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Supporting Information
Chameleon-Like Self-Assembling Peptides for
Adaptable Biorecognition Nanohybrids
Woo-jin Jeong, Sung-ju Choi, Jun Shik Choi, and Yong-beom Lim*
Translational Research Center for Protein Function Control and Department of Materials Science
& Engineering, Yonsei University, Seoul 120-749, Korea
*To whom correspondence should be addressed.
E-mail: yblim@yonsei.ac.kr
1
cyc-RCP-30
cyc-RCP-15
H2N
NH
H2N
HN
NH
H2N
NH
HN
HN
O
H
N
O
N
H
HN
O
O
HN
N
H
NH
O
O
NH
HN
NH
NH2
HN
NH
NH
O
O
O
NH
NH
NH
NH
H2N
N
H
NH
NH2
HN
HN
NH2
O
O
O
NH2
NH
H2N
HN
O
O
O
HN
HO
O
OH
NH
O
O
O
NH
O
O
O
NH2
NH
NH
O
OH
H
N
HN
HN
NH
O
NH
O
NH
NH2 HN
O
O
NH
HN
O
O
O
O
O
HN
N
H
O
H2N
NH
HN
O
H
N
N
H
O
O
HN
H2N
OH
H
N
HN
NH
NH2 HN
O
NH2
O
O
HN
NH2
HN
HN
NH
O
O
HN
HN
NH
H2N
NH
HN
NH2
NH
NH
O
NH
H2N
NH2
NH
H2N
H2N
N
H
O
O
NH
O
NH
O
H
N
N
H
O
HN
H2N
O
S
O
HO
O
O
HO
O
O
O
HN
NH
O
NH
O
HN
O
O
O
H
N
H
N
O
HN
N
H
O
NH
O
O
H
N
HN
O
H
N
O
O
N
H
O
O
S
O
HO
HO
O
NH
NH2
O
HN
O
NH2
O
H2N
NH2
HO
O
HN
H2N
NH
HN
O
H
N
N
H
O
N
H
O
N
H
O
suc-Rev-A4RK
H2N
H2N
H2N
NH
NH
H2N
NH
HN
HN
H2N
NH
HN
NH
HN
H2N
HO
NH
HN
O
O
O
H
N
-
O
O
N
H
O
H
N
O
N
H
O
H
N
O
N
H
O
H
N
O
N
H
O
OH
H
N
O
N
H
O
H
N
O
N
H
O
H
N
O
N
H
O
H
N
O
N
H
O
H
N
O
N
H
O
H
N
O
O
N
H
H
N
O
O
N
H
H
N
O
NH2
O
O
O
H2N
NH2
NH
NH
HN
HN
NH2
NH
HN
NH2
NH
HN
NH2
NH
HN
NH2
NH
H2N
HN
NH2
NH2
O
Sak
S
Figure S1. Chemical structures of the peptides.
2
Figure S2. MALDI-TOF MS spectra of the peptides. (a) cyc-RCP-30, (b) cyc-RCP-15, and (c)
suc-Rev-A4RK.
3
Figure S3. a) Structure of a Rev-ARM/RRE IIB RNA complex (Protein Data Bank accession
number, 1ETF). b) Structures of wild-type and mutant RRE IIB RNAs. The C46-G74 mutation
was previously shown to significantly decrease the Rev-ARM binding affinity and specificity.[1,2]
c) & d) EMSA results demonstrating that the wild-type RRE IIB RNA binds specifically to the
cyc-RCP-30/SWNT hybrid, whereas the mutant RRE IIB RNA cannot. The fluorescence from
RNA in the cyc-RCP-30/SWNT hybrid and RRE complex was quenched due to absorption of the
fluorescence emission by the SWNT. Figures in d) show the same gel with bright field (left) and
fluorescence (right) images. The image on the left side shows that the SWNTs are present in the
wells, possibly because the long and large SWNT hybrids and the SWNT complexes cannot pass
4
through the polyacrylamide gel. The results indicate that the wild-type RRE RNA could not be
detected in the gel because it made a strong ternary complex with the cyc-RCP-30/SWNT hybrid
and the ternary complex (cyc-RCP-30/SWNT/RNA) became retained in the well. In contrast, the
mutant RRE RNA could be detected in the gel because it was separated from the cyc-RCP30/SWNT hybrid, which was retained in the well during electrophoresis.
