Thymidine-based amphiphiles and their bonding to DNA

Article abstract of DOI:10.1039/c3nj41062e

To develop non-cationic vectors for gene delivery, a novel class of thymidine-based amphiphiles have been constructed. These molecules showed good biocompatibility. Their complexation with polyadenylic acid through hydrogen bonding and π-π stacking interaction was demonstrated by gel electrophoresis, capillary electrophoresis, transmission electronic microscopy (TEM) and molecular dynamics simulation. It was also found that their biocompatibility and binding ability with DNA varied inversely with the length of the alkyl chains.

Full text of DOI:10.1039/c3nj41062e

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Thymidine-based amphiphiles and their bonding
to DNA†
Cite this: NewJ.Chem., 2013,
37, 1122
Delin Pan, Chen Tang, Xinmeng Fan, Yating Li, Xiantao Yang, Hongwei Jin,
Zhu Guan, Zhenjun Yang* and Lihe Zhang
To develop non-cationic vectors for gene delivery, a novel class of thymidine-based amphiphiles have
been constructed. These molecules showed good biocompatibility. Their complexation with polyadenylic
acid through hydrogen bonding and p–p stacking interaction was demonstrated by gel electrophoresis,
capillary electrophoresis, transmission electronic microscopy (TEM) and molecular dynamics simulation.
It was also found that their biocompatibility and binding ability with DNA varied inversely with the
length of the alkyl chains.
Received (in Montpellier, France)
22nd November 2012,
Accepted 22nd January 2013
DOI: 10.1039/c3nj41062e
www.rsc.org/njc
Nucleoside-based amphiphiles, which have a double func-
tionality based on the combination of nucleic acids and lipid
Introduction
Gene therapy has gained significant attention as a potential
method for treatment of both acquired and inherited dis-
eases.1,2 Naked nucleic acids are easily degraded when used
either in vitro or in vivo, so vectors are employed in many
methods to protect nucleic acids and deliver them to targeted
cells. Viruses are efficient vectors for transportation of genes.
However, clinical applications of these vectors are restricted
due to their toxicity and immunogenicity.3 Consequently, var-
ious kinds of synthetic nonviral vectors have been investigated,
including cationic lipids, dendrimers, peptides, and polymers.4
A large number of novel cationic lipids have been designed and
assayed in transfection protocols, where nucleic acids bind to
cationic liposomes because of the charge interaction between
the negatively charged phosphate backbone of DNA and catio-
nic headgroup of the lipid.5 Cationic liposomes are of great
interest as gene transfection agents, but there are still signifi-
cant shortcomings because of their cytotoxicity and the possi-
ble binding to serum proteins, mostly negatively charged at
physiological pH.6 Although studies have exploited the fact that
divalent cations can mediate the interaction between DNA and
zwitterionic natural phospholipids (i.e., lecithins) leading to
the formation of Ca-mediated lipoplexes,7,8 to the best of our
knowledge there has been no report of natural or anionic lipids
used for gene delivery.
characteristics, have attracted much attention in the last
decades.9–12 These molecules constructed a large variety of
supramolecular systems.13–18 Interestingly, lipoplexes only
stabilized via base pairing interaction could be obtained by
molecular recognition between micellar systems made of short
chain nucleolipids and complementary polynucleotides.19,20
Besides, a thymine–lipoamino acid conjugate was reported,
which could form complexes with single-stranded DNA by
hydrogen bond networks in aqueous media,21 and a class of
hydrogelators containing nucleobases were also reported to be
able to bind and deliver nucleic acids.22 These studies have
opened the door for the design of novel classes of non-cationic
vectors for gene delivery, and the investigation of non-charge
interactions between nucleoside-based amphiphiles and nucleic
acids is still important for continued advancements in this
area.
0
´ ´
3 -O-Alkyl nucleotide was reported by Barthelemy and
co-workers many years ago, and has alkyl chain lengths of 16
or 20 carbon atoms (C20-30TMP and C20-30AMP, Fig. 1a and b).23,24
The authors investigated their supramolecular assemblies in
aqueous media and on a graphite surface. Results showed that
these nucleotide amphiphiles spontaneously assembled to
many nano-structures, but the application of them in gene
delivery was not investigated. Our group has been studying
novel gene delivery systems for many years.25–27 We considered
the potential application of 30-O-alkyl nucleotide as a non-
viral vector for genes, and the report20 that short-chain nucleo-
lipids realized molecular recognition with polynucleotides
encouraged us to construct short-chain 30-O-alkyl nucleotides
(TMPC8, TMPC12 and TMPC16, Fig. 1c–e), and to investigate
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, Xue Yuan Road 38, Beijing 100191, China.
