S1

Supplementary Information

Photo-regulation of Constitutive Gene Expression in Living Cells by Using of Ultrafast Photo-cross-linking Oligonucleotides

Takashi Sakamoto, Atsuo Shigeno, Yuichi Ohtaki, and Kenzo Fujimoto*

Electronic Supplementary Material (ESI) for Biomaterials Science.This journal is © The Royal Society of Chemistry 2014

S2

Experimental Procedures

Preparation of oligonucleotides: Oligoribonucleotides were purchased from Fasmac

(Japan). Phosphorothioate oligodeoxyribonucleotides were synthesized by an automatic

DNA synthesizer (3400 DNA synthesizer, Applied BioSystems) and purified by a

reversed-phase HPLC (JASCO PU-980, HG-980-31, DG-980-50, UV-970 system equipped

with an InertSustainTM C18 column (GL Science, 5 µm, 10 × 150 mm)). Preparation of

oligonucleotides was confirmed by MALDI-TOF-MS (see Supporting Information Table

S2). Phosphoramidite of CNVK was synthesized according to a method described in the

literature.1

Cell culture and transfection of AS-ODNs and siRNA: GFP stable cell line (GFP-HeLa)

was purchased from Cell Biolabs, Inc. (CA, USA). Cells were cultured in Dulbecco’s

MEM (10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin) in a

humidified chamber (37ºC, 5% CO2). Cells were trypsinized and resuspended in

Opti-MEM without antibiotics and transferred to 96-well plate at a density of 2×104 cells

per well in a volume of 100 µl and incubate for 24 h (37ºC, 5% CO2) before antisense

treatment. Transfection of K-ASs was carried out using Lipofectamine RNAi Max

(Invitrogen) according to the manufacture’s procedure. After the transfection, cells were

cultured in Dulbecco’s MEM (10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml

streptomycin) and in a humidified chamber (37ºC, 5% CO2).

Photoirradiation: Before the photoirradiation, cell culture medium was replaced to PBS.

Photoirradiation was performed by a UV-LED light source (1600 mW/cm2, Z-UV,

OMRON, Japan). After the photoirradiation, cell culture medium was replaced to

Dulbecco’s MEM (10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin)

and then cultured in a humidified chamber (37ºC, 5% CO2).

Quantification of mRNA: Two hours after the photoirradiation, total RNA was extracted

using CellAmpTM Direct RNA Prep Kit (Takara, Japan) and reverse transcription was

S3

performed by the PrimeScript® RT reagent Kit (Takara, Japan) according to the

manufacturer’s procedure. Resulting cDNA was subjected to real-time PCR using an

automated real-time PCR system (Smart Cycler®, Takara, Japan) with SYBR Premix Ex

Taq II perfect real time (Takara, Japan) and 0.4 µM of primers (For GFP: forward;

ATGGTGAGCAAGGGCGAG, reverse; GTGGTGCAGATGAACTTC (for AS-a),

forward; CAACAGCCACAACGTCTATATC, reverse;

AACTCCAGCAGGACCATGTGAT (for AS-b,c,d), For β-actin: forward; CTGGCACCACACCTTCTACA, reverse; AGCACAGCCTGGATAGCAAC, For SIN3B:

forward; TTCAACAACGCCATCAGCTA, reverse; GGCAGGAACTGTCCAAACTC).

Quantity of GFP, β-actin and SIN3B mRNA was estimated from the change in CT values

with the normalization using the amounts of GAPDH mRNA estimated from real-time

RT-PCR with a GAPDH primer set (forward; CATGCCAGTGAGCTTCCCGTT, reverse;

GTGGAGTCTACTGGCGTCTTC).

Confocal laser scanning fluorescence microscopy: After the transfection of AS-ODN, 10

sec of photoirradiation was performed and the cells were cultured for 24 h. Fluorescence

image of the cells were obtained by a confocal laser-scanning microscope (C2Si, Nikon,

Japan) with 488 nm laser excitation and 525 nm detection and 30 µm pinhole radius.

S4

Table S1. Sequence and MALDI-TOF-MS analysis of the synthetic oligonucleotides using this

study

a “X” and under bar indicate CNVK and photo-cross-linking site, respectively.

Name Target region Sequencesa Calcd.

