The effects of the 4-(4-methylpiperazine)phenyl group on nucleosides and oligonucleotides: Cellular delivery, detection, and stability

Article abstract of DOI:10.1002/asia.201000574

As drug candidates, one promising way to improve the cellular delivery efficacy of oligonucleotides is to introduce a cationic group. By introducing a cationic moiety into the oligonucleotide structure, they become capable of approaching the cell surface

Full text of DOI:10.1002/asia.201000574

DOI: 10.1002/asia.201000574
The Effects of the 4-(4-Methylpiperazine)phenyl Group on Nucleosides and
Oligonucleotides: Cellular Delivery, Detection, and Stability
Sun Min Park, Su-Jin Nam, Hyun Seok Jeong, Won Jong Kim, and Byeang Hyean Kim*[a]
Dedicated to Professor Eiichi Nakamura on the occasion of his 60th birthday
Abstract: As drug candidates, one
promising way to improve the cellular
delivery efficacy of oligonucleotides is
to introduce a cationic group. By intro-
ducing a cationic moiety into the oligo-
nucleotide structure, they become ca-
pable of approaching the cell surface
and also of crossing the cellular mem-
brane. In an effort to develop cell-per-
meable oligonucleotides, we examined
the piperazinephenyl-bearing 2’-deoxy-
uridine (PPU), which can be not only
cationic but also fluorescent as a cat-
ionic monomer for cationic oligonucle-
otides. Several modified DNA oligonu-
cleotides with different numbers of PPU
building blocks were synthesized and
evaluated for the effect on thermal sta-
bility and conformation by the intro-
duction of PPU. The cellular delivery of
modified oligonucleotides was different
depending on the number of PPU build-
ing blocks. Furthermore, these PPU-
modified oligonucleotides had suffi-
cient fluorescence that we were able to
identify the delivery results without the
use of conventional fluorescent tags.
They were predominantly localized in
the cell cytoplasm. In addition, they
were stable enough after 3 hours in the
presence of nuclease. These results
showed that a piperazinephenyl moiety
that is conjugated with nucleobase is
able to deliver and detect the oligonu-
cleotides, which suggests that this con-
cept of ꢀdual-function oligonucleotidesꢁ
might be utilized in diagnostics, thera-
peutics, and as a convenient biological
tool for probing the activity of oligonu-
cleotides inside cells.
Keywords: cations · drug delivery ·
nucleobases
stability
· oligonucleotides ·
Introduction
moieties to reduce the net negative charge. The most
common cationic moieties include guanidinium functionali-
ties[12–19] and several types of amino groups[20–24] that exhibit
a net positive charge over a wide pH range and under phys-
iological conditions. The other inherent weakness of thera-
peutic oligonucleotides is their low resistance to degradation
by nuclease (endo- and exonuclease). However, by employ-
ing structural variations (such as dumbbell or hairpin
shapes)[25–27] or nuclease-resistant modified nucleotide (such
as LNA nucleotide)[28], the enzymatic stability of oligonucle-
otides can be modified.
In the current study, we assess the potential of cationic
hairpin-structured oligonucleotides by incorporation of a
modified nucleotide in therapeutic applications such as a
hairpin oligonucleotide decoy. A new piperazinephenyl-ap-
pended nucleoside (PPU) was synthesized by a simple Suzuki
coupling reaction and incorporated into oligonucleotides to
achieve enhancement of cellular uptake (Scheme 1). This
building block (PPU) is designed to have a tertiary amine
moiety as the cationically charged part and suitable fluores-
cence obtained by simultaneous p conjugation between pi-
perazinephenyl and a nucleobase. Herein, we report the
characteristic features of PPU and its oligonucleotides for
Despite several decades of research, the potential of gene
therapy has been hindered by several unresolved issues.[1–5]
One of the main problems involves proper delivery of the
therapeutic agent. At present, several strategies utilize oli-
gonucleotides loaded onto specialized carrier systems such
as cationic lipids and dendrimers.[6–9] Or the terminal of an
oligonucleotide is conjugated to functional compounds and
polymers, such as cholesterol and poly(ethylene glycol)
(PEG), for better cellular uptake.[10] In addition, the nucleo-
tide monomer itself may be chemically modified. Highly
charged, anionic oligonucleotides cannot easily penetrate
into cell or endosomal membranes. Therefore, several stud-
ies[11] have utilized modified oligonucleotides with cationic
[a] S. M. Park, S.-J. Nam, H. S. Jeong, Prof. W. J. Kim, Prof. B. H. Kim
Department of Chemistry, BK school of Molecular Science
Pohang University of Science and Technology (POSTECH)
Pohang, 790-784 (Republic of Korea)
Fax : (+82)54-279-2115
E-mail: bhkim@postech.ac.kr
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201000574.
