A Janus-Type Phthalocyanine for the Assembly of Photoactive DNA Origami Coatings

Article abstract of DOI:10.1021/acs.bioconjchem.1c00176

Design and synthesis of novel photosensitizer architectures is a key step toward new multifunctional molecular materials. Photoactive Janus-type molecules provide interesting building blocks for such systems by presenting two well-defined chemical functionalities that can be utilized orthogonally. Herein a multifunctional phthalocyanine is reported, bearing a bulky and positively charged moiety that hinders their aggregation while providing the ability to adhere on DNA origami nanostructures via reversible electrostatic interactions. On the other hand, triethylene glycol moieties render a water-soluble and chemically inert corona that can stabilize the structures. This approach provides insight into the molecular design and synthesis of Janus-type sensitizers that can be combined with biomolecules, rendering optically active biohybrids.

Full text of DOI:10.1021/acs.bioconjchem.1c00176

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Article
A Janus-Type Phthalocyanine for the Assembly of Photoactive DNA
Origami Coatings
Asma Rahali,† Ahmed Shaukat,† Verónica Almeida-Marrero, Bassem Jamoussi, Andrés de la Escosura,
Tomás Torres,* Mauri A. Kostiainen,* and Eduardo Anaya-Plaza*
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ABSTRACT: Design and synthesis of novel photosensitizer
architectures is a key step toward new multifunctional molecular
materials. Photoactive Janus-type molecules provide interesting
building blocks for such systems by presenting two well-defined
chemical functionalities that can be utilized orthogonally. Herein a
multifunctional phthalocyanine is reported, bearing a bulky and
positively charged moiety that hinders their aggregation while
providing the ability to adhere on DNA origami nanostructures via
reversible electrostatic interactions. On the other hand, triethylene
glycol moieties render a water-soluble and chemically inert corona
that can stabilize the structures. This approach provides insight into
the molecular design and synthesis of Janus-type sensitizers that can be combined with biomolecules, rendering optically active
biohybrids.
zinc(II) Pcs (ZnPc) and DNA origami structures can be
assembled through electrostatic interactions, yielding enhanced
optical properties and origami protection against enzymatic
digestion.32 Thus, the development of Pc derivatives with
better architectural and functional control of their optical
properties is a desirable goal. Herein we describe the design
and synthesis of a Janus-type Pc with sharp differences in the
chemical nature of the functional groups placed at opposite
sides of the molecule. Furthermore, we show that the Pcs are
able to bind electrostatically on the surface of DNA origami
structures, which modulates their optical properties.
INTRODUCTION
■
Phthalocyanines (Pc) are porphyrinoid derivatives, composed
of four isoindole units connected through nitrogen atoms, and
an inner coordination site able to accommodate a wide variety
of metal atoms.1,2 The resulting extended aromatic surface is
responsible for their high extinction coefficient in the red/near-
infrared region of the UV−vis spectrum, which, together with
their rich electrochemistry and stability, makes them out-
standing candidates for a variety of applications in optoelec-
tronics,3−5 catalysis,6−8 sensing,2 and biomedicine.9−15 In the
latter, conjugation of Pcs and diverse biomolecules enhance
desired properties such as biocompatibility, biodistribution,
and targeting of photogenerated reactive oxygen species such
as singlet oxygen (1O2).16 Among the vast array of
biomolecules available, nucleic acid derivatives and, in
particular, DNA origami has been at the center of extensive
research in recent years.17−21 DNA origami preparation is
based on the folding of a long, circular single stranded DNA
(“scaffold DNA”) by customized short (15−60 nucleotides)
single stranded oligonucleotides (“staple strands”).22−24 This
technology yields almost any well-defined arbitrary nanoscale
shape in highly parallel fashion. Due to its multimodal nature
and nanometre-scale accuracy, DNA origami nanostructures
are directly relevant for drug delivery,25,26 imaging,21,27
biophysical studies,28,29 material science,30 and nanostan-
dards.31
RESULTS AND DISCUSSION
■
Design of the Janus-Type Moiety and DNA Origami.
