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Title: Study of the Molecular Recognition of Aptamers Selected through Ovarian Cancer Cell-SELEX
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Title: Study of the Molecular Recognition of Aptamers Selected through Ovarian Cancer Cell-SELEX
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Creator: Dimitri Van Simaeys
Dalia Lopez-Colon
Kwame Sefah
Rebecca Sefah
Elizabeth Jimenez
Weihong Tan
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Publication Date: 2010
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Resource Identifier: 10.1371/journal.pone.0013770

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OPEN ACCESS Freely available online


Study of the Molecular Recognition of Aptamers

Selected through Ovarian Cancer Cell-SELEX

Dimitri Van Simaeys1"2, Dalia L6pez-Col6n1"2, Kwame Sefah1"2, Rebecca Sutphen3, Elizabeth
Jimenez"2, Weihong Tan1"2'4*
1 Department of Chemistry, Center for Research at Bio/Nano Interface, University of Florida, Gainesville, Florida, United States of America, 2 Shands Cancer and Genetic
Research Center, Department of Physiology and Functional Genomics, University of Florida, Gainesville, Florida, United States of America, 3 Department of Pediatrics,
College of Medicine, University of South Florida, Tampa, Florida, United States of America, 4 Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of
America


Abstract

Background: Ovarian cancer is the most lethal gynecological malignancy, and the ovarian clear cell carcinoma subtype
(OCCA) demonstrates a particularly poor response to standard treatment. Improvements in ovarian cancer outcomes,
especially for OCCA, could be expected from a clearer understanding of the molecular pathology that might guide
strategies for earlier diagnosis and more effective treatment.

Methodology/Principal Findings: Cell-SELEX technology was employed to develop new molecular probes for ovarian
cancer cell surface markers. A total of thirteen aptamers with Kd's to ovarian cancer cells in the pico- to nanomolar range
were obtained. Preliminary investigation of the targets of these aptamers and their binding characteristics was also
performed.

Conclusions/Significance: We have selected a series of aptamers that bind to different types of ovarian cancer, but not
cervical cancer. Though binding to other cancer cell lines was observed, these aptamers could lead to identification of
biomarkers that are related to cancer.

Citation: Van Simaeys D, L6pez-Col6n D, Sefah K, Sutphen R, Jimenez E, et al. (2010) Study of the Molecular Recognition of Aptamers Selected through Ovarian
Cancer Cell-SELEX. PLoS ONE 5(11): e13770. doi:10.1371/journal.pone.0013770
Editor: Cameron Neylon, University of Southampton, United Kingdom
Received May 3, 2010; Accepted October 6, 2010; Published November 1, 2010
Copyright: 2010 Van Simaeys et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors thank National Institutes of Health (NIH) for support (R01GM079359, R01 CA133086, R01-CA106414). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
E-mail: tan@chem.ufl.edu
( These authors contributed equally to this work.


Introduction

Ovarian cancer is the fifth most common cancer in women [1],
and has the highest death rate of any gynecologic malignancy.
This disease is characterized by few early symptoms, presentation
at an advanced stage in the majority of cases, and poor survival
rates [2-8]. The prognosis is especially poor for patients with
ovarian clear cell adenocarcinoma (OCCA), which is often
resistant to standard platinum-based chemotherapy [6-10].
The most commonly used serum biomarker for clinical
diagnosis and prognosis is ovarian cancer antigen 125 (CA-125).
The CA-125 value is elevated in approximately 90% of late-stage
cases of epithelial ovarian cancer (stages 3 and 4). However, it is
only elevated in 50-60% of women with early stage disease and is
also elevated in a number of benign conditions [2,5,7,11-13].
The utilization of aptamers has great potential for the identifica-
tion of new biomarkers. Aptamers, which are probes capable of
specifically binding to cell surface markers expressed by targeted
tumor cells [14-21], are short single-stranded oligonucleotides of
about 100 nt. They are selected from large combinatorial pools of
sequences by Systematic Evolution of Ligands by Exponential
Enrichment (SELEX) for their capacity to bind to targets, which can