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FULL PAPERS
spectra (i.e., the excitation maxima), a fluorescence spec-
trum was obtained. Two emission peaks at 389 nm and
502 nm were observed, but the major is the latter one. As
observed by the emission spectrum, the green color of PPU
was detected under UV illumination. The quantum yield of
PPU in several selected solvents was almost 0.05 (CHCl3 in
neutral pH), and the fluorescence intensity was low relative
to conventional fluorophore-appended 2’-deoxyuridine, for
example,
FLU
(2’-deoxy-5-(2-ethynylfluorenyl)uridine;
Figure 1).[32–34] As the pH of solvent was decreased from 7
(neutral) to 4 (acidic), fluorescence quenching of PPU was
observed and was presumably due to the protonation of the
piperazine moiety (see the Supporting information).
To investigate the effect of PPU in the oligonucleotides,
hairpin-type DNA oligonucleotides (18-mer) were prepared
Scheme 1. Synthesis of 4-(4-methylpiperazine)phenyl-appended nucleo-
side. Reagents and conditions: a) 4,4’-dimethoxytrityl chloride (DMTrCl),
Et3N, pyridine, RT, 5 h, 90%; b) 1-methyl-4-[4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl]piperazine, [PdACTHNUGTRNEG(UN PPh3)4], NaOH, THF/H2O/
MeOH (2:2:1), 70–758C, 6 h, 82%; c) 2-cyanoethyl diisopropylchloro-
phosphoramidite, 4-methylmorpholine, CH2Cl2, RT, 1 h, 95%.
cellular delivery and detection. By using a piperazine
moiety, we have demonstrated that oligonucleotides that
contain PPU have increased thermal stability relative to un-
modified ones and can be delivered to the inside of a cell,
cytoplasm in particular. In addition, their presence in cyto-
plasm can be identified without fluorescent labeling by
using a confocal microscope. Finally, we have demonstrated
that the PPU-modified oligonucleotides are more stable than
natural ones against enzymatic degradation.
Results and Discussion
Methylpiperazine, a tertiary amine, was employed as a cat-
ionic modifier due to its affordable gene-transfer effect[29]
and basicity. The conjugate acid of methylpiperazine exhib-
its a relatively high pKa value (ꢀ10) relative to other
amines. To introduce the piperazine moiety, 4-(4-methylpi-
perazine)phenyl boronic acid was used to modify the C5 po-
sition of 2’-deoxyuridine by means of Suzuki coupling[30] to
yield
(
5-[4-(4-methylpiperazine)phenyl]-2’-deoxyuridine
PPU) in high yield. With this simple synthetic procedure,
PPU is able to have fluorescence by p conjugation between
the phenyl ring and uracil.
The new cationic monomer, PPU, was fluorescent. This
effect was likely due to delocalization of the nitrogen lone-
pair electrons through the p-conjugated system of para-
phenylene and uracil.[31] Therefore, PPU served as a “dual-
function oligonucleotide,” thus facilitating both delivery and
detection. Prior to the oligonucleotide synthesis, the spectro-
scopic characteristics of PPU were investigated by UV ab-
sorption and fluorescence measurements. Absorption peaks
were observed at 258 nm and 298 nm, which originated from
the nucleobase and the p-conjugated system of the (methyl-
piperazine)phenyl moiety with the nucleobase, respectively.
By using the information obtained from the UV absorption
Figure 1. a) UV spectrum of PPU. b) Emission spectrum of PPU. c) The
photo image and structure of PPU and FLU.
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Chem. Asian J. 2011, 6, 487 – 492

The Effects of the 4-(4-Methylpiperazine)phenyl Group on Nucleosides and Oligonucleotides
with varying numbers of PPU (X) and compared against a
natural unmodified sequence: natural 5’-dATA-
TACGTTTTCGTATAT-3’, (X)6 5’-dATAXACGXXXX-
CGXATAT-3’, (X)8 5’-dAXAXACG-XXXXCGXAXAT-3’.
To identify the effect of PPU on hybridization, the melting
temperature (Tm) of synthesized oligonucleotides was mea-
sured by UV/Vis absorption in pH 7 Tris buffer (Figure 2a).
duplex structure. In contrast, electrostatic effects due to the
base modification stabilized DNA duplex hybridization.