Figure S4. Formation of the cyc-RCP-30/CNT hybrids and solubilization of the SWNTs (in 150
mM NaCl).
5
Figure S5. An AFM image of the cyc-RCP-30/SWNT hybrids. Bar: 200 nm.
6
Figure S6. CD spectrum of cyc-RCP-15/MWNT in 150 mM KF. The diameter of the MWNTs
was 60−90 Å.
7
Figure S7. SDS-PAGE analysis of the peptide/CNT hybrids. Lane 1, peptide alone (12 nmol);
lane 2, cyc-RCP-30/SWNT hybrid (12 nmol/20 µg); lane 3, cyc-RCP-30/MWNT hybrid (12
nmol/20 µg); lane 4, peptide alone (12 nmol); lane 5, cyc-RCP-15/SWNT hybrid (12 nmol/20
µg); lane 6, cyc-RCP-15/MWNT hybrid (12 nmol/20 µg). The diameter of the MWNTs was
100−150 Å.
8
Figure S8. a) Temperature-dependent CD spectra and b) changes in the CD values at 222 nm for
the cyc-RCP-15/MWNT hybrid. The diameter of the MWNTs was 100−150 Å.
9
EXPERIMENTAL SECTION
MATERIALS
Fmoc-amino acids and the oligoethylene glycol-based linker, Fmoc-21-amino-4,7,10,13,16,19hexaoxaheneicosanoic acid (Fmoc-NH-(PEG)5-COOH, 22 atoms) were purchased from
Novabiochem. Carbon nanotubes (CNTs) were obtained from Hanhwa Nanotech. RNA oligos
were purchased from Integrated DNA Technologies.
METHODS
Peptide synthesis and macrocyclization reaction
The peptide was synthesized on Rink Amide MBHA resin LL (Novabiochem) using standard
Fmoc protocols on a TributeTM peptide synthesizer (Protein Technologies, Inc). Standard amino
acid protecting groups were employed except cysteine, in which an acid-labile methoxytrityl
(Mmt) group was used.
For cyclization, the peptide-attached resin (20 µmol in terms of N-terminal amine groups) was
swollen in N-methyl-2-pyrrolidone (NMP) for 30 min. Then, bromoacetic acid was first coupled
to the N-terminal part of the resin-bound peptide. Before addition to the resin, a mixture of
bromoacetic acid (28 mg, 200 µmol) and N,N′-diisopropylcarbodiimide (15.5 µL, 100 µmol) in
NMP was incubated for 10 min for carboxyl activation. Following addition of the mixture to the
resin, the reaction was continued for 1 h with shaking at room temperature, in a 6 mL
polypropylene tube with a frit (Restek). The resin was then washed successively with NMP and
dichloromethane (DCM). For orthogonal deprotection of the Mmt group from the cysteine, the
10
resin was treated with 1% trifluoroacetic acid (TFA) in DCM several times (1 min × ~7).
Intramolecular cyclization reaction was performed in 3 mL of 1% diisopropylethylamine
(DIPEA) in NMP overnight with shaking at room temperature. The resin was then successively
washed with NMP, DMF, and THF, and dried under reduced pressure. For cleavage and final
deprotection, the resin was treated with cleavage cocktail (TFA:1,2-ethanedithiol:thioanisole;
95:2.5:2.5) for 3 h, and was triturated with tert-butyl methyl ether (TBME). The peptides were
purified by reverse-phase HPLC (water-acetonitrile with 0.1% TFA). The molecular weight was
confirmed by MALDI-TOF mass spectrometry. The purity of the peptides was >95% as
determined
by
analytical
HPLC.
The
peptide
concentration
was
determined
spectrophotometrically in water/acetonitrile (1:1) urea using a molar extinction coefficient of
tryptophan (5,500 M-1cm-1) at 280 nm.
Figure S4. Peptide cyclization scheme.
CD spectroscopy (CD)
11
CD spectra were recorded using a ChirascanTM Circular Dichroism spectrometer equipped with
peltier temperature controller (Applied Photophysics., Ltd). CD spectra for peptides and
peptide/CNT biohybrids were recorded from 260 to 190 nm using a 2 mm path-length cuvette.