PLoS ONE | www.plosone.org


range from small molecules to proteins or polysaccharides, as well as
tumor cells [16-19,21-23]. Aptamers have well-defined tertiary
structures that dictate the selectivity for their targets.
The target specificity and affinity of aptamers are similar to
those of antibodies, but with several advantages over antibodies for
clinical use. Aptamers may be chemically synthesized in a short
time at relatively low cost, allowing better batch-to-batch
reproducibility and easier incorporation of chemical modifications.
Since aptamers for cells are selected without prior knowledge of
the target molecules, selected aptamers can be used to identify new
surface markers on cancer cells [14,16-18,20,24-27].
In this work, a total of 13 aptamers was selected for two model
ovarian cancer cell lines: the OCCA line TOV-21G [28] (10
aptamers) and the ovarian serious adenocarcinoma line CAOV-3
[29] (3 aptamers). The cell surface targets of the aptamers were
also briefly investigated. Preliminary investigation of the aptamers'
targets and binding characteristics was also performed.

Results and Discussion

Two model ovarian cancer cell lines were chosen for the
selection of ovarian cancer aptamers: the OCCA cell line TOV-


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Ovarian Cancer Cell Aptamers


21G and the ovarian serious adenocarcinoma cell line CAOV-3. In
order to identify aptamers that specifically bind to ovarian cancer
cells, the cervical cancer cell line HeLa was used for counter-
selection. The SELEX procedure for TOV-21G is described
briefly below. A detailed description is provided in the exper-
imental section.
To start the selection process, 20 pmol of naive library was
enriched by sequential binding to TOV-21G cell monolayers.
Sequences showing non-specific binding to general cell surface
markers were removed by ..I ,1.. 1, 11,,. enriched pool with HeLa
cells (rounds 2, 4, 5, 7, 8, 9, 12, 20, 21, 22). The eluted pool for
each round of SELEX was amplified 1, .... _1l PCR, after which
the ssDNA pools of interest were recovered and monitored for
enrichment toward TOV-21G by flow cytometry. As the selected
pools were enriched with sequences that recognize and bind to the
target cell line, an increase in fluorescence signal was observed
(Figure la). But attempts to omit counter-selection in rounds 13 to
19 led to enrichment for HeLa-binding sequences. The sequences
binding to HeLa cells were successfully removed by counter
selection in subsequent rounds, while the enrichment towards the
target cell line was maintained T;_ni. lb). After 22 rounds of
SELEX, an enriched pool that specifically bound to the model
OCCA cell line, but marginally to HeLa cells, was obtained
(Figure 2). Thus, the pool was successfully enriched for sequences
binding surface markers expressed by the model OCCA cell line,
but not by cervical cancer cells.
Following completion of the selection process, three pools were
chosen and submitted for sequencing: the final pool (round 22), the
previous pool (round 21) and a pool showing minimal enrichment
(round 13). Pool sequencing was used to help identify aptamer
candidates by generating large quantities of sequences. This
number of sequences (here a minimum of 2000 per pool) is large
enough to allow identification of aptamers that only have a small
representation in the pool (i.e. less then 1%). As can be seen in
Table 1, selected aptamers were indeed present as early as a
minimal enrichment was observed. AptTOVl's size percentage


