Supporting Information

Supporting Information
Wiley-VCH 2013
69451 Weinheim, Germany
DNA Aptamer-Mediated Cell Targeting**
Xiangling Xiong, Haipeng Liu, Zilong Zhao, Meghan B. Altman, Dalia Lopez-Colon,
Chaoyong James Yang,* Lung-Ji Chang, Chen Liu, and Weihong Tan*
anie_201207063_sm_miscellaneous_information.pdf
Materials: Unless otherwise stated, all solvents and chemicals were obtained from Sigma--Aldrich
and used without further purification. DNA synthesis reagents were purchased from Glen Research.
PEG phosphoramidite (DMT-Hexaethyloxy-Glycol phosphoramidite) was purchased from
ChemGenes Corporation (Wilmington, MA). CellTrackerTM Green CMFDA, CellTraceTM Far Red
DDAO-SE, Annexin V/Dead Cell Apoptosis Kit and the CellTraceTM CFSE cell proliferation kit
were purchased from Invitrogen. Interleukins were purchased from PeproTech. Purified Mouse AntiHuman Perforin was purchased from BD Biosciences.
Table^^1
Oligonucleotide sequences used in this work.
Probe Name
Sequence
Lipo-Sgc8
Lipo-TD05
LipoKK1B10
Lipo--DNA
5' Diacyllipid-(PEG)4-TTT
5' Diacyllipid-(PEG)4-AAC
5' Diacyllipid-(PEG)4-ACA
TGT-3'
5' Diacyllipid-(PEG)4-NNN
TTT TAT CTA ACT GCT GCG CCG CCG GGA AAA TAC TGT ACG GTT AGA-3'
ACC GGG AGG ATA GTT CGG TGG CTG TTC AGG GTC TCC TCC CGG TGA-3'
GCA GAT CAG TCT ATC TTC TCC TGA TGG GTT CCT ATT TAT AGG TGA AGC
NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN-3'
Cell Surface Labeling: Cells (200^^µL, 1×106^^cells^mL<M->1) were suspended in a 96-well plate
and incubated with lipo--DNA probes (1^^µm lipid--DNA, with or without fluorescent dye) in cell
culture medium at 37^°C for 2^^h. Cells were then washed three times with PBS to remove free
probes and resuspended in the desired buffer or cell culture medium.
Imaging of Lipo--DNA on Cell Surface. Ramos cells were incubated with TMR-labeled lipo-Lib
probes, as described above. Images were taken and collected in the perpendicular lateral (x-y) plane
by laser scanning confocal microscopy with a 488^^nm argon laser and a 543/633^^nm helium/neon
laser.
Homotypic and Heterotypic Cell Assembly. For homotypic cell assembly, Ramos cells labeled
with lipo-TD05-TMR or lipo-Lib-TMR were shaken at 300^^rpm for 20--30^^min at 25^°C. For
heterotypic cell assembly, Ramos cells were labeled with lipo-Sgc8-TMR or lipo-Lib-TMR first, and
a proper ratio of CEM cells was then combined in binding buffer and shaken at 300^^rpm for
30^^min at 25^°C. Aliquots were analyzed by laser scanning confocal microscopy. All experiments
were repeated 5 times.
Selective Cell Assembly in Cell Mixture. Green-stained Ramos cells and untreated CEM cells were
mixed together at a 1:1 ratio. K562 cells modified with either Lipo-TD05-TMR or lipo-Sgc8-TMR
probes were co-incubated with 5 equivalents of the above cell mixtures, respectively, and shaken at
300^^rpm for 30^^min at 25^°C. Aliquots were analyzed using laser scanning confocal microscopy.
Cell Assembly Treated with DNase I. After cell aggregates were formed, cells were centrifuged
and resuspended in 1×DNaseI buffer with 20U/mL DNaseI and incubated at 37^°C for 10^^min.
Aliquots were analyzed by laser scanning confocal microscopy.