The chemical design of novel Pcs results in derivatives with
enhanced performance for targeted applications. For instance,
axial or peripheral modification with hydrophilic groups
renders aqueous solubility of the otherwise hydrophobic
macrocycles.33,34 More refined architectures can provide
additional control on the interaction with the environment,
either by tuning their self-assembly or providing recognition/
targeting moieties. In this study, a Janus-type ZnPc (1) was
Received: April 6, 2021
Revised: May 11, 2021
Published: May 24, 2021
Hybrids of Pc with DNA origami have been scarcely
investigated despite the evident synergy between the moieties.
We have previously reported that symmetric octacationic
© 2021 The Authors. Published by
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designed, synthesized, and characterized. The synthesized
molecule displays a Janus-type architecture, presenting differ-
ent functional groups on opposite sides of the molecule: on
one side, a bulky and positively charged region, located on a
single isoindole; on the other side, a total of six triethylene
glycol chains on the remaining three isoindole moieties (Figure
1a), rendering a water-soluble, inert, and biocompatible
oligonucleotide staple strands.36 This yields a rigid cylindrical
nanostructure of 82 nm length and 16 nm diameter (Figure
1c). The 60HB synthesis was achieved adapting previously
reported methodology,37 using a 7249 nucleotide scaffold and
141 staple strands. This results in a brick-like structure with 20
nm height and 33 nm length (Figure 1d). For further details,
refer to SI.
Synthesis of the Janus-Type Molecule. The synthesis of
1 (Scheme 1) was achieved by statistical cyclotetramerization
of phthalonitrile A, containing four pyridyl groups, and B six
(neutral and biocompatible) triethylene glycol (TEG)
monomethyl ether chains. Briefly, A was prepared by
microwave-assisted nucleophilic aromatic substitution of 4,5-
dichlorophthalonitrile with 2,6-bis(3-pyridinyl)phenol.38 B was
prepared by Rosenmund−von Braun cyanidation of the
dibromo aromatic derivative (see SI for details). The statistical
cyclotetramerization of A and B was carried out at 140 °C for
12 h, in 2-dimethylaminoethanol (DMAE) with 1,8-diazabicy-
clo [5.4.0]undec-7-ene (DBU) as a base. The resulting AB3
derivative (2) was obtained in 15% yield, after separation from
the other ZnPc compounds formed (mostly, the B4 and the
ABAB/A2B2 derivatives) through column chromatography in
silica gel. A mixture of chloroform/methanol/pyridine
(100:5:1) was employed as eluent. Subsequent purification
was achieved by size exclusion chromatography performed in
Biobeads with chloroform as eluent. Last, methylation of the
pyridyl groups was performed with methyl iodide in dry DMF,
yielding 1 in 87% yield. Compound 1 was characterized by
means of 1H NMR in DMSO-d6, high-resolution mass
spectrometry (HR-MS), and infrared (IR) spectroscopy (see
SI).
Optical Characterization. To study the aggregation of 1,
the UV−vis absorption spectrum in DMSO, water, and 1x
phosphate-buffered saline (PBS) was recorded (see Figure 2a).
In DMSO, the spectrum showed an intense Q-band at 685 nm,
characteristic of nonaggregated monomeric ZnPc. In aqueous
medium, on the other hand, 1 presented a split Q-band, as
consequence of the asymmetric substitution pattern of the
macrocycle. Additionally, it shows a diminished absorption
coefficient (ε) and broader features of the Q-band than in pure
DMSO, with the absorbance maximum shifting to 690 and 694
nm in water and PBS, respectively. These spectral features are
indicative of molecular crowding due to the hydrophobic
nature of Pc, occurring to a different extent depending on the
conditions. However, no bathochromic shift to ca. 630 nm of
Figure 1. (a) Chemical structure of 1. (b) Schematic representation
of the bulky cationic groups hindering the cofacial aggregation.
