives can self-assemble on their own, e.g., into
2
9
31
reactions. For efficient catalysis to occur, anchoring of various
components in the membrane was essential. Metal-catalyzed,
photochemical conversion of DA precursors was demonstrated
on the surface of the vesicles using simple aliphatic hydro-
carbon chains (10 carbons long). Such a simple hydrophobic
moiety was sufficient for almost quantitative anchoring the
various functional components of the photocatalytic unit, i.e.,
the metal complex photosensitizer, ruthenium(II) tris-(bipyr-
layers at the interface between water and air or into
32
micelles. Furthermore, repulsive electrostatic interactions
between the negatively charged vesicle surfaces and the
amphiphilic nucleic acids could negatively impact their
insertion in the membranes.
To achieve the insertion of DNA in DA bilayers, two possible
types of anchoring moieties were considered (i) prototypical
DNA amphiphiles with multiple linear anchoring moieties (i.e.,
30
idine), and its relay donor, an 8-oxoguanine nucleobase.
DNA with 2 single hydrocarbon chains incorporated on two
29
31,32
According to the protocell concept, its reaction network
should be controlled by replicable information molecules. That
is, molecules containing an information sequence that can be
chemically copied, e.g., nucleic acid oligomers instead of a
single nucleobase (8-oxoguanine). To further develop our
model, our catalytic system therefore requires the tethering of
nucleic acids onto the membrane surface to allow for sequence-
specific functionalization of the electron relay. The stable
tethering of DNA oligomers, which are replicable and can
support the photochemistry in our system, would permit the
nucleobases
) and (ii) novel nucleic acid conjugates
consisting of an ssDNA and a bolaamphiphile anchoring
moiety. The nature of the bolaamphiphile tether could lead to a
better retention of the anchored DNA, because such an
amphiphilic molecule has a hydrophilic group at both ends of
33
its hydrophobic core. Thus, it could potentially interact with
the fatty acid headgroups on both sides of the bilayer
(transmembrane anchoring), provided the length of the
hydrophobic core was comparable to the thickness of the
bilayer: In the case of decanoic acid, estimates of the bilayer
29
34
18,35
realization of a complete protocell. Furthermore, it should
find applications in molecular targeting of nanocompartments
by bioaffinity as the DNA sequence would direct a specific
interaction with complementary strands, or even in the direct
delivery of chemicals encapsulated in the aqueous lumen of
thickness in the literature range from 22−26 Å to 28 Å.
To assess more rapidly the importance of various parameters
in the insertion process, a synthetic route via click chemistry
(Figure 1a) was chosen. This synthetic approach enabled us to
prepare different amphiphile and bolaamphiphile DNA
conjugates in a modular fashion by only employing one DNA
strand and various other organic moieties that can serve as an
anchor (Figure 1b). As the metal catalyst in this Cu(I)-
8
vesicles or that of the tethered oligomers themselves.
The tethering of nucleic acids onto the negatively charged
fatty acid vesicles posed a more complex challenge than the
B
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3
6−38
catalyzed [3 + 2] azide−alkyne cycloaddition (CuAAC)
tethered and a higher DNA fraction was associated with the
vesicles (Table 1) compared to nonamphiphilic salmon sperm
DNA (unspecific association). A relatively effective, specific
association of dU11 and dU20 with POPC vesicles was also
detected.
39
can be harmful to DNA, the copper(I) stabilizing ligand tris-
benzyltriazolylmethyl)amine (TBTA) was utilized to protect
40
(
the DNA from degradation during synthesis.
The incorporation of the chosen and synthesized DNAs
derivatized with hydrophobic moieties into DA vesicles was
assessed using two different methods for the insertion: post-
vesiculation insertion (classical method), where the conjugates
have to spontaneously insert into the bilayer themselves (i.e.,
pierce the bilayers), and insertion during vesiculation aimed at
promoting their association with the bilayers during self-
assembly. The insertion efficiency was gauged by using a
fluorescently labeled DNA with complementary sequence to
that of the bolaamphiphile DNA conjugates and determining
whether its hybridization could enable the direct visualization of
the DA membranes.
Table 1. Insertion Rate (%) of Prototypical Lipid DNA
Conjugates dU11 and dU20 and Unspecific Interaction of
a
Salmon Sperm DNA
lipid-DNA amphiphiles
(
%)
vesicle type
dU11
dU20
DNA (salmon sperm) (%)
Oleic acid
POPC
19.5
50
5.5
37
10.5
11.0
a
In all cases, 0.5 mg/mL DNA was used. Considering the amphiphile
concentrations, molar ratios of amphiphile to Lipid-DNA amphiphiles
were as follows: For dU11, 130:1 and 36:1 with oleic acid (17.9 mM)
and POPC (5 mM), respectively. For dU20, 230:1 and 130:1.
However, POPC and oleic acid concentrations (1:3) ensured
comparable vesicle surfaces (considering headgroup areas and
CVCs). Standard deviations: max 5% units.
