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Supporting Information
Self-assembly of hybridized peptide nucleic acid amphiphiles
Li-Han Liu, Ze-Yong Li, Lei Rong, Si-Yong Qin, Qi Lei, Han Cheng, Xiang Zhou,
Ren-Xi Zhuo, and Xian-Zheng Zhang*
Key Laboratory of Biomedical Polymers of Ministry of Education & Department of
Chemistry, Wuhan University, Wuhan 430072, China
*Corresponding author. Tel.: + 86 27 6875 5993; Fax: + 86 27 6875 4509.
E-mail address: [email protected] (X. Z. Zhang).
Materials
Fmoc/Bhoc PNA monomer was acquired from South Korea Panagene Company,
Rink Amide resin (Loading: 0.51 mmol g-1
), 2-(7-azo-benzotriazole)-N, N, N',
N'-tetramethyluronium hexafluorophosphate (HATU), 1-hydroxy-7-azo-benzotriazole
(HOAt), N-methylmorpholine (NMM), N-Fluorenyl-9-methoxycarbonyl (Fmoc)
protected L-amino acids (FMOC-Glu(OtBu)-OH), and triisopropylsilane (TIS) were
purchased from GL Biochem. Ltd. (Shanghai, China) and used as received.
Diisopropylethylamine (DIEA) was acquired from GL Biochem. Ltd. (Shanghai,
China) and used after distillation. Lauric acid, acetic acid, caproic acid, stearic acid,
trifluoroacetic acid (TFA), Tween 20, N,N-dimethylformamide (DMF),
N-methylpyrrolidone(NMP, methanol, dichloromethane (DCM) and anhydrous ether
were obtained from Shanghai Chemical Co. (China), TFA, DMF, NMP were used
after distillation, 3, 3’-diethylthiadicarbocyanine dye DiSC2(5) was purchased from
Alfa Aesar.
Synthesis of PNA-amphiphiles
PNAAs were synthesized manually by solid-phase peptide synthesis (SPPS) using
Fmoc/Bhoc protected PNA monomers on the Fmoc-protected Rink Amide resin in 10
µmol scale. One hundred milligrams of resin was soaked in DMF for half hour and
then deprotected twice with 20% piperidine in DMF for 10 min to remove the Fmoc
protecting group, then a DMF solution of the mixture of FMOC-Glu(OtBu)-OH (1
equiv.), HATU (0.9 equiv.), and DIEA (3 equiv.) was added. After stirring for 2 h at
room temperature, the reaction solution was drained off and the resin was washed
with DMF four times. After repetition of the deprotecting, acylating and deprotecting
reactions for amino acids loading, a NMP solution of the mixture of Fmoc/Bhoc
protected PNA monomer (4 equiv.), HATU (3.6 equiv.), and NMM (8 equiv.) was
added. After stirring for 80 min at room temperature, the reaction solution was
drained off and the resin was washed with DMF four times, unreacted sites were then
capped by a 2 min incubation of the resin with the a mixture of acetic anhydride and 2,
6-lutidine in DMF (v/v/v=5:6:89), and then treated the resin with 20% piperidine in
DMF for 2 min twice to remove the Fmoc protecting group in attached PNA
monomer. The reaction solution was drained off and the resin was washed with DMF
four times. Repeated the coupling steps, the capping steps and Fmoc deprotected steps
several times until the desired PNA peptide was synthesized. Acetic acid (caproic acid,
lauric acid, or stearic acid) was then coupled to the N terminus of the PNA peptide, in
a 5-fold excess. The resin was washed with DMF, methanol, DCM three times, and
then removed the remained DCM under vacuum. The resin was then soaked in a
mixture of TFA/m-cresol/TIS/H2O in the volume ratio of 93:3:2:2 for 1.5 h to cleave
the PNAA from the resin and remove OtBu and Bhoc side protecting groups. Once
cleaved from the resin, the TFA was removed by rotary evaporation, and the residue
was precipitated by the addition of cold dry ether. The precipitated PNAA was cooled
in a -20 °C freezer for 3 h to ensure complete precipitation. The solid was separated
from the ether by centrifugation for 1 min, then the top phase ether solution was
decanted off and the precipitate was resuspended with another addition of cold dry
ether. The dispersion and centrifugation processes were done in triplicate. Upon
completion, the PNAAs precipitates were dried under vacuum and the obtained solid
was conserved in a -20 °C freezer. The purity of the PNAAs were comfirmed by high
performance liquid chromatography (HPLC) with a C18 reversed phase column using
a linear gradient from 15 to 65% of acetonitrile/H2O containing 0.1% trifluoroacetic
acid (PNAA1, 3, 4, 5) at 1.0 mL min−1
for 30 min and 5 to 95% of acetonitrile/H2O
containing 0.1% trifluoroacetic acid (PNAA2) at 1.0 mL min−1
for 25 min. HPLC
chromatogram of PNAAs were recorded at absorbance of 220 nm. The molecular
weights of PNA-amphiphiles were analyzed by matrix assisted laser
desorption/ionization time of flight mass spectrometry (MALDI-TOFMS) in
H2O/acetonitrile (v/v=1:1) solution at a concentration of 0.1 mg mL-1
.