Triethylene glycol monomethyl ether chains and iodide counterions
are omitted for clarity. Structures and dimensions of (c) 24HB and
(d) 60HB.
surface. The charged substitution pattern indeed provides a
rigid and bulky frame that hinders the typical aggregation of
these derivatives in aqueous media (Figure 1b). This ensures
that the outstanding photophysical properties of Pcs are
maintained and not quenched upon self-assembly.35 Addition-
ally, the four densely packed charges are suitable to undergo
electrostatic recognition with negatively charged DNA origami
structures. Here we have studied two model structures: 24- and
60-helix bundles (24HB and 60HB, respectively). The 24HB
was obtained by one-pot thermal annealing, folding a long
scaffold of 7560 nucleotides with the help of 202
a
Scheme 1. Synthesis of ZnPc 1
a
Asymmetric ZnPc 2 was obtained after separation from statistical cyclotetramerization of A and B in the presence of Zn(OAc)2. Cationic ZnPc 1
was obtained after quaternization of the pyridinyl moieties in the presence of MeI.
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to undergo exciton coupling, while the chemical design hinders
cofacial aggregation or H-type stacking.
Photoactivity. Finally, the photoinduced 1O2 quantum
yield (φΔ) of 1 was determined in DMSO, using the relative
method (see SI for details). The photodegradation of a
molecular scavenger such as 1,3-diphenylisobenzofuran
(DPBF) was recorded as a function of the irradiation time
1
(Figure 2b). It shows a O2-induced bleaching at 414 nm
(Figure 2b inset), while absorption of 1 remains intact,
indicating no relevant photobleaching of the dye. ZnPc 1
shows a φΔ of 0.44, calculated with respect to the
nonsubstituted ZnPc as reference (φΔ(DMSO) = 0.67). This
value highlights the efficient performance of 1 as photo-
sensitizer, despite the bulky peripheral decoration.
Figure 2. (a) UV−vis (solid) and emission (dotted) spectra of the
Janus-type ZnPc 1 in DMSO (black), water (blue) and PBS (red). [1]
= 3.6 (UV−vis) and 1.8 μM (emission). λexc = 655 nm. (b) DPBF
absorption decrease at 414 nm, photoinduced by 1 in DMSO. Inset:
plot of the DPBF absorption decay induced by 1 (black) and
reference ZnPc (red).
Biohybrid Characterization. The interaction between 1
and DNA origami was studied by UV−vis spectroscopy in
DMSO/water (1:9). In particular, the absorption of 1 at a
constant concentration of 7.5 μM was recorded in the presence
of increasing concentrations of 24HB (Figure 3a) and 60HB
(Figure S7a). The ZnPc showed no significant band shift.
However, by plotting the absorbance at 690 nm against the
24HB concentration (Figure 3b), two clear trends were
observed (similar results were obtained for 60HB). First, the
addition of 24HB up to 1.5 nM (10 000 equiv) results in a
decrease in the apparent extinction coefficient (ε). This
behavior, named binding asymptote, is the consequence of an
excess of 1 binding stepwise to the DNA origami (Figure 3c,
top). The saturation point (Figure 3c, middle) represents the
complex with densely packed ZnPc, showing an apparent
minimum ε. A further increase of the 24HB concentration (up
to 7.5 nM, 50 000 equiv) resulted in an apparent ε increase
(decluttering asymptote). In this regime, the available DNA
origami surface area increases, diminishing the molecular
packing of 1 (Figure 3c, bottom). This was further studied by
repeating the above-mentioned experiments in the presence of
increasing amounts of NaCl (Figure 3d and Figures S6 and
S7). The binding asymptote was practically nonexistent above
50 mM of NaCl, because of the salt-induced aggregation of
free 1 (Figure 3d). On the other hand, the decluttering
asymptote was maintained, pointing toward a clear binding
resilience when the ionic strength of the medium is high. This
can be explained by the formation of a stable, kinetically
the absorption maximum was observed, as traditionally shown
in cofacial ZnPc aggregation.39 The same behavior was
observed in water with increasing concentrations of NaCl
(Figure S5).