E-mail: yangzj@bjmu.edu.cn; Fax: +86 10-8280-2503; Tel: +86 10-8280-2503
† Electronic supplementary information (ESI) available: Synthetic procedures and
characterization data, 1H, 13C and 31P NMR spectra, gel electrophoresis images.
See DOI: 10.1039/c3nj41062e
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and applied to a column of Dowex 50 WX4 (200–400 mesh) to
give the desired product.
Biocompatibility
Cell viability and proliferation were measured with a Cell
Counting KIT-8 (CCK-8, Dojindo, www.dojindo.cn). This system
consisted of WST-8 (2-[2-methoxy-4-nitrophenyl]-3-[4-nitro-
phenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt)
that produced a water-soluble formazan dye upon bioreduction
in the presence of an electron carrier.28 WST-8 is reduced by
dehydrogenase in cells to give a yellow product (formazan),
which is soluble in the tissue culture medium. The amount of
the formazan dye generated by the activity of dehydrogenases
in cells is directly proportional to the number of living cells.
8000 cells (HEK293) were seeded on a 96-well plate with DMEM
+ 10% FBS and incubated at 37 1C and humidified 5% CO2 for
18–24 hours until confluency reached B70%. Nucleotide
amphiphiles at different concentrations were added, and plates
were incubated for another 24 h. Then 10 microliters of thawed
CCK-8 solution was added to each well. Plates were incubated
for 2 h at the same incubator conditions, after which the
absorbance was read at 450 nm with a reference wavelength
of 600 nm. After deducting the absorbance of nucleotide
amphiphiles themselves (plates without cell), cell number was
correlated to optical density (OD). Cell viability was calculated as:
Fig. 1 Chemical structures of nucleoside-based amphiphiles.
their complexation with DNA in aqueous media. Sodium salts of
these nucleotides were designed to increase their water solubility.
Experimental
Synthesis
cell viability = (OD450(sample)/OD450(control)) ꢀ 100%
where OD450(sample) is the absorbance at 450 nm of cells added
Reagents were purchased in analytical grade or higher purity with nucleotide amphiphiles, and OD450(control) is the absor-
from Sigma-Aldrich or Alfa Aesar. Unless otherwise noted, bance at 450 nm of the negative control.
materials obtained from commercial suppliers were used with-
out further purification. 1H, 13C, and 31P NMR were recorded on
Gel electrophoresis
a Bruker AVIII-400 spectrometer. Infrared (IR) spectra were Nucleotide amphiphile was mixed with polyadenylic acid
obtained using a Spectrum One Fourier transform infrared (polyA, 20 bps) at a base ratio of 10/1, and the sample was
(FT-IR) spectrometer (Nexus 470, Thermo Nicolet Co. Ltd.). heated at 60 1C and equilibrated at 4 1C for about a week
Mass spectra were recorded using a Xevo G2 Q-TOF spectro- (annealing). A fraction (4 nmol of nucleotide amphiphile) of the
meter. High-resolution mass spectra were recorded using a sample was analyzed on a TBE-buffered, 40% polyacrylamide
APEX IV FT-MS (7.0T) spectrometer.
gel. The gel was stained using SYBR Gold (Invitrogen, Carlsbad,
General procedure for the synthesis of 30-O-alkyl nucleotide. California) for 15 min and then visualized using a UV trans-
A
solution of 30-phosphoramidites-50-protected thymidine luminator and CCD camera. UV images and relative quantifica-
(compound 1, 750 mg, 1 mmol) and 1H-tetrazolium (70 mg) tion analysis were carried out using the ChemiDoc XRS System
in 15 mL anhydrous acetonitrile was cooled to 0 1C. Aliphatic (Bio-RAD, USA).
alcohol (2 mmol) was added to this solution. The reaction
mixture was stirred for 2 h at room temperature under argon.
Capillary electrophoresis
After that, solvent was removed under vacuum. The residue Experiments were performed on a Beckman Proteomelabt
was re-dissolved in CH2Cl2 (15 mL), and t-BuOOH (0.2 mL) was PA800 system (Beckman Coulter, Fullerton, CA, USA) equipped
added to this solution. After stirring for 30 min, solvent was with UV and LIF detector as well as the 32 Karatt software
removed under vacuum, and the residue was re-dissolved in (Beckman Coulter). A capillary tube (Yongnian Optical Fiber,
CHCl2CO2H–CH2Cl2 (3%, 20 mL). The reaction mixture was Hebei, China) with i.d. of 100 mm was used. The total and
stirred for 30 min at room temperature, and purified by column effective lengths of the capillary were 30.2 and 20 cm, respectively.
