Simultaneous improvement of specificity and affinity of aptamers against
Streptococcus mutans by in silico maturation for biosensor development
Nasa Savory1,3, Yayoi Takahashi2,3, Kaori Tsukakoshi1, Hijiri Hasegawa1, Madoka Takase1,
Koichi Abe1, Wataru Yoshida1, Stefano Ferri1, Shizuko Kumazawa2, Koji Sode1 and Kazunori
Ikebukuro1,*
1
Department of Biotechnology and Life Science, Tokyo University of Agriculture and
Technology, 2-24-26 Naka-cho, Koganei, Tokyo 184-8588, Japan
2
Devices Development Center, TDK Corporation, 2-15-7 Higashi-Ohwada, Ichikawa, Chiba
272-8558, Japan
3
These authors equally contributed to this work.
*Corresponding author
Address: Department of Biotechnology and Life Science, Tokyo University of Agriculture and
Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan
E-mail: ikebu@cc.tuat.ac.jp
Tel/Fax: +81 42 388 7030
1
Supplementary methods
Flow cytometry
S. mutans ATCC 25175 cells were fixed in 70% (v/v) ethanol for 2 h on ice and washed in TBS
buffer. The cells were incubated with a concentration range of 5′-fluorescein-modified
SMa#G11-5 or SMa#G11-6 in TBS-T buffer for 30 min at r.t. After the cells were washed twice
in TBS-T buffer, flow cytometry was performed using FACSCalibur (Becton Dickinson, NJ). A
total of 20,000 events were counted for each analysis, using triplicate analyses of triplicate
incubations. Total fluorescence intensity was calculated using FACS station Ver. 2.0, and
autofluorescence intensity of S. mutans cells was subtracted as background signal. The KD values
for binding of aptamers to S. mutans were estimated by Scatchard plot analysis.
CD spectroscopy
Aptamers SMa#G11-5, SMa#G11-6 and the core sequence were heat treated as described above
in TBS buffer or Tris buffer (10 mM Tris-HCl, pH 7.4). The CD spectra of SMa#G11-5,
SMa#G11-6, and the core sequence (f.c. 5 µM) were measured with a J-720 spectropolarimeter
(Jasco) at 200–320 nm with a 0.1-cm path length cuvette at 25 C.
Native polyacrylamide gel analysis
For assessment of Gq formation, a BODIPY-labeled macrocyclic heptaoxazole (Gq ligand) (f.c.
10 µM) was incubated with SMa#G11-5, SMa#G11-6, or the core sequence (f.c. 10 µM) for 1 h
at r.t. The samples were then loaded onto a 10% polyacrylamide gel. DNA bands were detected
by fluorescence of the Gq ligand as well as staining with SYBR Safe DNA gel stain (Life
Technologies).
2
Fluorescence intensity (A.U.)
4000000
3500000
3000000
2500000
2000000
SMa#G11-5
1500000
SMa#G11-6
1000000
500000
0
-500000
0
100
200
300
400
Aptamer concentration (nM)
Fig. S1. Binding saturation curve of aptamer SMa#G11-5 and SMa#G11-6 to S. mutans cells. S.
mutans ATCC 25175 cells were incubated with varying concentrations of 5′-fluorescein-labeled
SMa#G11-5 or SMa#G11-6. After the cells were washed twice in binding buffer, flow cytometry
was performed. A total of 20,000 events were counted for each analysis, using triplicate analyses
of triplicate incubations. Total fluorescence intensity was calculated using FACS station Ver.
2.0, and cell autofluorescence intensity was subtracted as background signal. Error bars are
standard deviations (n = 3). The KD values for binding of aptamers to S. mutans were estimated
by Scatchard plot analysis.
3
ph
ilu
s
L.
ac
id
o
ut
an
s
m
S.