The cellular uptake of PPU-modified oligonucleotide by
HeLa cells was evaluated under confocal microscopy. The
PPU modifier is slightly fluorescent, and confocal microscopy
studies were performed without the use of fluorescent tags.
The conditions of cellular uptake were optimized by moni-
toring the HeLa cell response to three different concentra-
tions of oligonucleotide: 200, 400, and 1000 nm. Natural,
(X)6, and (X)8 were tested with and without a transfection
agent (Oligofectamine, Invitrogen, Carlsbad, CA, USA).
Cells incubated with (X)6 exhibited a relatively stronger
fluorescence in the cytoplasm than others at the 200 nm
level, independent of transfection agent (Figure 3 and the
Figure 2. a) Synthesized oligonucleotides and their Tm values. b) CD spec-
tra of ODN natural, (X)6, and (X)8.
Figure 3. Confocal images of a) HeLa cells. b) HeLa cells incubated with
a natural oligonucleotide. c) HeLa cells incubated with X6 with transfec-
tion agent. d) HeLa cells incubated with X6 without transfection agent.
A higher Tm was observed in DNA that contained PPU (in
the case of (X)8 or (PPU)8, it showed another transition at
288C), thereby signifying a stabilization of the DNA duplex,
presumably due to electrostatic interactions between the pi-
perazine component and the phosphate backbone in the
stem region of the hairpin.[19,20] The DNA oligonucleotides
used in this study showed somewhat higher Tm and, in the
case of (X)8, the sharpness of the melting curve decreased.
In general, hairpin-type DNA oligonucleotide has a higher
melting temperature than a simple duplex one. Even though
the results showed a more enhanced and broader range of
melting temperature, the thermal stability caused by the
electrostatic interaction with the number of PPU units on oli-
gonucleotides is demonstrated (see the Supporting informa-
tion for additional Tm of modified DNA or RNA duplex
with PPU). The thermal stability of synthesized oligonucleo-
tides was either much higher or was not determined when
they were measured in the presence of additional salt
(NaCl, KCl, and sodium cacodylate). The modified oligonu-
cleotides were further characterized by circular dichroism
measurements (CD). These results showed that the modified
oligonucleotides exhibited a conventional B-form DNA
(Figure 2b).[35] Therefore, the nucleic acid analogue of PPU
does not generate significant perturbation of the DNA
Supporting information). However, (X)8 showed weakened
fluorescence intensity without a transfection agent. Differ-
ent numbers of PPU modifier ((PPU)n, in which n=2, 4, 6, 8)
were also evaluated for cellular uptake; (X)6 was optimal.
Therefore, the lowest concentration of the modified oligonu-
cleotide, (X)6, induced the higher fluorescence when taken
in by the cell. The efficiency of cellular uptake was expected
to decrease with increasing amounts of modifier and concen-
tration due to aggregation, as suggested by related polypep-
tide studies.[36–38]
Further experiments were carried out to identify the exact
localization of PPU-modified oligonucleotide inside of the
cell in a simple single-strand system. Linear oligonucleotides
that had been treated with fluorescein, Flu-(TTT)6 and Flu-
(TXT)6, were prepared and incubated with HeLa cells as de-
scribed before. To address the localization, the nucleus was
stained by Hoechst 33258 and a significant green fluores-
cence was noticed from the cytoplasm in the case of Flu-
(TXT)6. When the fluorescence signals were merged togeth-
er to confirm the localization, an overlapped signal was not
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489

B. H. Kim et al.
FULL PAPERS
improved cellular delivery of PPU-modified oligonucleotides
originates from not only their cationic character but also en-
zymatic stability. Currently, more-potent modifiers are re-
quired to induce cellular uptake of gene therapeutic agents,
but these modifiers must also be easy to implement. The re-
sults reported here demonstrate that the modified PPU could
easily and readily be used as a modifier to enable intracellu-
lar oligonucleotide delivery without fluorescent tagging.
Experimental Section
General Methods of Chemical Synthesis
All chemicals were obtained from Aldrich Chemical Company or the
specified individual chemical companies and were used without further
purification. Each reaction was performed under an inert atmosphere of
dry argon and by using glassware that was flame-dried under vacuum.
Flash chromatography was performed on silica gel 60 (230–400 mesh;
ASTM). Melting points are uncorrected and were obtained with an Elec-
trothermal 1A 9000 series apparatus. FTIR spectra were recorded with a
Bruker FTIR PS55+ spectrometer. Low-resolution FAB+ mass spectra
were obtained with a JEOL JMS-AX505WA (FAB) spectrometer.