The fluorescence spectra of 1 was recorded in the same
three solutions (Figure 2a). In DMSO, 1 showed an emission
centered at 700 nm upon excitation at 665 nm. A fluorescence
quantum yield (φF) value of φF = 0.09 was calculated by
adapting the method of Williams (see Experimental Section),
irradiating the sample at 665 nm and with nonsubstituted
ZnPc as reference (φF = 0.18, Figure S1). In aqueous media,
both absorption and fluorescence decrease drastically. In water
(Figure 2a, blue), the absorbance/emission ratio is maintained
when compared to DMSO. This is a consequence of the
aggregation hindrance of the cationic groups, avoiding the
fluorescence quenching by exciton coupling. The same spectra
in 1x PBS buffer (Figure 2a, orange) show a clear decrease in
the fluorescence while revealing small changes in the
absorption spectrum. This effect can be explained by
agglomeration or crowding of 1 caused by the higher ionic
strength of 1x PBS. A similar effect was observed with pyrene-
substituted cationic ZnPcs.40 The molecules are close enough
Figure 3. (a) Absorption spectra of 7.5 μM 1 in the presence of 0−7.50 nM of 24HB. (b) Absorbance of 7.5 μM of 1 at 690 nm in the presence of
different 24HB concentrations. (c) Top to bottom: schematic representation of the 1−24HB hybrids at (A) 40 000, (B) 10 000 and (C) 2000
equiv of 1 per 24HB. (d) Absorbance of 7.5 μM of 1 at increasing amounts of 24HB and NaCl. The solvent used for all experiment is aqueous 10%
DMSO.
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trapped state promoted by the creation of a core−shell-like
structure. In this morphology, the charged interface between
ZnPc 1 and DNA origami is protected from the ionic strength
of the media by the hydrophilic yet neutral ethylene glycol
corona.
The stoichiometric ratio required for the complex formation
was determined by agarose gel electrophoretic mobility shift
assay (EMSA). A constant concentration of 24HB (2 nM) was
titrated with 1 to determine the maximum amount bound, i.e.,
the saturation ratio (Figure 4a). At increasing amounts of 1,
each positive charge of 1), 1−24HB complex formation
reverses, releasing intact 24HB as observed by the identical
mobility compared to intact 24HB (Figure 4c).
In this paper, the preparation of a novel Janus-type
photosensitizer has been reported. The chemical design,
presenting two well-defined chemical sites, renders a unique
structure with very specific functionalities. On one hand, the
bulky and positive moiety serves as an anchoring group for the
recognition of negatively charged biomolecules. At the same
time, it hinders the aggregation of ZnPc in a wide range of
aqueous media, including milli-Q water, high NaCl concen-
trations, or 1x PBS buffer. On the other hand, the water-
soluble yet neutral TEG chains provide aqueous solubility and
aid to maintain electrostatic interactions even in high ionic
strength, important for the future development of electrostati-
cally directed photoactive biohybrids and their stability in
biologically relevant media. When compared to nonsubstituted
ZnPc, 1 shows matching photophysical properties such as
1
fluorescence and O2 quantum yield.
ZnPc 1 shows efficient and reversible electrostatic binding to
DNA origami structures, which is a prerequisite for the
preparation of origami complexes relevant in photodynamic
therapy. The resulting biohybrids show remarkable resilience
toward disassembly triggered by the ionic strength of the
media. In summary, in this work we took the first steps toward
design and synthesis of Janus-type Pc materials with resilient
optical properties and targeted binding toward biomolecules.
Despite the complex stability in different aqueous media, the
binding occurs only in a narrow set of conditions, limiting their
application in biological media. Thus, refined structures are
desirable to, first, increase the binding affinity to biomolecules,
and second, to explore interactions other than electrostatics to
achieve better selectivity.
Figure 4. Agarose gel EMSA of (a) 24HB (2 nM) titrated with
increasing molar equivalents of 1. (b) 1−24HB complex (100 μM and
2 nM, respectively) in the presence of increasing NaCl concen-
trations. (c) 1−24HB complex (70 μM and 1.75 nM, respectively)
titrated with increasing amounts of heparin.
EXPERIMENTAL PROCEDURES
Materials. Chemicals and equipment are detailed in
Supporting Information.
