Supplementary Data

Pyrene-Labeled Deoxyguanosine as a Fluorescence Sensor to Discriminate Single

and Double Stranded DNA Structures: Design of Ends Free Molecular Beacons

Katsuhiko Matsumoto,a, b Yuta Shinohara,a Subhendu S. Bag, a

Yoshiki Takeuchi,a

Takashi Morii,b Yoshio Saito,*a and Isao Saito*a

a
Department of Materials Chemistry and Engineering, School of Engineering, Nihon University, Koriyama,

Fukushima 963­8642, Japan, and

 bInstitute of Advanced Energy, Kyoto University, Uji, Kyoto 611­0011, Japan

List of supplementary data

1 General Experimental

2 Post modification of ODNs

3 MALDI-TOFF Mass data

4 UV-visible spectra of ODNs

5 Fluorescence imaging for MB 2, MB 3 and MB 5

6 Thermal melting curves of ODNs

1
1 General Experimental

Oligonucleotide synthesis and characterization: All the reagents for DNA synthesis were purchased from Glen
Research. The ODNs were synthesized and characterized according to our previously published literatures 7,8
[ODNs were synthesized by a conventional phosphoramidite method by using an Applied Biosystems 392
DNA/RNA synthesizer]. ODNs were purified by reverse phase HPLC on a 5-ODS-H column (10 x 150 mm,
elution with 50 mM ammonium formate buffer (AF), pH 7.0, linear gradient over 45 min from 0% to 40%
acetonitrile at a flow rate 2.0 ml/min). ODNs containing modified nucleotides were fully digested with calf
intestine alkaline phosphatase (50 U/mL), snake venom phosphodiesterase (0.15 U/mL), and P1 nuclease (50
U/mL) at 37 oC for 3 h. Digested solutions were analysed by HPLC on a CHEMCO-BOND 5-ODS-H column (4.6
x 150 mm), elution with a solvent mixture of 50 mM ammonium formate buffer (AF), pH 7.0, linear gradient over
60 min from 0% to 50% acetonitrile at a flow rate 1.0 ml/min). The concentration of each ODNs were determined
by comparing peak areas with standard solution containing dA, dC, dG, and dT at a concentration of 0.1 mM. Mass
spectra of ODNs purified by HPLC were determined with a MALDI-TOF mass spectroscopy (Shimadzu, AXIMA-
LNR).

2 Post modification of ODNs

O
O
O N O
O
O O N
N NH NH
H2N N
N H N
N NH2 N NH2
O O
O aq. NaHCO3, O
Et3N,DMF
37 oC., 16 h
O O

NH2G PyIG

ODN 1 ODN 2
MB 1, 4 MB 2, 5

O O
N O
N O O N
NH O NH
H2N N
N N NH2 H N
O N NH2
O O
O aq. NaHCO3, O
Et3N,DMF
O 37 oC., 16 h
O

NH2G PyIIG

MB 1 MB 3

Scheme S1 Post synthetic modification of oligonucleotides

Preparation of ODN 2: To a solution of 8-hexylamino-2'-deoxyguanosine (NH2G) containing oligonucleotide

(ODN 1) (0.15 mol) in aq. NaHCO3 solution (1M, 300l) was added excess amount of pyrenecarboxylic acid
activated ester (1.0 mg, 2.9 mol) in anhydrous DMF (50 l). To The mixture was added anhydrous triethylamine

2
(10 l) and the solution was incubated at 37 oC for 16 hours. The reaction mixture was diluted with 1.0 ml AF
buffer and was filtered off (cosmonice filter, pore size: 0.45 m). The solution was purified by HPLC to afford PyI
G
containing oligonucleotide( ODN 2).(0.13 mol) Other PyI
G and PyII
G containing oligonucleotides were preparated
in a similar manner.

ODN2

ODN 1

0 10 20 30 40
retention time (min)

Fig. S1 HPLC analysis of the mixture after post modification of ODN 1.

3 MALDI-TOF mass data for the ODNs:

Table S1 MALDI-TOF mass data for the ODNs

ODNs MALDI-TOF mass MALDI-TOF mass

+
calc. [M + H] found [M + H]+
1 4043.83 4042.13
2 4272.08 4272.49
4 (MB 1) 8407.69 8406.75
5 (MB 2) 8635.94 8635.90
6 (MB 3) 8692.00 8692.85
7 (MB 4) 9125.26 9125.10
8 (MB 5) 9581.76 9580.81

3
4 UV-visible spectra of ODNs

(a) (b)
1.2 MB 2 (closed) MB 3 (closed)
MB 2/ ODN 9 (open) 0.8 MB 3/ ODN 9 (open)
1 0.08 0.04
0.06 0.6
0.8 0.03
0.04

Abs.
Abs.

0.02
0.6 0.02
0.4
0.01
0
0.4 300 350 400 0
0.2 300 350 400 450
0.2

0 0
220 270 320 370 420 470 220 270 320 370 420 470

Wavelength (nm) Wavelength (nm)

(c) 1
MB 5 (closed)
MB 5/ ODN 9 (open)
0.8 0.1
0.08
0.6 0.06
Abs.

0.04
0.02
0.4 0
300 350 400
0.2

0
220 270 320 370 420 470

Wavelength (nm)

Fig. S2 UV-visible spectra of (a) MB 2 and the duplex with ODN 9, (b) MB 3 and the duplex with ODN 9, (c) MB 5 and the duplex

with ODN 9 (2.5 M, 50 mM sodiumphosphate, 0.1M sodium chloride, pH 7.0, r.t.).

4
5 Fluorescence imaging for MB 2, MB 3 and MB 5

(a) (b) (c)

Fig. S3 Fluorescence image of (a) MB 2 in a closed form (left) and MB 2/ODN 9 in an opened form (right), (b) MB 3 in a closed form

(left) and MB 3/ODN 9 in an opened form (right), (c) MB 5 in a closed form (left) and MB 5/ODN 9 in an opened form (right).

Sample solutions (2.5 M, 50 mM sodiumphosphate, 0.1M sodium chloride, pH 7.0) were illuminated at ca 365 nm by

transilluminator.

6 Thermal melting curves of ODNs

(a) (b)

(c) (d)

5
6
 (e) (f)

(g) (h)

(i) (j)

Fig. S4 Thermal melting curves of (a) ODN 1/3 (b) ODN 2/3 (c) MB1 (d) MB1/ODN9 (e) MB2 (f) MB2/ODN9 (g) MB3 (h)

MB/3ODN9 (i) MB5 (j) MB5/ODN9 (2.5 M, 50 mM sodiumphosphate, 0.1M sodium chloride, pH 7.0).