Figure 4. The localization of modified oligonucleotides Flu-(TXT)6 in the
HeLa cells (FITC=fluorescein isothiocyanate, DAPI=4’,6-diamidino-2-
phenylindole).
1H and 13C NMR spectra were recorded with a Bruker Aspect 300 NMR
spectrometer. Chemical shifts (d) of these spectra are reported in parts
per million (ppm) downfield relative to the internal standard, tetrame-
thylsilane (TMS). Coupling constants are reported in Hertz (Hz). Spec-
tral splitting patterns are designed as s (singlet), d (double), dd (double
doublet), dt (distorted triplet), t (triplet), m (multiplet), and br (broad).
observed (Figure 4). However, Flu-(TTT)6 did not show any
fluorescence except the nucleus stained by Hoechst 33258.
Therefore, the cellular uptake of PPU-modified oligonucleo-
tides (X)6 and Flu-(TXT)6 occurs predominantly in the en-
dosome or whole cytoplasm regardless of their structure and
this would be expected for cationic driven internalization.
The sensitivity of oligonucleotides to enzymatic degrada-
tion is one of their major problems as a drug candidate.
Compared to the natural oligonucleotides, the stability of
the modified oligonucleotides was enhanced against the ac-
tivity of nuclease (Exonuclease III; Promega, Madison, WI,
USA).[39–41] Undigested, modified oligonucleotide (X)6 still
remained after 3 h, whereas the natural oligonucleotide was
completely digested (Supporting information). Undigested
residue of (X)6 was identified by UV visualizer without dye
because of its fluorescence. Therefore, the introduction of a
PPU monomer could considerably improve the enzymatic
stability of PPU-modified oligonucleotides. The increased en-
zymatic stability of PPU-modified oligonucleotides would be
more beneficial for biomedical applications.
Synthesis of Cationic Modified Nucleosides
1: 5-Iodo-5’-DMT-2’-deoxyuridine (300 mg, 0.46 mmol) was dissolved in
THF/H2O/MeOH (2:2:1; 25 mL), and then [PdACHTUNRTGNEGNU(PPh3)4] (53 mg,
0.046 mmol), 1-methyl-4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenyl]piperazine (152 mg, 0.506 mmol), and NaOH (366 mg, 9.2 mmol)
were added. The reaction mixture was heated at reflux for 6 h at 70–
758C. The reaction mixture was concentrated under reduced pressure.
The residue was purified by chromatography through a short column of
silica gel (CH2Cl2/MeOH, 70:1) to yield 1 (264 mg, 82%). m.p. 140–
1
1438C; H NMR (300 MHz, CDCl3): d=7.62 (s, 1H; NH), 7.33–7.397 (m,
ꢁ
ꢁ
ꢁ
2H; Ar H), 7.16–7.26 (m, 9H; Ar H), 6.73–6.74 (m, 4H; Ar H), 6.61–
ꢁ
6.64 (m, 2H; Ar H), 6.37 (t, J=6.77 Hz, 1H; C1), 4.51–4.53 (m, 1H;
C3), 4.07–4.08 (m, 1H; C4), 3.75 (s, 6H; OCH3), 3.33–336 (m, 2H; C5),
2.52–2.61(m, 10H; piperazine+C2), 2.34 ppm (s, 3H; CH3); 13C NMR
(75 MHz, CDCl3): d=162.2, 158.8, 151.1, 149.9, 135.5 130.2, 129.1, 128.1,
127.2, 115.8, 113.4, 86.9, 86.2, 85.3, 72.6, 63.7, 55.4, 48.9, 46.4, 46.3,
41.2 ppm; IR (NaCl): n˜ =3460, 3281, 3058, 2941, 2835, 2563, 1703, 1609,
1508, 1510, 1460, 1378, 1294, 1177, 1034, 1008, 922, 827, 791 cmꢁ1; HRMS
(FAB): m/z: calcd for C41H45N4O7: 705.3288 [M+H]+; found: 705.3287.