Synthetic Protocols. DNA Origami. 24HB and 60HB
were obtained, purified, and characterized as described in
Supporting Information.
the intensity of the free DNA origami band decreased
gradually, until an excess at a 1/DNA origami molar ratio of
ca. 40 000. A similar experiment was carried out with 60HB
(Figure S3), yielding similar results.
■
To verify that the complexation is indeed electrostatically
driven, different NaCl concentrations were added before
mixing 1 and 24HB. A 1/DNA origami molar ratio of
40 000 was selected for this experiment, to ensure full coverage
of the DNA origami. The resulting EMSA indicated that 1
binds to the DNA origami up to 50 mM NaCl (Figure S4b,d).
Increasing the NaCl concentration above 100 mM hindered
the complexation, as revealed by the reappearance of an
intense DNA origami band, thus confirming the electrostati-
cally driven assembly. In contrast, addition of NaCl after
mixing the two components showed no effect on the complex
even at 300 mM (Figures 4b, S4c), which is above the
biologically relevant concentrations (i.e., 150 mM). This is in
good agreement with the results obtained by spectroscopy,
reinforcing the hypothesis of a core−shell morphology with the
nonionic triethylene glycol chains protecting the electrostati-
cally bound interface.
The reversibility of this interaction was characterized by
EMSA of 1−24HB, titrated with negatively charged heparin
(Figure 4c). This biopolymer presents a high density of
negative charges (2.7 sulfate groups per disaccharide),41,42
establishing a competitive binding with 1. Thus, the employed
18 kDa heparin molecules contain, on average, 90 negative
charges, which can bind up to 23 tetracationic ZnPc units. At a
high excess of heparin (300 negative charges of heparin for
Phthalonitrile A. It was synthesized adapting previously
reported methods.38
Phthalonitrile B. Into a two-necked round-bottom flask,
equipped with a stirrer, were placed 4,5-dibromo-1,2-di(tri-
(ethylenelycol) monomethyl ether (3 g, 5.35 mmol) and
copper cyanide (3.83 g, 0.042 mol). Dry DMF (80 mL) was
added, and the reaction was stirred 24 h at 150 °C. After that,
200 mL of NH4OH was added, and the solution was stirred for
24 h. Then the solution was extracted with ether, washed three
times with water, dried over MgSO4, and filtered, and the
solvent was vacuum evaporated, to afford the pure compound
1
as a colorless oil. Yield: 0.48 g, 20%. H NMR (300 MHz,
DMSO-d6): δ (ppm) = 7.73 (s, 2H; Har), 4.3−4.2 (m, 4H;
HarOCH2), 3.8−3.7 (m, 4H; OCH2CH2O), 3.60 (m, 4H;
OC2H4OC2H4OCH3), 3.5 (m, 8H; OC2H4OC2H4OCH3), 3.4
(m, 4H; OC2H4OC2H4OCH3), 3.22 (s, 6H; OCH3). 13C
NMR (75 MHz, DMSO-d6): δ (ppm) = 151.79, 117.34,
116.25, 107.38, 71.25, 70.02, 69.80, 69.57, 69.06, 68.51, 58.02.
FT-IR (film): v (cm−1) = 2870, 2227, 1740, 1587, 1515. MS
(FB+): Calcd for C22H32N2O8Na [M + Na]+: m/z: 475.2,
found 475.2.