decreased as the SELEX continued, which is remarkable given the
high affinity of this aptamer. This behavior has also been observed
in other selections [30]. Other aptamers show a consistent increase
in size percentage as the enrichment increased. The results from
AptTOV6 are included to demonstrate that even relatively small
families can lead to aptamers.
Sequences were aligned into families according to sequence
homology. The number of homologues was compared across the
different sequenced pools to validate their enrichment 1-1 ,....1 the
selection procedure using basic bio- informatics. Ten sequences
showing the best homology 1 t.. .1 .....I the pools were selected as
aptamer candidates, synthesized and tested for binding to the
model ovarian cancer cell lines. All the candidates showed binding
to TOV-21G, with binding affinities in the pico- to nano-molar
range (Table 2). This demonstrates the potential of next
generation sequencing in SELEX to become a powerful and
reproducible method for the development of aptamers.
As shown in Table 2, aptamers aptTOV1 (Kd =0.25
0.08 nM) and aptTOV2 (0.900.25 nM) bind very ;_1.1 to
TOV-21G cells. As shown in Table 2, both aptamers can
distinguish TOV-21G from HeLa cells.
The binding of the selected aptamers was tested with different
adenocarcinoma cell lines, as well as other types of cancer cell
lines, as shown in Table 2. Five of the aptamers selected against
TOV-21G showed binding to CEM cells (acute lymphoblastic
leukemia), while none of the aptamers bound to Ramos cells
(Burkitt's lymphoma) or HL-60 (acute promyelocytic leukemia).
All of the obtained aptamers bind to colorectal adenocarcinoma
(HCT-116) and glioblastoma (A172). The aptamers obtained from
TOV-21G do not bind to DLD-1 (Dukes' type C colorectal
adenocarcinoma), while the aptamers coming from CAOV-3 did
bind to this cell line. This behavior is similar to that observed in
previous work conducted in our laboratory.
Since both selections took place at 4C, the selected aptamers
were tested at physiological conditions. The aptamers were
incubated with the target cell line at 37'C and 4C and their


HeLa Cells

Pool 13

Pool 19
Pool 22


101 102 10bo 1' ' '
Fluorescence Fluorescence


Figure 1. The binding assay of the enriched pools with TOV-21G and HeLa cells. A) The enrichment with TOV-21G cells B) The marginal
binding of the respective pools to HeLa cells. By doing counter selection, sequences binding to HeLa were removed.
doi:10.1371/journal.pone.0013770.g001


., PLoS ONE I www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e13770







Ovarian Cancer Cell Aptamers


TOV-21G

AptTOVI at 4C
AptTOV I at 37C


Fluorescence Fluorescence
C 25


6
[aptTOVl] (nM)


8 10 12


Figure 2. The binding of PE/cyS-labeled aptTOVl (250 nM in binding buffer) A) to TOV-21G at 40C and 37C; B) to HeLa at 40C. The
negative control in these binding assays was PE/Cy5-labeled random library. C) Cells were incubated with varying concentrations of PE-Cy5-labeled
aptamer in duplicate. The fluorescence intensity originating from background binding at each concentration was subtracted from the mean
fluorescence intensity of the corresponding aptamer.
doi:10.1371/journal.pone.0013770.g002


binding was measured. All aptamers showed similar binding at
4C and 37'C (e.g., aptTOV1 in Figure i suggesting that they
have potential for in vivo studies.



Table 1. The evolution of aptamers throughout the
sequenced pools.


Pool 13 (%) Pool 21 (%) Pool 22 (%)


aptTOVl
aptTOV2
aptTOV2a
aptTOV2all
aptTOV3
aptTOV6


Rows represent specific aptamers' percentage in the sequenced pools.
AptTOVall is the sum of both AptTOV2 and AptTOV2a. AptTOV are aptamers
from the SELEX for TOV-21G cells.
doi:10.1371/journal.pone.0013770.t001