Transmission electron microscopy (TEM)
One µL of sample was placed onto a carbon-coated copper grid and dried completely. Then 1 µL
of water were added for 1 min to eliminate salt crystals and were then quickly wicked off using
filter paper. The specimen was observed with JOEL-JEM 2010 instrument operating at 120 kV.
The data were analyzed using DigitalMicrographTM software.
Atomic force microscopy (AFM)
For AFM, typically 2 µL of the sample in salt containing solution was deposited onto a freshly
cleaved mica surface and dried completely. To eliminate salt crystals, several microliters of
distilled water were added to the specimen while quickly drying it using a stream of argon gas.
The images were obtained in tapping mode with a Nanoscope IV instrument (Digital
Instruments). AFM scans were taken at setpoint of 0.8-1 V and scanning speed was 1-2 Hz.
Dynamic light scattering (DLS)
DLS experiments were performed at room temperature using an ALV/CGS-3 compact
goniometer system equipped with a He-Ne laser operating at 632.8 nm. The detector optics
employed optical fibers coupled to an ALV/SO-SIPD/DUAL detection unit, which employed an
EMI PM-28B power supply and an ALV/PM-PD preamplifier/discriminator. The signal analyzer
was an ALV-5000/E/WIN multiple-tau digital correlator with 288 exponentially spaced channels.
12
The scattering angle was 90º. The size distribution was determined using a constrainedregularization method.
Quantification of the amount of adsorbed peptide in peptide/CNT hybrids
The arc-produced SWNTs were suspended in tetrahydrofuran (THF) at 250 µg/mL, and 20 µL (5
µg) was added to a microcentrifuge tube. THF was evaporated using a centrifugal evaporator.
After evaporating the THF, various concentrations of the cyc-RCP-30 in water (300 µL) was
added, and the mixture was sonicated for 30 min. Then NaCl was added to a final concentration
of 150 mM and the solution was further sonicated. The suspension was centrifuged at 16110 × g,
4 °C for 1 h and the pellet that contains the peptide/SWNT hybrids was discarded. The
supernatants (10 µL) were then subjected to 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis in Tris-tricine buffer systems at 100 V. The free peptide at
various concentrations was simultaneously electrophoresed as a reference for quantification. The
gel was stained with Brilliant Blue G (Sigma-Aldrich) and the densitometric analysis of the
peptide band intensity was performed with Adobe Photoshop software.
Electrophoretic mobility shift assay (EMSA)
Wild type RRE IIB RNA sequence:
5’-GAC CUG GUA UGG GCG CAG CGC AAG CUG ACG GUA CAG GCC AGG UC-3’
Mutant RRE IIB RNA sequence:
5’-GAC CUG GUA UCG GCG CAG CGC AAG CUG ACG GUA GAG GCC AGG UC-3’
13
The SWNTs were suspended in tetrahydrofuran (THF), and 20 µg was added to a
microcentrifuge tube. After evaporating the THF, an aqueous solution of the cyc-RCP-30 (40
µM, 300 µL) was added, and the mixture was sonicated for 30 min. For the cyc-RCP-30/SWNT
hybrid formation, NaCl was added to a final concentration of 150 mM and the solution was
sonicated. The suspension was centrifuged at 16,110 × g for 10 min and the supernatant was
removed. The pellet (the cyc-RCP-30/SWNT hybrid) was resuspended with 50 µL of 1 µM RNA
solution in 150 mM NaCl. The ternary complex (the peptide/SWNT/RNA) formed stable
suspension. The complex was incubated at room temperature for 30 min, and then at 4 °C for 30
min. Ten microliter of the ternary complex was electrophoresed on a non-denaturing
polyacrylamide gel (10% polyacrylamide, 0.5 × TBE) at 200 V, 4 °C. RNA was visualized with
a SYBR Green II RNA gel stain reagent (Invitrogen).
[1] Tan, R.; Chen, L.; Buettner, J. A.; Hudson, D.; Frankel, A. D. RNA Recognition by an
Isolated α Helix. Cell 1993, 73, 1031-1040.
[2] Bartel, D. P.; Zapp, M. L.; Green, M. R.; Szostak, J. W. HIV-1 Rev Regulation Involves
Recognition of Non-Watson-Crick Base Pairs in Viral RNA. Cell 1991, 67, 529-536.
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