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K. Kim et al. / Bioorg. Med. Chem. 15 (2007) 4311–4317
ꢀ
1
of ꢀ0.33 ꢁC min . The preparation of nucleo-cages was
mixtures were then centrifuged at 10,000 rpm for
10 min and the supernatants (30 lL) were diluted four
times. To determine the amount of unbound Rho-
PNA, fluorescence intensity was measured at
confirmed by CLSM observation.
4.5. Preparation of lactose-modified nucleo-cages (Lac-
NCs)
kem = 570 nm at 10 ꢁC (k , 530 nm).
ex
Aqueous solution of Lac-psoralen 4 (10 mM) was added
to a solution of intact nucleo-cages (1 + 2 + 3, 200 lL)
at the final concentration of [total ODN] = 20 lM,
Acknowledgments
[
was photo-irradiated (ca. 8 mW cm
NaCl] = 0.5 M, [4] = 300, 450 or 600 lM. The mixture
We thank Professor I. Hamachi (Kyoto University) for
the use of the Carl Zeiss LSM 510. This study was finan-
cially supported by research grants from the Association
for the Progress of New Chemistry, Grant-in-Aid for
Scientific Research (A) (16205028) from the Japan Soci-
ety for the Promotion of Science, and by a Grant for
21st Century COE Program from the Ministry of Edu-
cation, Culture, Sports, Science and Technology of
Japan.
ꢀ
2
for 240 min,
5
ꢁC) in a 1.0 mm quartz cell with the 365 nm bright line
using high-pressure mercury lamp (USHIO, USH-
00SC) and a monochromator (Jasco, CT-10). The
5
progress of photo-crosslinking was monitored by fluo-
rescence spectra. Absorption spectra of the aqueous
mixtures were also recorded before and after UV irradi-
ation, by diluting the reaction mixture for five times with
0
.5 M NaCl aqueous solution.
4
.6. Determination of lactose contents in Lac-NCs
References and notes
An aliquot (200 lL) of Lac-NCs solution in 0.5 M aque-
ous NaCl was filtered through a Millipore Microcon
Centrifugal Filter Devices (cut off Mw 3000) at
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0,000 rpm for 90 min. Then filtrates was diluted three
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.7. Melting behaviors of nucleo-cages
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as a function of temperature on a spectrophotometer
1
23, 8226.
4
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1
(
heating rate, 1.0 ꢁC min ).
4.8. CLSM observation of interaction between PNA and
nucleo-cages
5
Confocal laser scanning fluorescence microscopy
(
CLSM) was conducted on a Carl Zeiss LSM 510 instru-
ment with a 63· oil-immersion objective. Excitation was
made with argon and helium–neon lamp and the excita-
tion wavelength was selected with an interference filter.
Emission range was selected with interference filter
either with transmittance 505–530 nm or beyond
6
7
8
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3
1
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5
60 nm. An aliquot (30 lL) of nucleo-cages solution
2
was placed on a glass-bottomed dish and observed by
CLSM. In order to observe the interaction between
Lac-NCs and PNA, rhodamine labeled peanut lectin
2
krishnan, M.; Reishus, D.; Shaw, B.; Adleman, L. J. Am.
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(
Rho-PNA) was employed. After Rho-PNA was added
to Lac-NCs solution ([total ODN] = 20 lM,
NaCl] = 0.5 M, [4] = 300 lM, in 50 mM sodium phos-
[
phate buffer, pH 7.5), the solution was aged for 1 h
and YOYO-1 was added ([YOYO-1] = 1 lM).
4
.9. Binding curves of Rho-PNA to nucleo-cages
Various volumes of Rho-PNA stock solution (45 lM)
were added to Lac-NC solution and the mixtures (final
volume, 60 lL) were incubated at 10 ꢁC for 1 h. The
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