To investigate the nature of the target molecule of each
aptamer, binding of each aptamer was tested after treatment of the
target cells with the proteases trypsin or proteinase K. As can be
seen in Figure 3a, aptTOV1 shows a clear loss of binding to TOV-
21G after protease treatment. The same behavior was also
observed with all other aptTOV aptamers. Interestingly, for the
second model cell line, CAOV-3, DOV3 and DOV4 retained
their binding after protease treatment (Figure 3b), suggesting that
these aptamers may not be binding to cell surface membrane
proteins, but rather to another type of cell surface marker (i.e.,
carbohydrate or lipid). Neither DOV3 nor DOV4 binds to the
tested leukemia cells (Table 3).
In conclusion, we have selected a series of aptamers with high
affinity for ovarian cancer cells, including OCCA (TOV-21G) and
serious adenocarcinoma (CAOV-3). By counter selection against
HeLa cells, aptamers that can distinguish ovarian cancer from
cervical cancer were selected. In particular, AptTOV 1 showed
very high affinity towards TOV-21G, with a Kd of 250 pM.
Given the limited number of biomarkers for ovarian cancer
currently available, the aptamers obtained from these selections
have potential for improving diagnosis and treatment of this deadly
disease. Because the aptamers also bind benign cysts (Table 3), the


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,., PLoS ONE I www.plosone.org







Ovarian Cancer Cell Aptamers


Table 2. A compendium of the aptamers obtained by selection vs cancers TOV-21G (aptTOV) or CAOV-3 (DOV).


Name

aptTOV1


aptTOV2


aptTOV2a


aptTOV3


aptTOV4


aptTOV5


aptTOV6


aptTOV7


aptTOV8


aptTOV9


DOV 3


DOV 4

DOV 6


Sequence

5' ATC CAG AGT GAC GCA GCA GAT CTG TGT AGG ATC GCA GTG TAG TGG ACA TTT GAT ACG
ACT GGC TCG ACA CGG TGG CTT A 3'
5' ATC CAG AGT GAC GCA GCA TAA TCT CTA CAG GCG CAT GTA ATA TAA TGA AGC CCA TCC
ACC TGG ACA CGG TGG CTT A- 3'
5' ATC CAG AGT GAC GCA GCA CAA TCT CTA CAG GCG CAT GTA ATA TAA TGG AGC CTA TCC
ACG TCG ACA CGG TGG CTT A- 3'
5' ATC CAG AGT GAC GCA GCA CTC ACT CTG ACC TTG GAT CGT CAC ATT ACA TGG GAT CAT
CAG TCG ACA CGG TGG CTT A- 3'
5' ATC CAG AGT GAC GCA GCA GGC ACT CTT CAC AAC ACG ACA TTT CAC TAC TCA CAA TCA
CTC TCG ACA CGG TGG CTT A- 3'
5' ATC CAG AGT GAC GCA GCA CAA CAT CCA CTC ATA ACT TCA ATA CAT ATC TGT CAC TCT
TTC TCG ACA CGG TGG CTT A- 3'
5' ATC CAG AGT GAC GCA GCA CGG CAC TCA CTC TTT GTT AAG TGG TCT GCT TCT TAA CCT
TCA TCG ACA CGG TGG CTT A- 3'
5' ATC CAG AGT GAC GCA GCA CCA ACT CGT ACA TCC TTC ACT TAA TCC GTC AAT CTA CCA
CTC TCG ACA CGG TGG CTT A- 3'
5' ATC CAG AGT GAC GCA GCA CCA GTC CAT CCC AAA ATC TGT CGT CAC ATA CCC TGC TGC
GCC TCG ACA CGG TGG CTT A- 3'
5' ATC CAG AGT GAC GCA GCA GCA ACA CAA ACC CAA CTT CTT ATC TTT TCG TTC ACT CTT
CTC TCG ACA CGG TGG CTT A- 3'
5' -ACT CAA CGA ACG CTG TGG ATG CAG
AGG CTA GGATCT ATA GGT TCGGAC GTC GAT GAG GAC CAG GAG AGC A 3'
5' ACT CAA CGA ACG CTG TGG AGG GCA TCA
GAT TAG GAT CTA TAG GTTCGG ACA TCG TGA GGA CCA GGA GAG CA 3'
5' ACT CAA CGA ACG CTG TGG AAT GTT GGGGTA GGT AGA AGG TGA AGGGGT TTC AGT TGA
GGA CCA GGA GAG CA 3'