RESULTS AND DISCUSSION
■
Incorporation of Prototypical Lipid-DNA into Fatty
Acid and POPC Vesicles. The aim of this study was to tether
single- and double-stranded DNA to decanoic acid vesicle
membranes and gain insight into the parameters that influence
the stability of the newly formed complexes. To this end, we
first tested the incorporation of lipid-DNA, a prototypical DNA
amphiphile. In this system, hydrophobic units, dodec-1-yne
chains, were introduced at the 5-position of uracil. The
resulting modified nucleoside (Figure 2) could be incorporated
Thus, the hydrocarbon chain anchors of dU11 and dU20 did
not facilitate the insertion of the negatively charged DNA
sequence into the targeted DA fatty acid membranes, and even
led to the disruption of some vesicles. The insertion of the
amphiphilic DNA within the fatty acid structures depends on a
balance between the energy cost of solubilization in the buffer,
in particular, for the hydrophobic moiety of the conjugates
(
dodec-1-yne chains), and the entropy gained by the systems
when these hydrophobic chains reside in the hydrophobic core
of the DA bilayers (the hydrophobic effect). The repulsive
interactions between the negatively charged vesicle surface and
the oligomer polyelectrolyte backbone could, however, have
canceled out the entropic gain. The absence of functional
groups in the hydrophobic moiety might have also contributed
to the result, as it would preclude additional stabilizing
interactions between the fatty acid headgroups and the
anchoring moiety. The vesicle disruption, and to some extent
the precipitation, could then be consistent with the formation
of mixed “micelles”, which have already been reported for these
Figure 2. Prototypical DNA intended to associate with the exterior of
lipid containers through hydrophobic anchors (hydrocarbon chains, in
red over a gray background). (a) Structure of the precursor of
modified dU amidite. (b) DNA sequences of the two prototypical
DNA-lipids: the derivatized U residues are in bold with their
hydrocarbon chains schematically represented.
32
two DNA conjugates.
These orienting experiments clearly underlined the need for
a novel, better-suited anchoring moiety in the case of fatty acid
vesicles. We therefore designed and synthesized four different
DNA conjugates (oligo-1, -2, -3, and -4; Figure 1b), using click
chemistry, which was recently reviewed in the context of DNA
into oligonucleotides by standard solid phase synthesis
procedures and the hydrophobic residue did not impair the
ability of the DNA amphiphiles to undergo sequence specific
3
2
41
base pairing. As other DNA amphiphiles, lipid-DNAs self-
conjugation strategies. The bolaamphiphile chemical struc-
32
assemble into micelles in buffer medium or form layered
tures of oligo-1, oligo-2, and oligo-4 were chosen to match the
reported thickness of the DA bilayer (between 22 and 28
3
1
structures at the air/water interface. Two lipid DNA
molecules were investigated regarding their incorporation into
fatty acid vesicles. The first one was a 20mer DNA sequence
with two hydrophobically modified deoxyuridines (dU) placed
in the center of the DNA strand (dU20), which forms a
hairpin. The second molecule was a 11mer with two
hydrophobic Us at its 5′-terminus (dU11).
3
4,35
Å),
which equals 18 carbons between the headgroups.
They, however, differed in the nature of their hydrophobic core,
as well as in the chemical functionality of their potential
headgroups (Figure 1b), which could all potentially form
hydrogen bonds with the acid headgroups on both sides of the
bilayer.
Assuming that the resulting bolaamphiphile anchor spans the
whole DA bilayer, the polar triazole ring, which results from the
conjugation step, could either be buried in the hydrophobic
core of the DA bilayer (oligo-1 and oligo-2) or act as a
headgroup (oligo-3 and oligo-4) (Figure 1). The structure of
oligo-3 resembles that of both oligo-1 and oligo-2 in terms of
Insertion experiments were first carried out with DA vesicles
30 mM) and the DNA amphiphiles, but no significant
(
insertion yields could be detected. Moreover, a precipitate was
observed upon DNA addition, indicating that some DA vesicles
were disrupted by the conjugates (data not shown). By
contrast, for the oleic acid vesicles the short dU11 could be
C
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Scheme 1. Synthesis Scheme for the Preparation of Azide 4
the hydrocarbon chain length, but lacks a second hydrophilic
group. This design aimed at investigating the influence of the
were conducted to assess the validity of the bolaamphiphile
anchoring approach, two aiming at the investigation of the
preparation methods and their influence on the incorporation.
The third type aimed at gauging the extent and strength of the
insertion into vesicle bilayers over time, as well as other
parameters such as the feasibility of hybridization of a single
stranded DNA to its complementary sequence once anchored
in the vesicles.
“bola”-nature, i.e., the necessity of a second hydrophilic group,
on insertion and retention of the conjugates in the bilayer.
Additionally, it could also shed light on the influence of
interactions between anchors and headgroups when compared
to the lipid−DNA conjugates. In fact, it could use either the 5′-
phosphate or the triazole ring as headgroup, although the
former arrangement would force the triazole ring inside the
hydrophobic core of the membrane.
To detect the insertion and the retention of the derivatized
DNAs, two single stranded DNA sequences were labeled with
an Alexa-488 fluorescent dye at their 5′-end: master-1 oligomer
was a 15-mer ssDNA with complementary sequence to the
bolaamphiphile-DNA conjugate sequence, while the master-2
oligomer consisted of a noncomplementary sequence.
In each type of experiment, the presence of DA vesicles (total
concentration 27 mM DA, i.e., 5 mM amphiphiles forming
Synthesis of the Bolaamphiphile−DNA Conjugates.