Quantitative analysis of cell aggregates. CEM cells were stained with CellTrace Far Red DDAO-SE,
and Ramos cells were stained with CellTracker Green CMFDA. After washing, CEM cells were
incubated with various concentrations of lipo-Lib or lipo-TD05 (without any dye molecules),
washed, and then incubated with different quantities of Ramos cells, as described above. The
fluorescent signals from channels 1 (green) and 4 (far red) were determined by flow cytometry
(Accuri C6 flow cytometer). The thresholds of channels 1 and 4 were set by comparing the
fluorescent signals generated from unstained CEM and Ramos cells. The percentage of aggregation
was calculated as 100 times the ratio of the double positive (green and far-red) population (upper
right region in Supporting Information, Figure^^S6) to the total CEM cell (far-red) population
1
(upper region in Supporting Information, Figure^^S6). Each set of samples was analyzed in
triplicate.
Aptamer-assisted immune cell-killing assay. K562 or Ramos cells were washed with PBS buffer and
labeled with 1^^µm carboxyfluorescein succinimidyl ester (CFSE), suggested by the manufacturer,
and then aliquotted to a 96-well microtiter plate at 1×104 cells/well. Immune effector cells were
added to each well at the desired E:T ratio. The final reaction volume was adjusted to 200^^µL. The
plate was kept in a humidified atmosphere of 5^% CO2 and 37^°C for 2--3^^h. Before flow
cytometry (Accuri C6 flow cytometer) analysis, propidium iodide (PI) was added to each sample and
incubated at RT in the dark for 30^^min to label dead cells. The target cell death was calculated as
the number of CFSE- and PI-positive cells over the total number of CFSE-positive cells.
Lipo-DNA Synthesis and Materials. All DNA sequences were synthesized from 3’ to 5’ using the
ABI 3400 synthesizer on 1.0 micromolar scale. DMT-Hexaethyloxy-Glycol (PEG) phosphoramidite
was coupled to DNA by extended coupling time (900 seconds) on DNA synthesizer. Each DNA
probe was coupled with four PEG phosphoramidite units. Lipid phosphoramidite was synthesized by
following a previously published procedure (1,2) and coupled using the DNA synthesizer by
extended coupling time (900 seconds). After synthesis, the DNA was cleaved and deprotected from
the CPG and purified by reverse phase HPLC using a C4 column (BioBasic-4, 200mm x 4.6mm,
Thermo Scientific) with 100 mM triethylamine-acetic acid buffer (TEAA, pH 7.5) and acetonitrile
(0-30 min, 10-100%) as an eluent. All purified lipo-DNA probes were stored in DNase/RNase free
water.
General Cell Culture Conditions. Jurkat, K562, CCRF-CEM (CCL-119 T-cell, human acute
lymphoblastic leukemia) and Ramos cells (CRL-1596, B lymphocyte, human Burkitt's lymphoma)
were obtained from ATCC (American Type Culture Collection) and were cultured in complete
RPMI 1640 medium (ATCC) supplemented with 10% fetal bovine serum (FBS) (heat inactivated,
GIBCO) and 100 IU/mL penicillin−streptomycin (Cellgro). CMV-specific CD8+ cytotoxic T
lymphocyte (CTL) clone was established by immortalizing primary T cells using lentiviral vectors
and was cultured in complete RPMI with 20U/mL IL-2, 5ng/mL IL-7 and 20ng/mL IL-15. The
washing buffer contained 4.5 g/L glucose and 5 mM MgCl2 in Dulbecco's PBS (Sigma). Binding
buffer used for incubation was prepared by adding yeast tRNA (0.1 mg/mL) (Sigma) and BSA (1
mg/mL) (Fisher) into the washing buffer to reduce background binding. Proteinase K was purchased
from Fisher Biotech. DNaseI was purchased from BioLabs.
Lipo-DNA insertion study. Cells were incubated with FITC-labeled lipo-Lib probes for different
time periods with different probe concentrations. Fluorescent signals from labeled cells were
examined by FACS flow cytometry. Data were analyzed using WinMIDI flow software, and mean
fluorescent intensity from different incubation conditions was compared. The one that gave the
largest fluorescent intensity was selected for subsequent labeling conditions.