% in pool

2,53


18,62


doi:1 0.1371 /journal.pone.0013770.t002


aptamers cannot be used to identify ovarian cancer per se. However,
since the aptTOV apamers do not bind to a cancer of similar
etiology (CAOV3) and also not to HeLa, they still have the potential
to provide more insight into the i. ,11,. 1. .. of ovarian cancer. It has
been observed that there are significant differences in the proteome
of serious and clear cell ovarian cancer [31]. The targets for these
aptamers are most likely down regulated or silenced in these two cell


TOV-21G

aptTOV I
aptTOV I 30 min
protease treated cells


models. Additionally, the AptTOV aptamers show binding to
cancer cell lines from different non-related cancers (Table 3), and
some AptTOV aptamers also bind CEM cells. This result suggests
that the aptamers obtained from this SELEX can be used for
profiling the expression of membrane proteins of different cancers.
Identifying the targets of the selected aptamers is expected to shed
light on the underlying mechanisms involved.


CAOV-3


DOV3
DOV3 on 30 min
protease treated cells


10. 10 10' 10'
Fluorescence Fluorescence


Figure 3. A preliminary study of the nature of the targets. A) No binding was observed for aptTOV1 to trypsinized TOV-21G cells. B) The
binding of DOV 3 and 4 to CAOV-3 cells was not affected by protease treatment. All other aptamers showed the same behavior as represented in a).
doi:1 0.1371/journal.pone.0013770.g003



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Kd (nM)

0.2510.08


0.9010.25


11+3


30+9


20+5


4.5+1.2


29+7

6.612.3


17+3

26110


132132


40120

39120







Ovarian Cancer Cell Aptamers


Table 3. Relative binding of the selected aptamers to various cell lines.


TOV- 21G CAOV-3 HeLa BCC


H23 HT-29 HCT-116


A172 Ramos CEM HL-60


-++ -++


-++ + ++
-++ + ++
-n- -n- -H-
++ ++ ++-H
A-I-- ++ + ++
-++ ++ ++
-++ ++ ++
-H- + -H- ++
-++ + ++


aptTOV1
aptTOV2
aptTOV2a
aptTOV3
aptTOV4
aptTOV5
aptTOV6
aptTOV7
aptTOV8
aptTOV9
DOV3
DOV4
DOV6


A dash indicates no observed binding to the corresponding cell line. See supplemental data S4 for our guidelines for the amount of pluses.
doi:10.1371/journal.pone.001 3770.t003


The discovery of two aptamers that were insensitive to protease
digestion is intriguing. Additionally, their binding to all tested
adenocarcinoma cells, but not to any of the leukemia cell lines,
suggests the potential to further elucidate the underlying molecular
differences between these cancer types. Further investigation is
warranted to identify the targets of these aptamers and assess their
performance in clinical samples.


Materials and Methods

Instrumentation and reagents
All oligonucleotides were synthesized by standard phosphor-
amidite chemistry using a 3400 DNA synthesizer (Applied
Biosystems) and were purified by reversed-phase HPLC (Varian
Prostar). All PCR mixtures contained 50 mM KC1, 10 mM Tris-
HCI (pH 8.3), 2.0 mM MgC2,, dNTPs (each at 2.5 mM), 0.5 aM
of each primer, and Hot start Taq DNA polymerase (5units/pL).
PCR was performed on a Biorad Thermocycler and all reagents
were purchased from Takara. Monitoring of pool enrichment,
characterization of the selected aptamers, and identification of the
target protein assays were performed by flow cytometric analysis
using a FACScan cytometer (BD Immunocytometry Systems).
Trypsin and Proteinase K were purchased from Fisher Biotech.
The imaging of cells was performed with an Olympus FV500-
IX81 confocal microscope (Olympus America Inc., Melville, NY).
The DNA sequences were determined by the Genome Sequencing
Services Laboratory at the University of Florida with the use of
454 sequencing (Roche).