Hexyn-5-yn-1-O-(2-cyanoethyl-N,N-diisopropyl)phosphor-ami-
dite was synthesized in moderate yields according to El-Sagheer
4
2
et al. and was attached at the 5′-end of a 15-mer DNA
oligonucleotide (Figure 1a) by use of an automatic DNA
synthesizer using standard conditions. The coupling yield
corresponded to that of common DNA amidites.
18
structures if the CVC of 22 mM is considered ) was first
confirmed in all investigated samples using Nile red as staining
reagent. The insertion of the DNA conjugates was then
evaluated using the fluorescence of the Alexa-488 on the master
oligomers. A representative set of micrographs is shown in
Figure 3.
4
3
1
2-Azidododecanoic acid was synthesized from 12-
bromododecanoic acid in 93% yield using a procedure reported
4
4
45,46
by Diaz et al., while 11-azido-1-undecanol
and 1-
4
7
azidodecane were synthesized from 11-bromo-1-undecanol
and 1-bromodecane, respectively, according to literature
procedures. Yields were 78% and 98%, respectively.
20-Azidoicosane-1-ol (4) was synthesized in four steps from
eicosanedioic acid (Scheme 1). First, eicosanedioic acid was
converted to its dimethyl diester derivative 1 according to
48
literature procedures. 1 was then reduced to 1,20-icosanediol
4
8,49
(2)
using LiAlH in THF in moderate yields (59%). The
4
diol 2 was converted to 20-bromoicosan-1-ol (3) using aqueous
HBr (48% in H O) in petroleum ether (bp. 100−140 °C)
2
50
according to a literature procedure (yield = 36%). Finally, 20-
azidoicosane-1-ol (4) was synthesized by azidification of 3 with
NaN in DMF in 72% yield.
3
CuAAC click chemistry was carried out as described in the
51
“
click chemistry manual” by BaseClick using CuBr/TBTA in
52−54
the ratio 1:2. This is an optimized method
which also has
been proven successful for other oligonucleotides. The
synthesis yields for each conjugate are reported according to
Optical Density equivalents (OD) in Table 2. Analysis by RP-
Figure 3. Chemical systems for the detection of the anchoring of DNA
to DA vesicles: (a) master-1 oligomer is complementary to the
anchored DNA chain (vesicular structures are observed after
hybridization); (b) master-2 is not complementary and does not
hybridize with the anchored oligomer (dark “shadow” vesicles are
observed due to the contrast with fluorescent master-2 present in
solution). Scale bar 10 μm).
a
Table 2. Synthesis Yields for Oligos -1 to -4
starting DNA amount
recovered amount of DNA
conjugate (OD)
(OD)
%
oligo-1
oligo-2
oligo-3
oligo-4
15
15
15
15
9.7
11.7
12.4
12.2
65%
78%
83%
81%
The localization of the fluorescence in the micrographs
Figure 3) permitted a qualitative assessment of the DNA
(
a
The recovered amounts for each DNA conjugate were determined
conjugate insertion in the DA vesicles. In the case of oligo-4/
master-1 mixtures, the fluorescence was mostly associated with
DA bilayers compared to a darker background. The negative
control with master-2 was successful, as the fluorescence
remained localized in the buffer, without any significant
accumulation on the surface of the structures. In fact, the
structures could only be observed because the master-2 was
added in the external medium and did not diffuse into the
structure’s aqueous lumina. When comparing the micrographs
after the desalting procedure.
HPLC and MALDI-TOF mass spectrometry confirmed the
successful 1,3-dipolar Huisgen cycloaddition between alkyne
and azide (see Supporting Information, Table SI-1 and
spectra).
Incorporation of Bolaamphiphile−DNA Conjugates
into Decanoic Acid Vesicles. Three types of experiments
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Figure 4. Representative images obtained with the incubation of DA vesicles with different bolaamphiphile DNA anchors (oligo-1, 2, 3, and 4) and
different reporter oligomers (master-1 and 2). Two different methods of preparation, “classical” and “dehydration−rehydration”, are presented. Scale
bar 10 μm.
of DA vesicle sample incubated with only one of the master
sequences, in the absence of DNA conjugates, the same overall
characteristics were observed (data not shown). That is,
unspecific interactions between the bilayers and DNA, which
were previously observed with the salmon sperm sample, could
be excluded here.
Thus, the label location ensured the possibility of detecting
only a positive bolaamphiphile insertion that does not
compromise the structure stability. Furthermore, these results
indicate that this method can be applied to gauge insertion
yields, as a good insertion would lead to a fluorescent
highlighting of the self-assembled structures. To better
understand the insertion dynamics of the DNA conjugates,
two preparation methods were investigated: classic and
dehydration−rehydration.
conjugate and reporter oligomer. The microscopic investigation
of the samples revealed that oligo-1, oligo-2, and oligo-3 did not
show any incorporation into DA vesicles, as only dark circular
“shadows”, the aqueous lumen of the vesicles, could be
observed in a fluorescent background (Figure 4). By contrast,
membranous structures were highlighted by fluorescent labeling
in samples containing oligo-4 as DNA membrane anchor. As
expected, the incubation of oligo-1, oligo-2, oligo-3, and oligo-4
with master-2 (noncomplementary sequence) did not result in
the fluorescent labeling of the membranous structures.