Initial library
(N60-L1)
Fig. S2. Aptamer blotting assay for binding of the random DNA library (N60-L1) to S. mutans
and L. acidophilus cells. 1 × 1010 CFU of S. mutans ATCC 25175 and 1 × 108 CFU of L.
acidophilus cells were spotted on Hybond-ECL nitrocellulose membrane. After incubation with
5′-FITC-modified the library N60-L1, oligonucleotide binding was detected using anti-FITC
antibody HRP conjugate.
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Fig. S3. CD spectra of (A) SMa#G11-5, (B) SMa#G11-6, and (C) the core sequence in 10 mM
Tris-HCl buffer (pH 7.4) or TBS buffer. Each oligonucleotide was folded in the respective
buffer, and the CD spectra of DNA at 25C were measured at 200–320 nm using a J-720
spectropolarimeter (JASCO) with a 0.1-cm path length.
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Fig. S4. Native PAGE (10% polyacrylamide gel) analysis of SMa#G11-5, SMa#G11-6, and the
core sequence incubated with or without BODYPY-labeled Gq ligand. Gq ligand bound to
DNAs was visualized with (A) fluorescence and (B) DNAs were stained with SYBR Safe.
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Table S1. Oligonucleotide sequences of ssDNA libraries and respective primers used for CellSELEX experiments.
Library set
Library
Forward primer
Reverse primer
N30-L1
5'ATACCAGTCTATTCAAT
T(N30)AGATAGTATGTG
CAATCA-3'
5'ATACCAGTCTATTC
AATT-3'
5'TGATTGCACATACT
ATCT-3'
N60-L1
5'ATACCAGCTTATTCAAT 5'T(N60)AGATAGTAAGAG ATACCAGTCTATTC
CAATGT-3'
AATT-3'
5'ACATTGCTCTTACT
ATCT-3'
N60-L2
5'GTACCAGCTTATTCAAT
T(N60)AGATAGTATGTT
CATCAG-3'
5'GTACCAGCTTATTC
AATT-3'
5'CTGATGAACATACT
ATCT-3'
N60-L3
5'ATAGTCTATCCATCAAT
T(N60)AGATAGCAAGTG
TATTCA-3'
5'GTACCAGCTTATTC
AATT-3'
5'TGAATACACTTGCT
ATCT-3'
N30 and N60 represent 30- and 60-nt randomized regions, respectively.
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Legends and footnotes of supplementary tables submitted as an MS Excel file.
Table S2. Aptamer sequences used as parent sequences for in silico maturation to improve
binding ability and specificity for S. mutans.
Footnote:
The sequence name represents selection number which aptamers were selected from (see Table
S4) followed by clone numbers and also indicates partial sequence of identified aptamers. For
example, SMa#1-4-P2 represents a partial sequence of an aptamer selected by selection 1 (see
Table S4) with clone number 4.
*Quantitative data for SMa#4-19-P2 were not available. This sequence was chosen according to
a result of aptamer blotting assay in which SMa#4-19-P2 showed binding to S. mutans as well as
SMa#4-19-P1.
Table S3. All aptamers generated during in silico maturation process and their relative binding
ability and specificity assessed by aptamer blotting assays.
Footnote:
Relative binding ability was normalized by binding signal of a parent aptamer SMa#3-6-P1 as
1.00. Relative binding specificity is a ratio of binding signal for S. mutans to that for L.
acidophilus of each aptamer. Each aptamer name represents generation number and sequence ID.
For example, SMa#G1-2 represents sequence ID 2 of generation 1 (G1) sequences. We also
assessed guanine rich parts of some sequences which are indicated as Gq-1 or -2 following its
original sequence name.
Table S4. Aptamer sequences selected in five independent Cell-SELEX experiments for S.
mutans.
Footnote:
Binding ability of each aptamer to S. mutans was assessed by aptamer blotting assays. The
sequence name represents selection number which aptamers were selected from followed by
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clone numbers and also indicates partial sequence of identified aptamers. For example, SMa#14-P2 represents a partial sequence of an aptamer selected by selection 1 with clone number 4.
9