[(M+H)+] Found

cORN-a 14 – 38 3’ UGGUGGGGCCACUUGUCGAGGAGCG

5’

AS-a 5’ ACCACCCCGGTGAACAGCTCCTCGC

3’ 7913.73 7913.48

K-AS-a1 5’ AXCACCCCGGTGAACAGCTCCTCGC

3’ 8020.77 8020.51

K-AS-a2 5’ ACCACCCCGGTGAXCAGCTCCTCGC

3’ 7996.76 7997.45

K-AS-a3 5’ ACCACXCCGGTGAACAGCTCCTCGC

3’ 8020.77 8018.87

K-AS inv 5’ CGCTCCTCGACAAGTGGCCCCACXA

3’ 8020.77 8019.69

cORN-b 471 – 495 3’ UCAAGUGGAACUACGGCAAGAAGAC

5’

AS-b 5’ AGTTCACCTTGATGCCGTTCTTCTG

3’ 7995.72 7996.88

K-AS-b1 5’ AXTTCACCTTGATGCCGTTCTTCTG

3’ 8037.74 8037.41

K-AS-b2 5’ AGTTCACCTTGAXGCCGTTCTTCTG

3’ 8062.75 8063.08

cORN-c 542 – 566 3’ UACCCCCACAAGACGACCAUCACCA

5’

AS-c 5’ ATGGGGGTGTTCTGCTGGTAGTGGT

3’

8211.74 8216.00

K-AS-c1 5’ AXGGGGGTGTTCTGCTGGTAGTGGT

3’ 8303.78 8300.48

cORN-d 592 – 616 3’ UGACCCACGAGUCCAUCACCAACAG

5’

AS-d 5’ ACTGGGTGCTCAGGTAGTGGTTGTC

3’ 8140.74 8140.82

K-AS-d1 5’ AXTGGGTGCTCAGGTAGTGGTTGTC

3’ 8247.78 8247.92

K-AS-d2 5’ ACTGGGTGCTCAXGTAGTGGTTGTC

3’ 8207.77 8209.54

S5

Table S2. Melting temperature (TM) of K-ASs with its complementary ORNs.a

Duplex TM / ºC Duplex TM / ºC Duplex TM / ºC

AS-a/cORN-a 69.4 ± 0.6 AS-b/cORN-b 55.6 ± 0.6 AS-d/cORN-d 59.3 ± 0.6

K-AS-a1/cORN-a 67.0 ± 0.5 K-AS-b1/cORN-b 52.3 ± 0.4 K-AS-d1/cORN-d 58.6 ± 0.4

K-AS-a2/cORN-a 64.5 ± 0.5 K-AS-b2/cORN-b 51.1 ± 0.4 K-AS-d2/cORN-d 57.6 ± 0.6

K-AS-a 3/cORN-a 65.8 ± 0.8 AS-c/cORN-c 67.3 ± 0.0

K-AS inv/cORN-a n.d.b K-AS-c1/cORN-c 65.4 ± 0.3 aTM values were determined from UV melting curves of the duplex ([Duplex] = 0.5 µM in 50 mM soduim cacodylate (pH 7.4) containing 100 mM NaCl). bNot determined.

S6

Figure S1. Denaturing PAGE analysis of the photo-cross-linking reaction of K-AS-a1, -a2, -a3 and

inv with cORN-a. PAGE analyses were performed on 15% polyacrylamide gel containing 7M urea

and 25% formamide. “M” indicates 10 bp ladder marker. Hetero-duplexes ( [AS-ODN] = [cORN] =

2 µM in PBS) were irradiated (366 nm) at 37ºC.

20#nt�

30#nt�

40#nt�50#nt�

0######0.1#####0.5######1#########5#######10######30�

ORN###################.#########+########+#########+#########+########+########+########+########+�cnvK.AS#(a.6.G)##################+########.#########+#########+#########+########+########+########+########+�

60#nt�

UV#Irradia@on#(sec)�

DNA##Ladder#

20#nt�

30#nt�

40#nt�50#nt�

0######0.1#####0.5######1#########5#######10######30�

ORN###################.#########+########+#########+#########+########+########+########+########+�cnvK.AS#(a.2.invert)#################+#########.#########+#########+#########+########+########+########+########+�

60#nt�

UV#Irradia@on#(sec)�

DNA##Ladder#

K-AS-a1�

K-AS-a2�

K-AS-a3�

K-AS inv�

60�50�40�30�

20�

bp�

cORN

-a�

AS-O

DN�

Photoirradiation time (s)�

0� 30�10�1� 5�0.1� 0.5�Photodimer�

Photodimer�

M�

60�40�30�20�60�50�40�30�

20�60�50�40�30�

20�

S7

Figure S2. Secondary structure of GFP mRNA calculated by mfold2 and the target site of the

AS-ODNs.