The different behavior of the various amphiphile anchors
could have been caused by different insertion rates in the
bilayer, due to both the nature of the hydrophobic cores or that
of the headgroups (acid versus hydroxyl group). Studies of the
flip−flop rates of fatty acid molecules in bilayers have
5
5,56
Post-Vesiculation Insertion−Classic Method. The in-
corporation of bolaamphiphile−DNA conjugates into the DA
vesicle membrane was first studied post-vesiculation. DA
vesicles prepared by pH vesiculation were incubated with a
conjugate solution that consisted of the prehybridized DNA
established that only neutral species (protonated
or
57
associated with cations ) are able to cross the hydrophobic
cores of bilayers. Thus, by analogy, protonation of the oligo-2
acid group should in principle improve its spontaneous
insertion into the bilayers. As an equilibrium between the
E
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deprotonated and protonated species existed at the exper-
imental pH, a prolonged incubation period could have
improved the insertion. However, measuring after 24 and 96
h incubation periods, the only noticeable change was the
progressive disappearance of the shadow vesicles, as DA
vesicles fragmented and reformed and the labeled oligo-2
diffused into the vesicular aqueous lumina. Only the oligo-4
system still showed clearly outlined membranes on a dark
background (Figure 5).
The results obtained with both preparation methods can be
summarized as follows: oligo-4 could spontaneously insert in
the bilayers of preformed vesicles and be stably incorporated
during vesiculation. By contrast, the other investigated
anchoring systems were not incorporated to such an extent
that bilayers could be visualized by the fluorescence on the
tethered DNA system.
22
From the oligo-4 chemical structure, its extensive incorpo-
ration can be explained by the presence of a long hydrocarbon
chain that fits well in the hydrocarbon core of the DA bilayer as
expected from their similar number of carbons. As a result, the
triazole ring and hydroxyl functions were correctly positioned
to interact with the acid headgroups of the DA bilayer, probably
leading to additional stabilization (Figure 1b). The hydroxyl
headgroup was expected to be advantageous for the
spontaneous insertion, as it is not charged at any pH (unlike
the acid). In addition, it should have a constructive interaction
with the fatty acid headgroups, as the stabilization of fatty acid
59−61
vesicles by fatty alcohols has been previously reported.
Figure 5. DA vesicles containing oligo-4 and master-1 visualized over
different periods of time. Scale bar 10 μm.
The different behavior of the other anchors can be explained
with similar arguments. The shorter hydrocarbon chain of the
azido-moiety, used as starting material in the click synthesis of
oligo-1 and -2, likely resulted in positioning of the triazole
group in the hydrophobic core of the bilayers. The charged 5′-
phosphate of the DNA and the hydroxyl or acid headgroup
should then be able to interact with the DA headgroups. In
additional experiments where the bolaamphiphile−DNA
conjugates and master-1 were added at two different stages of
the preparation, i.e., the conjugate during dehydration/
rehydration procedure and the master sequence after the
vesicle formation, the results with oligo-1 and oligo-2 indicated
that a slightly better stabilization could be achieved with an acid
headgroup (data not shown). Assuming that the anchors
Upon addition of Nile red to the suspension containing
oligo-1, vesicles and tubes were revealed (Figure 6) that were
invisible using the master-1 oligomer as sole fluorescent marker.
experienced a similar increase of their pK within the structures
a
as fatty acids do, a large fraction of their acid functions should
be deprotonated at the pH used in all experiments (pH 7.2).
The charged headgroup would then have slowed down the
dissociation from the bilayers. However, it would also have
decreased the rate of insertion during spontaneous insertion
experiments.
Figure 6. DA vesicles containing oligo-1 and master-1. Left: in green
light sensitive channel showing “shadow” vesicles. Right: in red light
sensitive channel (with addition of Nile red). Scale bar 25 μm.
As any stable insertion into the bilayers relies on the correct
balance between the stabilization of the hydrophobic moiety of
the amphiphile and the cost of its solvation in the aqueous
medium, it seems that the triazole function within the bilayer
weakened the hydrophobic interactions and led to the
dissociation of the bolaamphiphile−DNA conjugate from the
bilayers. Thus, the disruption of the packing of bilayer
membranes could not or only partially be counterbalanced by
the stabilization due to headgroup interactions. The hydro-
phobic incompatibility (mismatch) of triazole inside the
hydrophobic core of the membrane was already reported for
Insertion during VesiculationDehydration−Rehy-
dration Method. The “dehydration/rehydration” preparation
5
8
method was employed to force the association of the
bolaamphiphile−DNA conjugate with DA bilayers. Because of
the bilayer fragmentation that occurs during the procedure, the
association of the conjugates with charged headgroups should
have been facilitated and their retention improved during the
rehydration procedure, thereby leading to higher insertion
yields.
The results of this preparation procedure confirmed the
extensive insertion of oligo-4 into the DA vesicles (Figure 4).
The other DNA conjugates (oligo-1, oligo-2, oligo-3) employed
during the procedure yielded interesting results. Contrary to
the post-vesiculation method, bright structures, whose sizes and
shapes could have corresponded to vesicles (Figure 4), were
observed in all cases. However, an unambiguous observation of
the vesicle bilayer structure (bright circumferences of the
vesicle with a darker internal lumen) cannot be reported. That
is, the presence of these DNA conjugates likely led to the
formation of new structures, such as oil droplets, micelles, or
even crystals.