2: 2-Cyanoethyl diisopropylchlorophosphoramidite (120 mL, 0.537 mmol)
was added dropwise to a solution of compound 1 (301 mg, 0. 419 mmol)
and 4-methylmorpholine (140 mL, 1.27 mmol) in CH2Cl2 (12 mL) at room
temperature. After the reaction reached completion (1 h), the mixture
was concentrated in vacuo and purified by chromatography through a
short column of silica gel (CH2Cl2/MeOH, 70:1) to yield 2 (360 mg,
Conclusion
In summary, 5-[4-(4-methylpiperazine)phenyl]-2’-deoxyuri-
dine (PPU) was synthesized by a simple Suzuki coupling re-
action. PPU was amenable to cellular uptake in HeLa cells
and exhibited an intrinsic fluorescence, thereby affording si-
multaneous delivery and detection. Although the experi-
ments performed here did not cover a wide range of condi-
tions and give effective results, the results are still meaning-
ful since cells inoculated in 200 nm X6 exhibited similar fluo-
rescent intensities regardless of the addition of a transfec-
tion agent. In addition, PPU-modified oligonucleotides
showed more enhanced enzymatic stability than natural oli-
gonucleotides. Based on these results, it is assumed that the
1
95%). m.p. 78–808C (decomp.); H NMR (300 MHz, CDCl3): d=7.67 (d,
ꢁ
ꢁ
J=12.3 Hz, 1H), 7.34–7.37 (m, 2H; Ar H), 6.69–6.73 (m, 4H; Ar H),
ꢁ
6.55–6.59 (m, 2H; Ar H), 6.37–6.43 (m, 2H; H-1’), 4.61 (m, 1H; H-3’),
4.15–4.20 (m, 1H; H-4’), 3.73 (s, 6H; OCH3), 3.56–3.66 (m, 2H; H-5’),
ꢁ
ꢁ
3.34–3.44 (m, 1H; NCH), 3.24–3.29 (m, 1H; NCH), 3.13 (s, 4H; piper-
azine-H), 2.59–2.61 (m, 6H; CH2 and piperazine-H), 2.41 (t, J=6.5 Hz,
2H; CH2), 2.31 (s, 3H; CH3), 1.06–1.43 ppm (m, 12H; NCHCH3);
13C NMR (75 MHz, CDCl3): d=162.6, 158.7, 150.8, 150.2, 144.5, 135.6,
130.1, 129.2, 128.2, 127.1, 123.2, 116.0, 113.3, 85.8, 85.2, 63.3, 55.3, 54.9,
48.7, 46.0, 43.5, 40.3, 24.8, 20.3, 14.4 ppm; 31P NMR (121 MHz, CDCl3):
d=150.2, 149.7 ppm; IR (NaCl): n˜ =3037, 2935, 2874, 2837, 2800, 2751,
1705, 1685, 1609, 1511, 1458, 1249, 1155, 1080, 1034, 920, 864, 730 cmꢁ1
;
490
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Chem. Asian J. 2011, 6, 487 – 492

The Effects of the 4-(4-Methylpiperazine)phenyl Group on Nucleosides and Oligonucleotides
HRMS (FAB): m/z: calcd for C50H62N6O8P: 905.4367 [M+H]+; found:
seeded at a density of 2.5ꢃ105 cells per well, each well of which con-
905.4370.
tained 10% FBS-supplemented DMEM (2 mL), and were incubated for
4 h.
HeLa cells were transfected in the absence of serum with 200 nm modi-
fied oligonucleotides using oligofectamine (Invitrogen), or without any
transfection reagent. The cells were allowed to incubate at 378C in the
presence of oligonucleotides for 6 h in a CO2 incubator followed by re-
placement of DMEM (2 mL) that contained 10% FBS.
Oligonucleotide Synthesis
All reagents for oligonucleotide synthesis were purchase from Glen Re-
search. Natural and modified oligonucleotides were synthesized by
means of the standard solid-phase b-cyanoethylphosphoramidite method
with a PolyGen 12 column DNA synthesizer (Germany) and purified
under standard conditions. The concentration of oligonucleotides was de-
termined by ultraviolet (UV) absorption. Oligonucleotide sequences
used for experiments have been listed in Table 1.
For preparation, 24 h after transfection, cells were washed with Dulbec-
co’s phosphate buffered saline (DPBS), and then cover glasses were de-
tached from the bottom of the plate. The cells on the cover glass were
fixed with 3.5% paraformaldehyde solution. After additional washing
with DPBS, the cover glasses were transferred onto slide glass. Confocal
images were obtained from the fixed and nonfixed cell with an Olympus
FluoView FV1000 confocal microscope (Olympus Optical Co., Ltd.,
Tokyo).
Table 1. DNA sequences used in this study.