ZnPc 2. Compound B (0.2 g, 0.44 mmol), compound A
(0.041 g, 0.06 mmol), and Zn(OAc)2 (0.022 g, 0.12 mmol)
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3
were placed into a sealed tube, equipped with a stirrer, and
DMAE (2 mL) and DBU (0.1 mL) were added. The reaction
was stirred 36 h at 140 °C. After that, the solvent was
evaporated, and the crude product was triturated with ether
and filtered. Then ZnPc 2 was purified on a chromatographic
column of silica gel, using CHCl3/MeOH (10:0.5) as eluent
with 1% of pyridine, followed size-exclusion chromatography
with Biobeads using CHCl3 as eluent, to afford a dark green
sticky solid. Yield: 0.015 g, 12%. 1H NMR (500 MHz, CDCl3):
δ (ppm) = 8.90−8.86 (bs, 4H; H2′′), 8.79−8.68 (bs, 4H;
H6′′), 8.07 (s, 2H; H3, H6), 8.02 (d, J = 4 Hz, 4H; H3′, H5′),
7.99 (s, 2H; H4′), 7.51 (s, 6H; Har), 6.74 (dd, J = 5 Hz, J = 8
Hz, 4H; H5′′), 4.7−4.5 (m, 12H; HarOCH2), 4.1−4.0 (m,
12H; OCH2CH2O), 3.9−3.8 (m, 12H; OC2H4OC2H4OCH3),
3.7 (m, 12H; OC2H4OC2H4OCH3), 3.6 (m, 12H;
OC2H4OC2H4OCH3), 3.5 (m, 12H; OC2H4OC2H4OCH3),
3.3−3.2 (m, 18H; OCH3). FT-IR (film): v (cm−1) = 2870,
1720, 1602, 1489. MS (HR-MALDI TOF): Calcd for
it was bubbled with O2 for 1 min. A solution of ZnPc 1 with
0.1 au of absorbance at the Q-band was then added. Using a
halogen lamp of 300 W, the mixture was irradiated under
stirring, for defined intervals of time, for a decrease of the
DPBF absorbance of 3−4% at 414 nm. A filter was used to
remove light under 530 nm (Newport filter FSQ-OG530), and
neutral density filters to remove light (FBS-ND03 or FB-
ND10) were used when it was necessary. Experiments were
performed in triplicate, expressing the final results as an
average. φΔ of ZnPc 1 was calculated using the following
equation:
kSIaRT
kRIaST
ϕS = ϕR
Δ
Δ
where φΔ is the fluorescence quantum yield, S is the sample, R
is the reference, k is the slope of a plot of ln(A0/At) versus
irradiation time, and A0 and At the absorbance of DPBF before
and after irradiation time (t) respectively. IaT is the total
amount of light that the dye absorbs and is calculated as a sum
of intensities of the absorbed light Ia at wavelengths from the
filter cutoff to 800 nm (step 0.5 nm). Ia at one determined
wavelength can be determined by the Lambert−Beer law:
C
106H120N12O26Zn [M]+: m/z: 2040.7723, found 2040.7746.
UV−vis (DMSO): λmax (nm) (log ε) = 679 (4.66), 614 (3.84),
365 (4.27).
ZnPc 1. ZnPc 2 (0.012 g, 5.8 μmol) was placed into a sealed
tube, equipped with a stirrer, and distilled DMF (2 mL) was
added under argon atmosphere. Then CH3I was added
carefully, and the reaction was stirred 12 h at 120 °C. After
that, the solvent was evaporated, and the crude product was
triturated with ether, filtered, and washed with MeOH, to
afford a green sticky solid. Yield: 0.013 g, 87%. 1H NMR (500
MHz, DMSO-d6): δ (ppm) = 9.33 (s, 4H; H2′′), 8.97 (s, 4H;
H6′′), 8.83 (s, 2H; H3, H6), 8.60 (s, 2H; H4′), 8.49 (d, J = 6
Hz, 4H; H3′, H5′), 8.28 (d, J = 8 Hz, 4H; H4′′), 7.78 (m, 6H;
Har), 7.69 (t, J = 8 Hz, 4H; H5′′), 4.7 (m, 12H; HarOCH2),
4.18 (s, 12H; CH3), 4.07 (s, 12H; OCH2CH2O), 3.8 (m, 12H;
O C 2 H 4 O C 2 H 4 O C H 3 ) , 3 . 7 − 3 . 6 ( m , 1 2 H ;
OC2H4OC2H4OCH3), 3.6 (m, 12H; OC2H4OC2H4OCH3),
3.5−3.4 (m, 12H; OC2H4OC2H4OCH3), 3.2 (m, 18H;
OCH3). FT-IR (film): v (cm−1) = 3435, 3009, 2926, 2778,
2562, 2504, 2459, 2424, 1715, 1632, 1604. MS (HR-MALDI
TOF): Calcd for C110H132N12O26Zn [M]4+: m/z: 525.2161,
found 525.2171; calcd for C110H132N12O26ZnI [M + 3I]3+: m/
z: 742.5898, found 742.5909; Calcd for C110H132N12O26ZnI2
[M + 2I]2+: m/z: 1177.3373, found 1177.3394. UV−vis
(DMSO): λmax (nm) (log ε) = 685 (4.79), 614 (4.05), 365
(4.80).