6
2
phospholipids by Smith’s group. Furthermore, recent
1
8,63,64
investigations
of the insertion of polycylic aromatic
hydrocarbons in fatty acid vesicles have established that only
minute amounts of these molecules can be inserted into these
bilayers, before the molecules in excess are expelled into the
medium. Finally, the correct positioning of these anchors (i.e.,
the triazole embedded within the bilayer hydrophobic core and
the 5′-DNA phosphate interacting with the acid group of DA)
would have brought the negatively charged phosphates of DNA
backbones into closer proximity to the negatively charged
vesicle surfaces, thereby further weakening the anchor insertion.
F
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Oligo-3 lacked a second headgroup and could not insert into
the membrane even though it could have in principle interacted
through either its phosphate or its triazole group with bilayer
hydrophilic headgroups (Figure 1b). That is, the improvement
of the electrostatic interactions with the hydrophilic interface of
the bilayers (compared to the lipid-DNAs dU11 and dU20)
was not sufficient to shift the association/dissociation
equilibrium of the conjugate. This result underlined the
importance of anchor hydrophobic length in relation to the
bilayer thickness (i.e., the importance of the hydrophobic effect
and van der Waals interactions): The hydrophobic chain of C10
was too short to stabilize the insertion of such a 15mer ds DNA
polyelectrolyte system in the highly dynamic DA vesicle
membrane.
Our data however do not exclude the possibility of the
formation of micelle-like structures or even large oil-like
systems around the amphiphile anchors as the results of the
dehydration−rehydration methods with oligo-1 to 3. This
formation is even more plausible if the results of experiments to
separate nonassociated bolaamphiphile−DNA conjugates from
vesicles are taken into consideration (data not show). Indeed,
the free DNA conjugates could not even pass through 30 kDa
pores.
Hybridization of Oligonucleotides to DNA Amphi-
philes Inserted into DA Vesicles. Oligo-4 showed good
insertion into DA vesicles. The microscopic images of this
system clearly depicted membranous structures of various
shapes and sizes (Figures 4 and 5, Supporting Information
video 1) showing that hybridization of oligo-4 easily integrated
into fatty acid bilayers.
In the next step, experiments elucidating the ability of the
fluorescently labeled DNA to hybridize to the anchored
complementary DNA were performed. When master-1 solution
was added to preformed DA vesicles containing oligo-4
molecules, we initially observed dark “shadow” vesicles.
However, with time, master-1 molecules migrated to vesicle
surfaces and hybridized to the complementary oligo-4
sequence, giving rise to good microscopic images of vesicular
structures. This process is illustrated in Supporting Information
video 2. The prolonged incubation also highlighted the
retention of anchors within the vesicle bilayers (Figure 5).
Such a system could be used to observe the hybridization
process in real time.
containers after their formation) and, in the case of nucleic
acids, the preservation of the hybridization capabilities will be
important. Functionalized vesicle surfaces will allow for
molecular recognition. Thus, such nanoreactors should find
application in synthesis of chemicals and/or their delivery, as
well as in protocell models.
EXPERIMENTAL PROCEDURES
■
Syntheses. General Materials and Methods in Synthesis.
All reactions were monitored by analytical thin layer
chromatography (TLC) using precoated TLC plates DC-
Alufolien 60 F254 from Merck. UV-light or a stain of
(
NH ) Mo O ·4H O/Ce (SO ) (50:1) in 5% sulfuric acid
4 6 7 24 2 2 4 3
was used for visualization. Column chromatography was
performed using Merck Kieselgel 60 (0.040−0.060 mm).
Anhydrous THF was distilled over sodium benzophenone
immediately prior to use. CH Cl was distilled prior to use.
2
2
Anhydrous pyridine and DMF were dried over molecular sieves
4 Å), while anhydrous MeCN was dried over 3 Å sieves. All
(
other reagents and solvents came from commercial sources and
were used as received. Melting points (mp) were determined
̈
on a Buchi 535 melting point apparatus and are uncorrected.
High resolution electrospray ionization (ESI) mass spectra
were recorded on an Applied Biosystems/MDS Sciex Q-star
pulsar spectrometer. NMR spectra were recorded on a Bruker
Avance III instrument using the deuterated solvent as lock.
Chemical shifts are reported in ppm relative to tetramethylsi-
1
lane as internal standard for H NMR or the deuterated solvent
13
CDCl (δ 77.00) as internal standard for C NMR.
3
Synthesis of the Novel Bolaamphiphile−DNA Conjugates.
The synthetic pathway of 20-azidoicosane-1-ol (4) is presented
in Scheme 1.
Dimethyl icosanedioate (1) and 20-bromoicosan-1-ol (3)
4
8,50
were synthesized according to literature procedures.