Output of sir_graph (©)mfold_util 4.6

Created Wed Sep 18 02:20:51 2013

dG = -243.25 [Initially -260.90] GFP mRNA

AU

GG

UG

A

GC

A

AG

GG

CG

AG

GAGC

UG

UU

CAC

CG

G

GG

UG

GUG

CC

CA

UC

CU

GG

UC

GA

GC

UG

GA

C

GG

CG

AC

G

UAAA

CG

GC

CAC

A AG

UU

CA

GC

GU

GU

CC

GGC G

A

GG

GC

GAG

GG

CGA

UG

CC

ACCUA

C GG

CA

AGC

UG

A C C C UG

AA

GU

UC

AU

CUGC

ACCA

CC

GGC

AAG

CU

GC

C

C

GU

GC

CC

UG

GC

CCA

C

CC

UC

G

UG

ACC

A

CC

CUG

AC

CUA

CG

GC

GU

G

CA

G

UGCUUCAGCCGC

UA

CC

CC

G A C CA

CA

U G A A G C AGC

A CG

AC

UUC

UU C

AA

GU

CC

GC

CAU

GC

CCG

A A

GG

CU

AC

GU

CC

AG

GA

GCG

CA

C CA

U

CU

UC

UU C

AA

GG

AC

G

A

C

GG

CA A C U A

CA

AGA

C

C

CG

CG

CC

G

AG

GU G

A AGUUCGA

GG

GCG

A

CAC

CC U

G

GUG

AAC

CGC

A

UCGAGCU

G

A

AG

GG C A U C

GACU

UC

A AGG

AG

G AC

GG

C

AA C

AU

CCU

GG

GG

CA

C A A GC

UG

GA

GU

A

C AAC

UA

CA

AC

AG

CCA C

AA

CG

UC

UA

UA U

CA

UG

GC

CG

ACAAG

CA

G AA

GAA

CG

G CA

UC

AA

GG

UG

AA

CU

UC

A AG

A

U

C

CG

CC A C A A

CA

U C G AG G A

C G G C A G C G U G C A G C U C G C C G A

CC

A C U A CC

AGCA

GA

ACACCC

CC

A

UCGGCGA

CG

GCCCCGUGCUGCUGCCCGAC

AA

CC

AC

UAC

C

UG

AG

CA C C

CAG

UC

CG

CC

CU

GA

GC A

AAG

AC

C C CA

AC

G

A

G

A

AG

CG

CG

AU

CAC

AU

GG

UC

CU

GC

UGGA

GUUCGU G

ACCG

CC

GCCGGGA

UCA

C

UC

UC

GG

CA

UG

G

AC

GA

GC

UGUAC

AA

GUAA

5’

3’

40

80

120

160

200

240

280

320

360

400

440

480

520

560

600

640

680

3’