Cell culture and buffers
The CAOV-3, HeLa, H ._1 1 and TOV-21G cell lines
where obtained from the American Type Cell Culture (ATCC).
The CAOV-3 and TOV-21G ovarian cancer cell lines where
maintained in culture with MCBD 105: Medium 199 (1:1); the
HeLa cell line was cultured in RPMI-1640; and the H ._: I
cell line was cultured in Dulbecco's Modified Eagle's Medium
(DMEM). All media where supplemented with 10% FBS and
100 UI/mL Penicillin-Streptomycin. Other cell lines used for
selectivity assays included CEM (T cell leukemia), Ramos
(Burkitt's Lymphoma), HCT-116, DLD-1, HT-29 (colorectal


SPLoS ONE I www.plosone.org


adenocarcinoma), NCI_H23 (non-small cell lung Cancer) and
A172 (glioblastoma), all of which were cultured according to
ATCC specifications. All cell lines where incubated at 37C in a
5% CO2 atmosphere.
During the selection, cells were washed before and after
incubation with wash buffer 1\\ 1: containing 4.5 g/L glucose
and 5 mM MgC12 in Dulbecco's phosphate buffered saline with
calcium chloride and magnesium chloride -1.... Binding buffer
(BB) used for selection was prepared by adding yeast tRNA
(0.1 mg/mL) '1..... and BSA (1 mg/mL) (Fisher) to the wash
buffer to reduce background binding.

SELEX library and primers
The HPLC-purified library contained a segment of randomized
sequence of 40 nucleotides (nt) flanked by 20-nt primer
hybridization sites:
(5'- ATC CAG AGT GAC GCA GCA .N ., TGG ACA CGG
TGG CTT AGT-3') and (5'-ACT ACC AAC GAG CGA CCA
CT N ., AGA GTT CAG GAG AGG CAG GT-3'). The
forward primers were labeled with 5'-FITC and the reverse
primers were labeled with 5'-biotin.

In Vitro cell-SELEX
In this study, TOV-21G was used as the target cell line and
HeLa was used for counter-selection. For the first round, the cells
were incubated with 20 pmol of naive ssDNA library dissolved in
BB. For later rounds, 50 pmol of enriched pool were used for
incubation, also dissolved in BB. Before incubation, the ssDNA
pool was denatured by heating at 95 C for 5 min and was cooled
rapidly on ice for 5 min, allowing each sequence to form the most
stable secondary structure.
The cells were washed twice (2 min) with WB and incubated
with the DNA pool on ice in an orbital shaker for 30 min. In later
selection rounds, the cells were washed with increased stringency
to remove weakly binding sequences (a larger number of washes
and increased washing time, up to 5 min). The bound sequences
were eluted in 500 liL BB by heating at 95C for 15 min, cooled
on ice for 5 min and centrifuged at 14,000 rpm for 2 min.
The supernatant containing the DNA sequences was then
incubated with a negative cell line to perform a subtraction of


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A-+++ +
A-+++ +


.. .--+ .. .. .-.


DLD-1


++++ ++I +


- +++
++







Ovarian Cancer Cell Aptamers


general sequences, as described above. The remaining sequences
were amplified by PCR using the FITC- and biotin-labeled
primers. Amplifications were carried out at 95C for 30 s, 60C
for 30 s, and 72'C for 30 s, followed by final extension for 3 min
at 72'C. The selected sense ssDNA was separated from the
biotinylated antisense ssDNA by streptavidin-coated sepharose
beads (Amersham Bioscience). The ssDNA was eluted from the
sepharose beads by melting in a 0.2M NaOH solution.
The enrichment of specific sequences was assayed using flow
cytometry as explained below. When the level of enrichment
reached a plateau, pools of interest were submitted for sequencing.
The aptamer selection for the CAOV3 cell line was performed
using the same protocol, however a 1 minute trypsinization step
was used to suspend the cells before adding the pools.
Supplemental data S1 and S2 contain the clustal data of the
alignments of each selection (final pool).