Icosane-1,20-diol (2). LiAlH (2.05 g, 54.0 mmol, 2 equiv)
4
was suspended in anhydrous THF (100 mL) under N on an
2
ice bath. Dimethyl icosanedioate (1, 10.0 g, 27.0 mmol) was
dissolved in anhydrous THF (60 mL) and added dropwise. The
icebath was removed and the reaction mixture was stirred at rt
overnight. The reaction was quenched by carefully adding H O
2
(2.0 mL), 15% NaOH solution (2.0 mL), and H O (6.0 mL)
2
dropwise, while cooling on an ice bath. The resulting slurry was
filtered through a pad of Celite and washed with CHCl . The
3
In this study, the incorporation of two types of amphiphilic
DNA conjugates into single hydrocarbon chain amphiphile
bilayer structures was investigated. The anchoring of 15mer ds
DNA into decanoic acid vesicles was achieved even though
these structures are extremely dynamic systems. From the 6
candidates (2 prototypical lipid-DNA and 4 DNA conjugates
whose synthesis by click chemistry is reported here), only one,
oligo-4, showed strong association with the bilayers. The
anchoring was strong enough to visualize the vesicle structures
using the fluorescent label attached to the complementary
DNA strand. The presence of two headgroups on oligo-4, a
triazole and a hydroxyl group, flanking a hydrophobic core
whose length was adjusted to match the thickness of the fatty
acid bilayer, was clearly crucial for its stable incorporation into
the bilayer. As such our modular synthetic strategy has been
shown to be easily adaptable to other bilayer types, because the
hydrophobic part of the amphiphile can be easily adjusted to its
counterpart in the membrane.
organic phase was washed with H O and the solvent was
2
removed under reduced pressure to give 2 as a white solid (5.03
49
g, 16 mmol, 59%). Mp 94−99 °C (lit. 95 °C). NMR spectra
4
8,49
in accordance with literature data.
337.3078 (MH ).
MS (ESI): m/z =
+
20-Azidoicosane-1-ol (4). To a solution of 20-bromoicosan-
1-ol (3, 0.250 g, 0.66 mmol) in anhydrous DMF (5 mL) was
added NaN (0.085 g, 1.30 mmol, 2 equiv). The reaction
3
mixture was stirred at 80 °C for 40 h under N . After cooling to
2
rt, water (10 mL) was added and the mixture was extracted
with Et O (3 × 15 mL). The combined organic phases were
2
evaporated and the residue was taken up in CH Cl /H O (3:2,
2
2
2
25 mL). The phases were separated, and the aqueous phase was
extracted with CH Cl (2 × 15 mL). The combined organic
2
2
phases were dried (Na SO ), evaporated under reduced
2
4
1
pressure, and dried in vacuo. White solid (0.162 g, 72%). H
NMR (400 MHz, CDCl ): δ 1.26−1.36 (m, 32H, 16 × CH ),
3
2
1.64−1.52 (m, 4H, 2 × CH ), 3.25 (t, 2H, J = 7.0 Hz, CH N ),
2
2
3
13
The demonstrated spontaneous insertion of polyelectrolytes
into fatty acid vesicles (i.e., the modification of the vesicular
3.63 (t, J = 6.6 Hz, 2H, CH OH). C NMR (101 MHz,
2
CDCl ): δ 25.72, 26.69, 28.81, 29.13, 29.42, 29.46, 29.52, 29.58,
3
G
dx.doi.org/10.1021/bc500289u | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
9.60, 29.64, 29.66, 32.79 (12 × CH ), 51.47 (CH N ), 63.05
Article
2
corresponding nucleoside building block are depicted in Figure
2. Two amphiphilic DNA strands were synthesized; one was a
20mer sequence with two central lipid-modified nucleotides
(dU20) and another 11mer with two terminal hydrophobic
modifications (dU11).
Assessment of the Incorporation of Amphiphile−
DNA Conjugates into Membranes. General Materials and
Methods for Preparation of Vesicles. Sodium hydroxide
solution (1 M), hydrochloride solution (1 M), decanoic
2
2
3
+
(
(
CH OH). HRMS (ESI): m/z calcd for C H N ONa
2 20 41 3
+
MNa ): 362.3142, found 362.3140.
Oligonucleotide Synthesis. All modified oligonucleotides
were synthesized using standard automated solid-phase
̈
phosphoramidite coupling methods on an AKTA oligopilot
plus (GE Healthcare) DNA synthesizer in 50 μmol scale. All
solvents and reagents for oligonucleotide synthesis were
purchased from Novabiochem (Merck, UK) and SAFC
(
Sigma-Aldrich, Netherlands). Solid supports (Primer Sup-
(
capric) acid (DA), cholic acid, oleic acid, and Nile red were
purchased from Fluka, Switzerland. Phosphate buffer
Na HPO ) was obtained from Sigma, USA. 1-Palmitoyl-2-
portTM, 200 μmol/g) from GE Healthcare were used for the
synthesis of DNA. Oligonucleotides were purified by anion
exchange high pressure liquid chromatography (HPLC) using a
HiTrap Q HP 5 mL column (GE Healthcare) through custom
gradients using elution buffers A: 25 mM Tris-HCl pH 8.0 and
B: 25 mM Tris-HCl pH 8.0 and 1.0 M NaCl. Fractions were
desalted using centrifugal dialysis membranes (MWCO 3000,
Sartorius Stedim). Afterward the oligonucleotides were
characterized by MALDI-TOF mass spectrometry (MS) using
a 3-hydroxypicolinic acid matrix. Spectra were recorded on an
ABI Voyager DE-PRO MALDI TOF (delayed extraction
reflector) Biospectrometry Workstation mass spectrometer.
The concentrations of the DNA were measured on a
SpectraMax M2 spectrophotometer (Molecular Devices,
USA) using 1 cm light-path quartz cuvette.
(
2
4
oleoyl-sn-glycero-3-phosphocholine (POPC) was purchased
from Avanti Polar Inc. (USA).