Ia = I0(1 − e−2.3A
)
where A is the absorbance of the photosensitizer at the
determined wavelength, and I0 the transmittance of the filter at
the same wavelength.
Biohybrid Formation. Electrophoretic Mobility Assay.
The ZnPc−DNA origami complexes were prepared by mixing
the DNA origami solution (final concentration of 2.0 nM) with
increasing amounts of ZnPc, rendering ZnPc/DNA origami
ratios from 500 to 50 000. The final DMSO concentration was
adjusted to 10% for all samples. The mixtures were incubated
at room temperature for 45 min. The total sample volume was
20 μL, and 4 μL of gel loading dye (6×) was added before
loading 22 μL of the total sample into the gel pockets. The gel
was visualized using a BioRad ChemiDoc MP Imaging system,
and the gel was excited at 532 nm (Alexa 546) and 633 nm
(Alexa 647) (Figure S3).
UV−Vis Spectroscopy. The change in the absorbance
spectra of ZnPc in the presence of DNA origami structures
and NaCl was studied with UV−vis spectroscopy. The samples
were initially prepared using 100 μL PCR tubes and then
transferred to a clear flat-bottom 96-well plate. The ZnPc
concentration was kept constant at 7.5 μM, while the DNA
origami concentration varied between the samples to obtain
ZnPc/DNA origami ratios of 1000, 2000, 5000, 10 000,
20 000, 30 000, 40 000, and 50 000 which correspond to final
DNA origami concentrations of 7.5, 3.75, 1.5, 0.75, 0.38, 0.25,
0.19, and 0.15 nM, respectively. Samples without DNA origami
structures were also prepared as reference. First, the DNA
origami solution was diluted in their respective 1× FOB to
obtain the desired DNA origami concentration, after which
ZnPc was added. The final DMSO concentration was adjusted
to 10% for all samples. The samples were incubated for 45 min
before NaCl was added in final concentrations of 50, 100, 200,
and 500 mM.
Methods. Fluorescence quantum yield was measured by
adapting the method of Williams,43 using the following
equation:
2
i
j
j
j
j
j
j
y
z
z
z
z
z
z
i
j
j
y
z
z
z
z
z
η
Quot
QuotR
S
F
R
F
S
S
ϕ = ϕ j
j
2
j
k
ηR
{
k
{
where ΦF is the fluorescence quantum yield, S is the sample,
and R is the reference. Quot is the quotient of the integrated
fluorescence intensity and the absorption at the excitation
wavelength of the sample, and η is the refractive index of the
solvent. ΦF of ZnPc in DMSO is 0.18. ZnPc 1 and ZnPc were
irradiated at 665 nm.
Singlet Oxygen Quantum Yield. φΔ has been calculated
using a relative method, where 1,3-diphenylisobenzofuran
Reversible ZnPc−DNA Origami Binding. To recover the
DNA origami and dissociate the binding process, heparin
sodium salt was used. The 1−24HB complexes were prepared
by mixing the 24HB solution (final concentration of 1.75 nM)
with 1 (final concentration of 70 μM) to obtain a ZnPc 1/
1
(DPBF) decomposes by the presence of O2. Nonsubstituted
ZnPc was used as reference, that presents a φΔ(DMSO) = 0.67.