Affinity studies: Flow cytofluorometric analysis for the
determination of binding affinity
To determine the binding affinities of the aptamers, the target
cells (5 x105) were incubated with various concentrations of 5'-
biotin labeled aptamers on ice for 20 min in 100 pL of BB. Cells
were then washed twice with 500 gL of BB, and suspended in
100 gL of BB containing streptavidin-PE-Cy5.5. Cells were then
washed twice with 500 gL of WB, and were suspended in 200 gL
of BB for flow cytometric analysis, using a 5'-biotin labeled
random sequence as the negative control. All the experiments for
binding assays were repeated at least 2 times. The specific binding
intensity was calculated by subtraction of the mean fluorescence
intensity of the background binding from the mean fluorescence
intensity of the aptamers. The equilibrium dissociation constant
(Kd) of the fluorescent ligand was obtained by fitting a plot of the
specific binding intensity versus (Y) the aptamer concentration (X)
to the equation Y = B mX/(Kd+X) using SigmaPlot. Jandel, San
Rafael, CA). Supplemental data S3 contains the flow data for all
experiments presented in this article.

Selectivity and specificity
To determine the cell specificity of the selected aptamers, cell
lines including HeLa, K562, H23, H69, A172, HL-60. HT-29,
Ramos and CEM were used in binding assays by flow cytometry as
described above.

Effect of temperature on aptamer binding
The aptamer selection process and all of the binding assays were
performed on ice. It has been observed that some of the aptamers



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S PLoS ONE | www.plosone.org


selected at lower temperatures may not bind well at 37 C [26],
leading to poor performance under physiological conditions. In
order to verify binding stability, aptamers were incubated with the
target at 37'C, and fluorescence intensity was determined by flow
cytometry. Aptamers incubated on ice were used as the positive
control.

Protease digestion assay
Target cells (5 x105) were detached using non-enzymatic cell
dissociation solution. After resuspension, the cells were washed
with 3 mL of PBS and then incubated with 1 mL of 0.05%
trypsin/0.53 mM EDTA in HBSS or 0.1 mg/mL proteinase K in
PBS at 37'C for 1, 5, 15, 30 and 60 minutes. Pure FBS was added
to quench the proteinases. After washing with 2 mL of BB, the
treated cells were used for binding assays as described above.

Supporting Information

Data S1 Allignment of the final TOV pool.
Found at: doi:10.1371/journal.pone.0013770.s001 (1.13 MB
TXT)

Data S2 Allignment of the final CAOV3 pool.
Found at: doi:10.1371/journal.pone.0013770.s002 (0.20 MB
TXT)

Data S3 This file contains the flow data from the figures
presented in this article.
Found at: doi:10.1371/journal.pone.0013770.s003 (3.69 MB ZIP)

Data S4 Legend to the amount of pluses. This figure was our
guideline to determine the amount of pluses for table 3.
Found at: doi:10.1371/journal.pone.0013770.s004 (0.04 MB
PDF)

Acknowledgments

We thank Drs. Parag Parekh and Tahir Bayrac for the many useful
scientific discussions that contributed to this work, Mr. George Ansoaunoor
for technical help. We also thank Dr. Kathryn Williams for her critical
review of the manuscript. We thank the DNA sequencing core, ICBR, at
the University of Florida.

Author Contributions
Conceived and designed the experiments: DVS DLC RS WT. Performed
the experiments: DVS DLC EJ WT. Analyzed the data: DVS DLC KS
WT. Contributed reagents/materials/analysis tools: DVS DLC KS EJ RS.
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Ovarian Cancer Cell Aptamers


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