chromatography over silica with dichloromethane/methanol Before using, the new capillaries were rinsed with NaOH
(20/1) as the eluting solvent to give compound 2. 200 mg of solution (0.1 mol Lꢁ1) for 20 min, and subsequently with
compound 2 was dissolved in methanol–ammonia (20 mL) and deionized water for 5 min.
stirred at room temperature for 2 h. After that, the solvent was
PEG of molecular weight 35 000 (PEG 35 000) was purchased
evaporated to give a yellow residue. The residue was purified by from Fluka (Buchs, Switzerland). Acrylamide, Tris base, TEMED
column chromatography (Sephadex LH 20, DCM/MeOH 1/1), (99%), 3-(methacryloyloxy)propyltrimethoxysilane (g-MAPS, 98%)
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and ammonium persulfate (APS, 498%) were obtained from were applied to calculate the nonbonded interactions. A cutoff
Sigma (St Louis, MO, USA). Other reagents were all of analytical of 10 Å was used for the Lennard–Jones interactions. The final
grade and from Beijing Chemical Reagent Factory (Beijing, China). structure of TMPC12–polyA complex was produced from the
Bare fused-silica capillaries were permanently coated with 1000 steps of minimized averaged structure of the last 2.0 ns
polyacrylamide according to the method described in ref. 29, in of MD.
which 4% linear polyacrylamide (LPA) and g-methacryloxy-
propyltrimethoxysilane were used. Running buffer was prepared
by dissolution of Tris, boric acid and EDTA-2Na (TBE) to the
Results and discussion
appropriate concentrations. Prior to use, the buffer was
degassed for 10 min in an ultrasonic bath and filtered through
a 0.45 mm filter. Polymer solution was prepared by dissolution
of PEG 35 000 in the above corresponding buffer to the appro-
priate concentrations, followed by stirring and degassing
before use. Samples were introduced by electrokinetic mode
at ꢁ10 kV for 5 s. The UV detection wavelength was 254 nm.
The best separation of the samples was obtained at PEG
concentration of 20% and TBE concentration of 25 mmol Lꢁ1
(pH 8.0), and the optimal separation voltage was ꢁ20 kV.
Commercially
available
30-phosphoramidites-50-protected
thymidine (1) was used as the starting material, and the
synthetic approach was somewhat different to the literature
(Scheme 1).23 Intermediate 2 was obtained after the reaction of
compound 1 with aliphatic alcohol, deprotection and oxidation
in one pot. This intermediate was reacted in methanol–ammonia
solution, and applied to an ion exchange column to give the
desired product.
To verify the biocompatibility of nucleotide amphiphiles, we
added TMPC8, TMPC12 or TMPC16 to the culture of Human
Embryonic Kidney 293 cells, and cell proliferation was
measured with a Cell Counting KIT-8 (CCK-8). As shown in
Fig. 2, after being incubated with TMPC8 or TMPC12 at 100 mM
for 24 h, the viability of HEK 293 cells remained at nearly 100%,
indicating that these molecules are fully biocompatible. How-
ever, TMPC16 exhibited bad biocompatibility, and this result
also encouraged us to investigate the biological application of
short chain 30-O-alkyl nucleotides.
TEM image
To prepare TEM sample, a drop of the aqueous solution
(100 mM) of was allowed to air-dry onto a formvar-carbon-coated
230 mesh copper grid. The TEM experiments were performed using
a Philips Tacnai G2 20 S-TWIN microscope operating at 200 kV.
Molecular dynamics (MD) simulation
MD simulation was performed with AMBER 11 molecular
simulation package.30 The AMBER99 force field was used to
describe the TMPC12-polyA complex. To obtain molecular
mechanical parameters for the TMPC12, ab initio quantum
chemical methods were employed using the Gaussian 09
program.31 The geometry was fully optimized and then the
electrostatic potentials around them were determined at the
HF/6-31G* level of theory. The RESP strategy32 was used to
obtain the partial atomic charges.
The starting model of TMPC12–polyA complex was built
using Discovery Studio 2.5 software. The model was solvated
in TIP3P water using a octahedral box, which extended 8 Å away
from any solute atom. To neutralize the negative charges of
simulated molecules, Na+ counterions were placed next to each
phosphate group.
In order to determine whether the nucleotide amphiphiles
bind nucleic acids, we performed gel electrophoresis (GE) assay
with polyadenylic acid (polyA, 20 bps) as a model system.