Three milliliters of a stock solution of DPBF (OD ca. 1) in
DMSO was placed into a 10 × 10 mm quartz optical cell, and
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24HB ratio of 40 000. The mixture was incubated at room
temperature for 45 min. After the incubation, different
amounts of heparin sodium salt were added to obtain final
concentrations of 933 and 1400 μM, which corresponds to
approximately 300- and 450-times higher charge concentration
than that of ZnPc 1. After addition of heparin, loading dye (6
μL) was added before loading the sample into the gel. The
ZnPc−DNA origami complexes were prepared by mixing the
DNA origami solution (final concentration of 2.0 nM) and the
required amount of NaCl to prepare samples with final NaCl
concentrations of 20, 50, 100, 150, 200, and 300 mM. The final
DMSO concentration was adjusted to 10% for all samples.
After that, ZnPc (final concentration of 100 μM) was added at
a ZnPc/DNA origami ratio of 50 000 before the mixture was
incubated at room temperature for 90 min (Figure S4a,b). In
another gel, only the order of mixing was changed. NaCl was
added after the formation of the complex, i.e., after the initial
incubation of ZnPc−DNA origami complex for 45 min,
different amounts of NaCl (same as above) were added. The
sample was then incubated for an additional 45 min. The total
sample volume was 20 μL, and 4 μL of gel loading dye (6×)
was added before loading 22 μL of the total sample into the gel
pockets (Figure S4c,d).
Bassem Jamoussi − Department of Environmental Sciences,
Faculty of Meteorology, Environment and Arid Land
Agriculture, King Abdulaziz University, 21589 Jeddah, Saudi
Arabia
Andrés de la Escosura − Department of Organic Chemistry,
Universidad Autónoma de Madrid (UAM), 28049 Madrid,
Spain; Institute for Advanced Research in Chemical Sciences
(IAdChem), Universidad Autónoma de Madrid (UAM),
28049 Madrid, Spain; orcid.org/0000-0002-0928-8317
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.bioconjchem.1c00176
Author Contributions
†These authors contributed equally.
Funding
We acknowledge the Academy of Finland (286845, 308578),
the Magnus Ehrnrooth Foundation, and the Marie Sklodow-
ska-Curie grant (794536). MINECO-Feder funds (CTQ2017-
85393-P (TT), CTQ-2014-53673-P and CTQ-2017-89539-P
(AdlE), and PCIN-2017-042/EuroNanoMed2017-191, TEM-
PEAT (TT)).
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.bioconjchem.1c00176.
■
sı
ACKNOWLEDGMENTS
■
We acknowledge the provision of facilities and technical
support by Aalto University Bioeconomy Facilities and
OtaNano − Nanomicroscopy Center (Aalto-NMC) and
funding from the Jane and Aatos Erkko Foundation. The
authors also thank Sofia Julin and Heini Ijäs from Department
of Bioproducts and Biosystems, Aalto University, for providing
DNA origami samples. IMDEA Nanociencia also acknowl-
edges support from the “Severo Ochoa” Programme for
Centres of Excellence in R&D (MINECO, grant SEV-2016-
0686).
Chemicals, equipment, DNA origami folding protocols,
biohybrid formation and dissociation, EMSA gels, and
absorption spectra (PDF)
AUTHOR INFORMATION
Corresponding Authors
Eduardo Anaya-Plaza − Department of Bioproducts and
Biosystems, Aalto University, 02150 Espoo, Finland;
orcid.org/0000-0001-9944-6907;
■
ABBREVIATIONS
■
Email: eduardo.anaya@aalto.fi
1
Mauri A. Kostiainen − Department of Bioproducts and
Biosystems, Aalto University, 02150 Espoo, Finland;
orcid.org/0000-0002-8282-2379;
Pc, phthalocyanine; O2, singlet oxygen; HB, helix bundle;
DPBF, diphenylbenzofurane; φF, fluorescence quantum yield;
φΔ, singlet oxygen quantum yield
Email: mauri.kostiainen@aalto.fi
Tomás Torres − Department of Organic Chemistry,
Universidad Autónoma de Madrid (UAM), 28049 Madrid,
Spain; Institute for Advanced Research in Chemical Sciences
(IAdChem), Universidad Autónoma de Madrid (UAM),
28049 Madrid, Spain; IMDEA-Nanociencia, 28049 Madrid,
Spain; orcid.org/0000-0001-9335-6935;
Email: tomas.torres@uam.es
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