Evaluation of Cellular Cytotoxicity of Lipo-DNA. The cytotoxicity of lipid-Lib probe was tested
by standard MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) cell proliferation assay (Promega). K562, CEM and Ramos cells were incubated
with lipo-Lib, as previously described, and seeded into a 96-well cell culture plate. After 2 days of
incubation, the cell culture medium was replaced by MTS-containing medium. After 4 hours of
incubation, the absorbance at 490 nm from each sample was recorded by a microplate
spectrophotometer (Molecular Devices). Control samples were cells without lipo-Lib.
2
Proteinase Treatment of Cells. After washing with 2mL of washing buffer, Ramos cells were
incubated with 0.1mg/mL proteinase K in PBS at 37 °C for 20 min. To quench the proteinase
digestion, the sample was quickly mixed with 200 µL of PBS and placed on ice. Then the treated
cells were washed with 2mL of binding buffer and used for imaging.
Cytosolic Stain. CellTracker Green CMFDA (Invitrogen) and CellTrace Far Red DDAO-SE were
first dissolved in DMSO to 10 mM and further diluted to a final working concentration of 1µM and
10µM, respectively, in serum-free medium. Fresh cells were washed twice in PBS buffer and then
incubated in stain solution for 15-30 min at 37 °C. Labeled cells were washed twice in PBS buffer
and resuspended in cell culture medium for another 30 min at 37 °C before use.
(1)
(2)
Liu H, Zhu Z, Kang H, Wu Y, Sefah K, Tan W (2010) DNA-based micelles: synthesis, micellar
properties and size-dependent cell permeability. Chemistry 16:3791-3797.
Gold L, Janjic N, Schmidt P, Vargeese C (2001) Vascular endothelial growth factor (VEGF)
nucleic acid ligand complexes. U.S. patent 6,168,778.
3
Fig. S1. Lipid-DNA probe can insert into cells. a) FITC signal coming from CEM cells increased with
incubation concentration b) CTL and c) NK cells can be modified with lipo-DNA probes.
Fig. S2. Aptamer-mediated homotypic assembly of CEM cells. a) Aggregation of CEM cells after
treatment with lipo-Sgc8-TMR; b) CEM cells treated with lipo-Lib-TMR; c) Ramos cells treated with
lipo-Sgc8-TMR. (Scale bar: 100 µm)
4
Fig. S3. Sequence-specific heterotypic assemblies between CEM and Ramos. a) 1:10 mixture of lipoTD05-TMR-modified CEM (red fluorescence) and Ramos (nonfluorescent) cells. b) 1:10 mixture of lipoLib-TMR-modified CEM (red fluorescence) and Ramos (nonfluorescent) cells. (Scale bar: 100 µm)
Fig. S4. Sequence-specific heterotypic assemblies between Jurkat and CEM/Ramos cells. a) 1:10 mixture
of lipo-Sgc8-TMR- modified Jurkat (red fluorescence) and CEM (nonfluorescent) cells. b) 1:10 mixture
of lipo-Lib-TMR-modified Jurkat (red fluorescence) and CEM (nonfluorescent) cells. c) 1:10 mixture of
lipo-TD05-TMR-modified Jurkat (red fluorescence) and Ramos (nonfluorescent) cells. d) 1:10 mixture of
lipo-Lib-TMR-modified Jurkat (red fluorescence) and Ramos (nonfluorescent) cells. (Scale bar: 100 µm)
5
Fig. S5. Sequence-specific heterotypic assemblies between K562 and CEM/Ramos cells. a) 1:10 mixture
of lipo-Sgc8-TMR-modified K562 (red fluorescence) and CEM (nonfluorescent) cells. b) 1:10 mixture of
lipo-Lib-TM-modified K562 (red fluorescence) and CEM (nonfluorescent) cells. c) 1:10 mixture of lipoTD05-TMR-modified K562 (red fluorescence) and Ramos (nonfluorescent) cells. d) 1:10 mixture of lipoLib-TMR-modified K562 (red fluorescence) and Ramos (nonfluorescent) cells. (Scale bar: 100 µm)
6
Fig. S6. Sample flow cytometry data for quantification of CEM aggregation. CEM (far red, FL4)
incubated with 1µM lipo-Lib (a) or lipo-TD05 (b) and incubated with 10 equivalent Ramos (green, FL1)
cells. The percentage of aggregation was counted as the number of cells in the upper right region over
total upper region.