16
Vesicles were prepared by pH vesiculation for the fatty
acids (DA in 100 mM phosphate buffer, pH 7.2, and oleic acid
in 100 mM Trizma buffer, pH 9.0) or film hydration for POPC
100 mM Trizma buffer, pH 9.0). For studies of prototypical
lipid-DNA insertion into fatty acid and POPC vesicles, the
vesiculation was followed by size homogenization by extrusion
hand-held mini-extruder from Avanti Polar Inc., USA,
sequential use of 400 nm polycarbonate filters from Whatman
plc, UK).
(
(
In brief, to prepare 1 mL DA (30 mM) vesicle suspension in
phosphate buffer (100 mM), 3.81 mg DA was weighed and
suspended in 500 μL phosphate buffer (200 mM, pH 7.0). 1 M
NaOH solution was used to reach pH above 9.5, and the
resulting sample was briefly heated to 43 °C. Finally, the sample
was acidified with 1 M HCl to a pH 7.2 and Milli-Q water was
added to obtain the final suspension volume of 1 mL.
Microscopy. All coverslips and glass slides were treated with
dimethyldichlorosilane (PlusOne Repel-Silane ES, GE Health-
care Life Sciences) in order to improve the hydrophobicity of
the surface. A Nikon Eclipse TE2000-S inverted fluorescence
microscope (Nikon, Japan), with a mercury lamp as the light
source and a filter set, as well as a 100× oil-immersion
fluorescence APOCHROMAT objective coupled with a
multiplication factor of 1.6× on the camera was used. The
open-source software package μManager was used. Movies
were obtained using the Multi-Dimensional Acquisition
window, each comprising 100 images taken at 50 ms intervals.
The stage position list window was used if the same coverslip
position had to be found more than once.
Microscopy assessment with Nile red (excitation and
emission filters: 540−585/600−650 nm) was used to confirm
the presence of DA vesicles, especially prior to further
experiments with a vesicle sample. 5 μL vesicle solution was
pipetted onto a glass slide and 0.5 μL Nile red was applied to a
coverslip.
Post-Oligo Synthesis. Click chemistry on oligonucleotides
was carried out according to the “Click chemistry manual” by
51
BaseClick GmbH, Germany.
The “click solution” (0.1 M CuBr/0.1 M TBTA (1:2) in
DMSO/t-BuOH (3:1)) was prepared by mixing the following
two freshly prepared solutions: CuBr (1 mg) in 70 μL DMSO/
t-BuOH (3:1) and 140 μL of the TBTA-solution (54 mg in 1
mL DMSO/t-BuOH (3:1)).
To the freshly prepared “click solution” (30 μL) a 2 mM
DNA solution (0.1 μmol in 50 μL H O) was added followed by
2
the azide (12-azidododecanoic acid, 11-azido-1-undecanol, 1-
azidodecane or 4; 1.0 μmol in 50 μL DMSO). The mixture was
thoroughly mixed and shaken at rt for 4 h using an Eppendorf
Mixmate. The reaction was subsequently quenched with 3 M
NaOAc (100 μL) and the DNA was precipitated from cold
EtOH (1 mL) in a freezer overnight. The vial was centrifuged,
the supernatant was removed, and the pellet was washed with
cold EtOH (2 × 1 mL). The pellet was dried in a centrifugal
evaporator, Speedvac, (RVC-18 CD plus, Martin Christ
Gefriertrocknungsanlagen GmbH, Germany with a chemically
resistant membrane pump vacuubrand MZ 2C NT, from
Vacuubrand GmbH, Germany), redissolved in pure water (1
mL), and desalted on a NAP-10 resin column from GE
Healthcare. After drying in a speedvac, the oligo was
redissolved in double deionized water. The identities of click-
modified oligonucleotides were confirmed by MALDI-TOF MS
on a Bruker Daltonics Microflex LT using a 3-hydroxy picolinic
acid matrix and by analytical RP-HPLC carried out on a Waters
65
Spontaneous Membrane Association of dU11, dU20, and
Unspecific DNA. The prototypical DNA amphiphiles dU11 and
dU20 or sonicated DNA (sonicated salmon sperm DNA
resulting in on average 500 nt long fragments) were incubated
600 system with a Waters 600 controller, a Waters 2996 PDA
(
final concentration: 0.5 mg/mL) with the vesicle suspension
detector, and a Waters 717 autosampler on an XBridge OST
C18 2.5 μm, 19 × 100 mm column with an XBridge Prep C18
(final concentrations: 30 mM DA, 17.9 mM oleic acid or 5.0
mM POPC) at 23 °C for 24 h, before being subjected to a gel
separation column (Bio-Gel A-1.5m from Bio-Rad Laborato-
ries, Inc., USA) to remove the free DNA from the suspension.
The vesicles were destroyed by addition of 1.8 molar excess of
cholate (150 mM stock solution) or by increasing the pH to 11
for the POPC and fatty acid systems, respectively. UV
measurements were corrected for residual scattering.
5
5
μm, 10 × 10 precolumn using a 38 min linear gradient of 0−
3% MeCN in 0.05 M triethylammonium acetate (pH 7.4) at a
flow rate of 2.50 mL/min.
Synthesis of the Prototypical Lipid−DNA Conjugates
(
dU11 and dU20). Lipid-DNA was prepared as described
31,32
earlier.
