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: xz-zhang@whu.edu.cn (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).