Fig. S7 1:5 mixture of lipo-TD05-TMR-modified CEM (red) cells and Ramos (green) cells imaged right
after mixing. CEM and Ramos cells remained apart, and only a few small aggregates were observed.
Scale bar: 100 µm.
7
Fig. S8. 1:5 mixture of a) lipo-Lib-TMR- or b) lipo-TD05-TMR-modified CEM (red) cells and Ramos
(green) cells after 25 min incubation. Scale bar: 100 µm.
8
Fig. S9. Aggregates of Ramos cells disappear after treatment with proteinase K. a) homotypic aggregates
of Ramos cells after modification with lipo-TD05-TMR; b) the same assembled cells after incubation at
37oC in the presence of Proteinase K. (Scale bar: 100 µm)
a)
CTL only
Viability of CTL
CTL + lipo-DNA
100%
80%
PI
60%
83.34%
81.82%
40%
20%
0%
CTL only
CTL + lipo-DNA
Annexin V
Fig. S10 Cytotoxicity of the lipo-DNA probe. a) Apoptosis and cell death staining of unmodified and
lipo-DNA modified CTL. The lower left square represented healthy cell population.
b) Cell proliferation assay. Cells labeled with lipid-DNA (grey bars) showed no significant difference in
proliferation rate compared with cell-only control (black bars), indicating that lipid-DNA is not toxic to
cells at 1 µM concentration. Data are means of three measurements. Bars are standard deviations.
9
Fig S11. Sample flow cytometry data of CTL-Ramos killing assay. CFSE- and PI-positive cells were
dead Ramos cells. A01: Ramos only; B01: unmodified CTL and Ramos; C01: lipo-Lib-modified CTL
and Ramos; D01: lipo-TD05-modified CTL and Ramos. The percentage of dead Ramos was calculated as
the number of CFSE- and PI-positive cells over the number of CFSE-positive cells.
Table S1: Aggregation percentage of CEM cells
Lipid-DNA concentration
CEM to Ramos Ratio
Sample 1
Sample 2
Lipo-Lib
Sample 3
Mean
SD
Sample 1
Sample 2
Lipo-TD05
Sample 3
Mean
SD
1:1
4.51
7.04
3.63
5.06
1.77
49.98
31.62
33.57
38.39
10.08
500nM
1:5
6.84
5.15
5.81
5.93
0.85
68.75
60.56
63.00
64.10
4.21
1:10
5.47
6.56
5.47
5.83
0.63
65.15
65.92
69.72
66.93
2.45
1:1
4.33
3.40
3.57
3.77
0.50
79.49
77.12
80.04
78.88
1.55
1µM
1:5
5.11
4.47
7.77
5.78
1.75
91.64
84.54
91.18
89.12
3.97
1:10
5.39
4.30
5.52
5.07
0.67
93.73
91.18
93.46
92.79
1.40
1:1
3.32
4.38
3.55
3.75
0.56
86.25
81.19
83.95
83.80
2.53
2µM
1:5
4.78
5.39
4.72
4.96
0.37
96.38
96.29
94.89
95.85
0.84
1:10
5.67
4.28
4.92
4.96
0.70
93.82
94.31
95.71
94.61
0.98
1:1
5.24
3.96
3.45
4.22
0.92
78.57
86.16
85.34
83.36
4.17
5µM
1:5
6.11
6.90
6.70
6.57
0.41
97.31
95.14
94.71
95.72
1.39
10
1:10
6.40
6.69
7.19
6.76
0.40
97.68
95.22
95.85
96.25
1.28
11

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