Determination of Critical Micelle Concentration (CMC)
Fluorescence spectra using pyrene hydrophobic fluorescent probe were recorded on a
LS55 luminescence spectrometer (Perkin–Elmer). 50 µL of pyrene solutions (0.12
µM in acetone) were added to containers, after the acetone evaporated, 1 mL aqueous
solution of PNAA at particular concentration varying from 10-8
to 3*10-4
M was
added to the container. The sample solutions containing pyrene residues were kept at
room temperature for 24 h to reach the equilibrium of pyrene partition between water
and micelles. For the pyrene excitation spectra, the emission wavelength was set to
393 nm, and the excitation spectra of samples were recorded ranging from 300 nm to
360 nm, the intensity ratio I341/I337 was analyzed as a function of logarithm of the
PNAA concentration.
Micelle Formation
The micelles of PNAAs were prepared by directly dissolved in aqueous solutions.
Transmission Electron Microscopy (TEM)
The morphology of the self-assembled PNAAs was examined on transmission
electron microscopy (TEM, JEM-2010, Japan). For the TEM observation, the PNAA
containing solutions were placed on the copper grids with Formvar film. Then
samples were negatively stained on phosphotungstic acid aqueous solution (2 wt%)
and naturally dried before observations.
Size Distribution Measurements
Zeta Sizer Nano ZS (Malvern Instruments) was exploited to determine the size
distribution of self-assembled PNAAs micelles. The micelle-contained solutions were
passed through 0.45µm pore size filters before measurement.
Circular dichroism (CD) spectroscopy
Circular dichroism was performed on a J-810 spectropolarimeter (Jasco, Japan). CD
samples were prepared at 10 mM sodium phosphate buffer (pH 7.4) at the
corresponding concentrations in a 1 mm quartz cell. UV-Melting curve was obtained
at 260 nm by J-810 spectropolarimeter (Jasco, Japan) with a digital circulating water
bath using a 5 mm quartz cell. The hybrid sample (PNAA3 at 10 µM) reported was
incubated at 90 oC for several minutes first, then slowly cooled to 10
oC. The sample
was heated at 1 oC/min to a higher target temperature.
Hybridization Experiment
The base pairing of PNAAs was studied by UV-VI spectroscopy (Lambda Bio40) at
the concentration of 10 µM containing 15 µM DiSC2(5) in 10mM sodium phosphate
buffer (pH 7.4) with 10% methanol by volume. This solution should be stored and
used in the dark to avoid photo-bleaching of the DiSC2(5) dye.
Concentration dependence UV-Vis absorbance Experiment
UV-Vis absorbance was obtained at 450-750 nm for DiSC2(5) contained PNAA5
solutions in 10 mM sodium phosphate buffer (pH 7.4) with 3% methanol by volume
using UV-VI spectroscopy (Lambda Bio40).
Figure S1. MALDI-TOF spectra and structure of corresponding PNAAs. a,
C2-ctgactga-E4 (PNAA1), expected molecular weight: 2743.06, observed molecular
weight: [M+H+]=2745.56; b, C6-ctgactga-E4 (PNAA2), expected molecular weight:
2799.12, observed molecular weight: [M+H+]=2800.96; c, C12-ctgactga-E4 (PNAA3),
expected molecular weight: 2883.22, observed molecular weight: [M+H+]=2885.98; d,
C18-ctgactga-E4 (PNAA4), expected molecular weight: 2967.31, observed molecular
weight: [M+H+]=2968.99, [M+Na
+]=2991.02; e, C12-ctgactga-E2 (PNAA5), expected
molecular weight: 2625.13, observed molecular weight: [M+H+]=2626.49,
[M+Na+]=2648.52.
Figure S2. HPLC analysis of PNAAs.
Figure S3. The intensity ratio as a function of logarithm of PNAA concentration, and
the calculated CAC value for PNAA1 and PNAA2.
Figure S4. CD spectra of the cyanine dye DiSC2(5) bound to parallel PNAA duplexes
(16 µM, 16 µM, 10 µM, 16 µM, 10 µM, respectively) and the control is the PBS
solution of cyanine dye DiSC2(5) (15 µM) without additions.
Figure S5. CD spectra of the self-assembled PNAA3 micelles at a concentration of 10
µM, 100 µM, 300 µM.
Figure S6. UV-Vis absorption of DiSC2(5) (15 µM) and DiSC2(5) (15 µM) with
Tween 20 (5 mg/mL).
Figure S7. UV melting curve for PNAA3.
Figure S8. Concentration dependence UV-Vis absorbance of PNAA5 solutions with
corresponding 1.5 equiv. of DiSC2(5).