TMPC8, TMPC12 or TMPC16 was mixed with polyA at a base
ratio of 10/1, and the samples were heated at 60 1C and
equilibrated at 4 1C for about a week (annealing). The annealing
treatment was also performed on the aqueous dispersions of
TMPC8, TMPC12, TMPC16 and polyA. After that, the samples
were run on non-denaturing polyacrylamide gel and stained by
SYBR Gold,33 a commercially available nucleic acid gel stain.
In Fig. 3, the bright band for the free polyA in column (a)
disappeared entirely for the TMPC8–polyA mixture (column b),
darkened obviously for the TMPC12–polyA mixture (column d),
but changed insignificantly for the TMPC16–polyA mixture
(column f). Columns c and e do not contain any polyA and as
expected show no indication of the presence of SYBR Gold.
These results indicated that a full complexation occurred for
Molecular dynamics (MD) simulation was carried out by
using the SANDER module of AMBER 11. The calculations
began with 500 steps of steepest descent followed by 500 steps
of conjugate gradient minimization with a large constraint of
500 kcal molꢁ1 Åꢁ2 on the TMPC12–polyA complex. Then 1000
steps of steepest descent followed by 1500 steps of conjugate
gradient minimization with no restraints on the TMPC12-polyA
complex were performed. Subsequently, after 20 ps of MD,
during which the temperature was slowly raised from 0 to
300 K with weak (10 kcal molꢁ1
Å
ꢁ2) restraints on the
TMPC12–polyA complex, the final unrestrained production
simulations of 3.0 ns for the molecule was carried out at
constant pressure (1 atm) and temperature (300 K). In the
entire simulation, SHAKE was applied to all hydrogen atoms.
Periodic boundary conditions with minimum image conventions
Scheme 1 (i) ROH, 1H-tetrazolium, anhydrous acetonitrile, 0 1C to r.t., argon,
2 h; (ii) t-BuOOH, CH2Cl2, r.t., 30 min; (iii) CHCl2CO2H–CH2Cl2 (3%), 30 min;
(iv) NH3–MeOH, r.t., 2 h, Dowex 50 WX4 (200–400 mesh).
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Fig. 2 HEK 293 cell viability test for 24 h of TMPC8, TMPC12 and TMPC16
.
Fig. 4 Electropherograms of (a) polyA; (b) TMPC8–polyA (10/1), (c) TMPC8; (d)
TMPC12–polyA (10/1), (e) TMPC12; (f) TMPC16–polyA (10/1) and (g) TMPC16
,
respectively. The separation system is described in the ESI.† The marked peaks 1,
2, 3 and 4 represent polyA, TMPC8, TMPC12 and TMPC16, respectively.
Table 1 Relative peak area of remained polyA and nucleotide amphiphiles after
complexationa
TMPC8–polyA
TMPC12–polyA
TMPC16–polyA
Fig. 3 Electrophoretic mobility of (a) polyA; (b) TMPC8–polyA (10/1); (c) TMPC8;
(d) TMPC12–polyA (10/1); (e) TMPC12; (f) TMPC16–polyA (10/1) and (g) TMPC16
respectively.
,
polyA
NAb
0
0.33
0.15
0.31
0.44
0.52
a
Peak areas relative to corresponding external standards (Fig. 3; peak 1
of spectrum a, peak 2 of spectrum c, peak 3 of spectrum e, and peak 4 of
b
spectrum g were used as external standards, respectively). NA =
the TMPC8–polyA mixture, and the complexation decreases nucleotide amphiphile.
with the growth of the alkyl chain of nucleotide amphiphile.
However, the bands of the complexes of polyA with nucleotide
amphiphiles were not detected in this experiment. FAM Unfortunately, the complexes of polyA with nucleotide amphi-
(5-carboxyfluorescein)-labeled polyA was used to track the philes were not detected in these electropherograms, either.
complex formation (see the ESI†). Based on the experimental
The supramolecular assemblies of TMPC8, TMPC12 and
results, we believe that the complexes have dispersed in the TMPC16 were investigated by transmission electron microscopy
column and the concentration was at too low a level to be (TEM). Ribbon-like structures were observed in the aqueous
´ ´
detected. Interestingly, it was observed that TMPC16 was also dispersion of TMPC16, as reported by Barthelemy and co-workers
stained by SYBR Gold (Fig. 3, columns f & g), indicating that its (Fig. 5a).23 However, similar structures were not observed in
supramolecular assembly resembled DNA in architecture. It aqueous dispersions of short-chain nucleotide amphiphiles. As
was supposed that the tendency of self-assembly inhibited the shown in Fig. 5b, TMPC12 formed conglutinant vesicles at a
complexation of TMPC16 with DNA.
dimension of approximately 30 nm in aqueous solution. It was
As a more sensitive analysis tool, capillary electrophoresis supposed that the different hydrophobicity of alkyl chains,
(CE) was also used to investigate the complexation of nucleo- and the different phase transition temperatures, caused the
tide amphiphiles with polyA. Samples were prepared the same diversity on supramolecular assemblies of TMPC16 and TMPC12.
way as GE assay, and performed on a no-gel CE.34 Products were In addition, a supramolecular structure was not observed in the
qualitatively determined by contrasting with standard sample, aqueous dispersion of TMPC8, and that was because TMPC8 is
and quantitatively determined by calculating the peak areas. completely soluble in aqueous media to form a true solution.