The sequences of the two DNA amphiphiles with
hydrophobic units attached to the uracil base and the
H
dx.doi.org/10.1021/bc500289u | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
Spontaneous Membrane Association of Bolaamphiphile−
DNA Conjugates. To evaluate the association of the
bolaamphiphile−DNA conjugates (oligo-X), master-1 and
master-2 oligonucleotides were purchased from Invitrogen,
USA. The sequence of master-1 was complementary to the
incorporated 15mer and was 5′-CGC GAT CGC GCC TTT-3′
with fluorochrome Alexa-488 (ex 495/em 519) attached to the
AUTHOR INFORMATION
Corresponding Author
Fax: +(45) 6615 8760; Tel: +(45) 6550 4437; E-mail:
■
*
monnard@sdu.dk.
Author Contributions
Michael C. Wamberg and Rafał Wieczorek have decided to
publish this paper with shared first authorship (in alphabetical
order).
5
′ end to qualitatively assess the association by fluorescence
microscopy (excitation and emission filters: 455−490/499−540
nm). The sequence of master-2 was noncomplementary to the
incorporated 15mer and was 5′-GCG CTA GCG CGG AAA-3′
with fluorochrome Alexa-488 attached to the 5′ end.
Notes
The authors declare no competing financial interest.
Post-Vesiculation InsertionClassic Method. 9 μL of the
ACKNOWLEDGMENTS
■
3
0 mM DA vesicle suspension was incubated for half an hour at
We are grateful for many fruitful discussion and suggestions
from colleagues at the University of Southern Denmark,
especially Anders Utoft Madsen, Philipp M.G. Loffler, and
̈
a slightly elevated temperature (30 °C) with the mixture of two
oligomers: 1.8 μL (57 mM) bolaamphiphile−DNA conjugate
(
oligo-1, -2, -3, or -4) and 1.7 μL (57 mM) detection oligomer
Pernille Lykke Pedersen. We thank Tina Grubbe Hansen,
BioNEC Research Center at University of Southern Denmark
for MALDI-TOF and RP-HPLC analysis of the oligonucleo-
tides. The research leading to these results has received funding
from the Danish National Research Foundation through the
Center for Fundamental Living Technologies (FLinT, headed
by center leader prof. Steen Rasmussen), from the European
Community’s Seventh Framework Programme (FP7/2007-
2013) under grant agreement no. 249032 (MATCHIT), and
from the European Community’s Seventh Framework Pro-
gramme (FP7/2007-2013) under Grant Agreement no.
222422, ECCell − Electronic Chemical Cell (a project in the
EU FP7-IST-FET Open Initiative). A.H. acknowledges
financial support from the EU through the ERC starting
grant NUCLEOPOLY and the ChemThem grant from NWO.
(
master-1 or 2). This procedure was carried out to ensure
enough time for the association process with the membranes.
The final concentrations were 8.9 mM of a bolaamphiphile−
DNA conjugate, 8.4 mM master-1, and 22 mM decanoic acid.
This solution was then used directly in microscopic studies.
Alternatively, oligo-X and detection oligomer were first
incubated in an Eppendorf tube for 30 min at 30 °C in order to
facilitate hybridization. Subsequently, 9 μL 30 mM DA were
added to the Eppendorf tube, followed by incubation for 30
min at 30 °C. The final concentrations in all cases were 8.9 mM
oligo-1, 8.4 mM master-1, and 22 mM decanoic acid. This
solution was then used directly in microscopic studies.
58
Dehydration−Rehydration Method. Samples were pre-
pared similarly to the post-vesiculation method, using 0.5 mL
Eppendorf tubes. Nitrogen gas was then gently applied through
the tubes for about half an hour, until all liquid had evaporated.
Milli-Q water was then added to reach the former volume.
Finally, the samples were sonicated for 5 min, vortexed,
centrifuged, and left for half an hour for equilibration at 30 °C.
This solution was then used directly in microscopic studies.
Time-Resolved DNA Hybridization on Functionalized
Decanoic Acid Vesicles. The solution of DA vesicles was
prepared as described above, but only with oligo-4
bolaamphiphile DNA without reporter DNA oligo (master).
ABBREVIATIONS
■
dd water, double deionized water; dsDNA, double stranded
DNA; C10, alkane hydrocarbon chain with the length of 10
carbon atoms; CuAAC, Copper-catalyzed azide−alkyne cyclo-
addition; CVC, critical vesicle concentration; DA, decanoic
acid; DMF, dimethylformamide; EtOH, ethanol; OD, optical
density equivalents; MALDI-TOF, matrix assisted laser
desorption/ionization−time-of-flight; MeCN, acetonitrile;
MeOH, methanol; pK , logarithmic acid dissociation constant;
a
3
μL of vesicle solution was added to the glass slide and 0.4 μL
RP-HPLC, reverse phase high-performance liquid chromatog-
master-1 solution (57 mM) was applied to the coverslip. The
final concentrations were ∼8 mM oligo-4, ∼7 mM master-1,
and ∼22 mM DA. This setup was then observed under a
microscope in the green channel (master-1 emission) as
described above. Within the first 2 min the first visible
structures were observed and recorded as time point 0.
Subsequently, the sample was observed for the total duration of
raphy; ssDNA, single stranded DNA; TBTA, tris-
(
benzyltriazolylmethyl)amine; t-BuOH, tert-butyl alcohol
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