Oligonucleotide
(X=PPU)
Sequence
(5’!3’)
MS (m/z)
calcd found
N
ACHTUNGTRENNUNG
Hoechst 33258 Nuclear Staining
Each fluorescein isothiocyanate (FITC)-labeled oligonucleotide was di-
luted in serum-free OptiMEM media to a final concentration of 1000 nm.
Then the cells were incubated with oligonucleotide solutions for 3 h.
After incubation, the cells were washed three times with PBS and then
fixed with 3.5% (w/v) paraformaldehyde (Sigma) at room temperature
for 12 min.
natural
(X)6
(X)8
d(ATATACGTTTTCGTATAT)
d(ATAXACGXXXXCGXATAT)
d(AXAXACGXXXXCGXAXAT) 6761.9 6764.1
fluorescein-
d(TTTTTTTTTTTTTTTTTT)
fluorescein-
5479.9 5480.7
6441.4 6441.4
Flu-(TTT)6
5950.4 5946.1
Flu-(TXT)6
6911.9 6920.8
d(TXTTXTTXTTXTTXTTXT)
After washing three times with PBS, the nucleus of cells was stained with
Hochest 33258 dyes diluted in 1% BSA (bovine serum albumin). Then
the coverslips were placed on a glass slide and then sealed. The fluores-
cent confocal images were captured with an Olympus FluoView FV1000
confocal microscope (Olympus Optical Co., Ltd., Tokyo).
Photophysical Properties of PPU
The absorption and emission wavelength of PPU were determined with a
Cary 100 Conc UV/Vis spectrophotometer and Cary Eclipse Fluores-
cence spectrophotometer. The absorbance of PPU (75 mm in CHCl3) was
monitored at room temperature. Emission spectra and quantum yield
were obtained using quinine sulfate as a standard for nucleoside PPU. The
quantum yield of PPU was estimated using Equation (1):
Digestion Experiment of Oligonucleotides with Exonuclease III
Prepared oligonucleotides (0.23 OD) were mixed with 10ꢃ Exonuclea-
se III buffer (3 mL; final concentration: 66 mm Tris-HCl, pH 8.0, 0.66 mm
MgCl2) and the final volume was adjusted with H2O (30 mL). Exonuclea-
se III (Promega, 200U) was added at room temperature and each aliquot
(5 mL) was taken at certain time intervals (10, 30, 60, 120, 180, and
240 min). The enzyme activity of aliquots was quenched by 0.5m EDTA
(2 mL) and PAGE loading buffer (formamide; 5 mL). Using the 15%
PAGE (7m urea, 140 V), the digested samples by Exonuclease III were
analyzed. The gel of the natural oligonucleotide was stained by StainsAll
(Sigma), but the (X)6 gel images were obtained with UV visualizer with-
out dye.
2
FFðXÞ ¼ ðAS=AXÞðFX=FSÞðnX=nSÞ FFðSÞ
for which S is standard, X is unknown; A is the absorbance at the excita-
tion wavelength, F is the fluorescence intensity, n is the refractive index
of the solvent, and F is the quantum yield).
Thermal Stability
UV melting-temperature experiments were performed with a Cary 100
Conc UV/Vis spectrophotometer on 18-mer DNA hairpin-type oligonu-
cleotides. The absorbance of the 0.5 OD (optical density) sample in 5 mm
Tris buffer (pH 7) was monitored at 260 nm from 3 to 918C at a heating
rate of 1.08Cminꢁ1. Tm values were determined by means of the maxi-
mum value of the first derivate plots of absorbance versus temperature.
Acknowledgements
We thank Professor Sung Key Jang (POSTECH, South Korea) for cell
nuclear staining experiments and Eun Mi Jeon for selective cell experi-
ments. We are grateful to the NRF for financial support through the
Gene Therapy R&D program (M10534000011-05N3400-01110) and the
CRI-Acceleration Research program.
Circular Dichroism (CD) Measurements
CD spectra were collected with a JASCO J-810 spectropolarimeter with
a temperature controller. The cell holder was flushed with dry nitrogen
gas. For each sample, five spectrum scans were accumulated over the
200–500 nm wavelength range at room temperature. The 0.5 OD of oligo-
nucleotides were used in 5 mm Tris buffer (pH 7).
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Cell Culture
HeLa cells were cultured in Dulbeccoꢁs modified Eagle medium
(DMEM; HyClone), supplemented with 10% fetal bovine serum (FBS;
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Chem. Asian J. 2011, 6, 487 – 492
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Received: August 16, 2010
Published online: December 14, 2010
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