As shown in Fig. 4, after the addition of TMPC8, TMPC12 or Actually, it has been observed that the sodium salt of TMPC8 is
TMPC16, the peak of polyA disappeared or decreased corre- highly hygroscopic in atmospheric conditions.
spondingly. Relative peak areas were given in Table 1, which
To investigate the supramolecular structure variation of
suggested that TMPC8 has fully bound the polynucleotides, TMPC16 and TMPC12 after the addition of polyA, TEM images
TMPC12 has bound most of the polynucleotides, and TMPC16 of these complexes were observed. Ribbon-like structures were
has bound only part of the polynucleotides. This result coincided also observed in TMPC16–polyA mixture solution (Fig. 5c),
with GE assay, indicating that the complexation decreases with which was almost the same as the TEM image of TMPC16
the growth of the alkyl chain of nucleotide amphiphile. aqueous solution. This result coincided with GE and CE
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and energy terms. The calculated root-mean-square deviation
(RMSD) values stabilized and remained stable during the last
2.0 ns period of simulation. As shown in Fig. 6a and b,
calculation results demonstrated a dsDNA-like complex in
aqueous solution based on p–p stacking and Watson–Crick
base-pairing interactions. Taking the 10th deoxyadenosine
(from the 50 end) and the corresponding TMPC12 molecule
bound to it for an example, the length of N–H and O–H
hydrogen bonds were respectively 1.885 Å and 1.852 Å
(Fig. 6c), which are close to those in the canonical Watson–
Crick base-pairing modes (1.832 Å and 1.930 Å, respectively).35
However, for the 20th deoxyadenosine and the corresponding
TMPC12 molecule, the distances are much longer (Fig. 6d, 4.469
and 3.790 Å for N–H and O–H, respectively) because of edge
effects, which decrease the stability of the complex.
Conclusions
In conclusion, we have developed a novel class of thymidine-
based amphiphiles, and investigated their complexation with
single-stranded DNA through hydrogen bonding and p–p stacking
interactions. Experimental results from gel electrophoresis,
capillary electrophoresis, transmission electron microscopy
and molecular dynamics simulation supported the binding of
these nucleotide amphiphiles with polyadenylic acid. Further-
more, it was found that their biocompatibility and binding
ability with DNA varied inversely with the length of the alkyl
chains. It is obvious that these molecules are negatively
charged, and this research provided further evidence for the
use of non-cationic interactions to manipulate DNA. The devel-
opment of amphiphiles possessing affinity to DNA through
more specific interactions, as opposed to more general electro-
static forces, will be a benefit for the design of novel classes of
non-viral carriers for gene delivery. Further study on the
application of these amphiphiles in gene delivery is ongoing
in this laboratory.
Fig. 5 TEM image of (a) TMPC16 (bar = 500 nm), (b) TMPC12 (bar = 100 nm),
(c) TMPC16–polyA mixture (bar = 500 nm) and (d) TMPC12–polyA mixture (bar =
200 nm) in aqueous solution.
experiments, indicating a low binding interaction between
TMPC16 and poly A.
On the other hand, the TEM image of TMPC12 changed
dramatically after the addition of polyA. As shown in Fig. 5d,
‘‘worm’’-like structures were observed in the mixed solution of
TMPC12 and polyA, in contrast to the vesicle structures of TMPC12
aqueous solution. This variation on supramolecular architecture
indicated the binding of nucleotide amphiphiles with polyA.
Molecular dynamics simulation was used to study the thermo-
dynamic stability of TMPC12–polyA complex. A 3.0 ns MD
simulation of the complex led to a stable trajectory of geometry
Acknowledgements
This research was supported by Ministry of Science and
Technology of China (No. 2012CB720604) and NNSFC (No.
20932001), which are gratefully acknowledged. The authors
thank Prof. Dr Yu Liu, Prof. Dr Yong Chen, Dr Ying-Ming Zhang
and Yang Yang of Nankai University for their advice and help in
this study.
Notes and references
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c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem., 2013, 37, 1122--1127 1127