Output of sir_graph (©)mfold_util 4.6

Created Wed Sep 18 02:20:51 2013

dG = -243.25 [Initially -260.90] GFP mRNA

AU

GG

UG

A

GC

A

AG

GG

CG

AG

GAGC

UG

UU

CAC

CG

G

GG

UG

GUG

CC

CA

UC

CU

GG

UC

GA

GC

UG

GA

C

GG

CG

AC

G

UAAA

CG

GC

CAC

A AG

UU

CA

GC

GU

GU

CC

GGC G

A

GG

GC

GAG

GG

CGA

UG

CC

ACCUA

C GG

CA

AGC

UG

A C C C UG

AA

GU

UC

AU

CUGC

ACCA

CC

GGC

AAG

CU

GC

C

C

GU

GC

CC

UG

GC

CCA

C

CC

UC

G

UG

ACC

A

CC

CUG

AC

CUA

CG

GC

GU

G

CA

G

UGCUUCAGCCGC

UA

CC

CC

G A C CA

CA

U G A A G C AGC

A CG

AC

UUC

UU C

AA

GU

CC

GC

CAU

GC

CCG

A A

GG

CU

AC

GU

CC

AG

GA

GCG

CA

C CA

U

CU

UC

UU C

AA

GG

AC

G

A

C

GG

CA A C U A

CA

AGA

C

C

CG

CG

CC

G

AG

GU G

A AGUUCGA

GG

GCG

A

CAC

CC U

G

GUG

AAC

CGC

A

UCGAGCU

G

A

AG

GG C A U C

GACU

UC

A AGG

AG

G AC

GG

C

AA C

AU

CCU

GG

GG

CA

C A A GC

UG

GA

GU

A

C AAC

UA

CA

AC

AG

CCA C

AA

CG

UC

UA

UA U

CA

UG

GC

CG

ACAAG

CA

G AA

GAA

CG

G CA

UC

AA

GG

UG

AA

CU

UC

A AG

A

U

C

CG

CC A C A A

CA

U C G AG G A

C G G C A G C G U G C A G C U C G C C G A

CC

A C U A CC

AGCA

GA

ACACCC

CC

A

UCGGCGA

CG

GCCCCGUGCUGCUGCCCGAC

AA

CC

AC

UAC

C

UG

AG

CA C C

CAG

UC

CG

CC

CU

GA

GC A

AAG

AC

C C CA

AC

G

A

G

A

AG

CG

CG

AU

CAC

AU

GG

UC

CU

GC

UGGA

GUUCGU G

ACCG

CC

GCCGGGA

UCA

C

UC

UC

GG

CA

UG

G

AC

GA

GC

UGUAC

AA

GUAA

5’

3’

40

80

120

160

200

240

280

320

360

400

440

480

520

560

600

640

680

3’

Output of sir_graph (©)mfold_util 4.6

Created Wed Sep 18 02:20:51 2013

dG = -243.25 [Initially -260.90] GFP mRNA

AU

GG

UG

A

GC

A

AG

GG

CG

AG

GAGC

UG

UU

CAC

CG

G

GG

UG

GUG

CC

CA

UC

CU

GG

UC

GA

GC

UG

GA

C

GG

CG

AC

G

UAAA

CG

GC

CAC

A AG

UU

CA

GC

GU

GU

CC

GGC G

A

GG

GC

GAG

GG

CGA

UG

CC

ACCUA

C GG

CA

AGC

UG

A C C C UG

AA

GU

UC

AU

CUGC

ACCA

CC

GGC

AAG

CU

GC

C

C

GU

GC

CC

UG

GC

CCA

C

CC

UC

G

UG

ACC

A

CC

CUG

AC

CUA

CG

GC

GU

G

CA

G

UGCUUCAGCCGC

UA

CC

CC

G A C CA

CA

U G A A G C AGC

A CG

AC

UUC

UU C

AA

GU

CC

GC

CAU

GC

CCG

A A

GG

CU

AC

GU

CC

AG

GA

GCG

CA

C CA

U

CU

UC

UU C

AA

GG

AC

G

A

C

GG

CA A C U A

CA

AGA

C

C

CG

CG

CC

G

AG

GU G

A AGUUCGA

GG

GCG

A

CAC

CC U

G

GUG

AAC

CGC

A

UCGAGCU

G

A

AG

GG C A U C

GACU

UC

A AGG

AG

G AC

GG

C

AA C

AU

CCU

GG

GG

CA

C A A GC

UG

GA

GU

A

C AAC

UA

CA

AC

AG

CCA C

AA

CG

UC

UA

UA U

CA

UG

GC

CG

ACAAG

CA

G AA

GAA

CG

G CA

UC

AA

GG

UG

AA

CU

UC

A AG

A

U

C

CG

CC A C A A

CA

U C G AG G A

C G G C A G C G U G C A G C U C G C C G A

CC

A C U A CC

AGCA

GA

ACACCC

CC

A

UCGGCGA

CG

GCCCCGUGCUGCUGCCCGAC

AA

CC

AC

UAC

C

UG

AG

CA C C

CAG

UC

CG

CC

CU

GA

GC A

AAG

AC

C C CA

AC

G

A

G

A

AG

CG

CG

AU

CAC

AU

GG

UC

CU

GC

UGGA

GUUCGU G

ACCG

CC

GCCGGGA

UCA

C

UC

UC

GG

CA

UG

G

AC

GA

GC

UGUAC

AA

GUAA

5’

3’

40

80

120

160

200

240

280

320

360

400

440

480

520

560

600

640

680

3’

Output of sir_graph (©)mfold_util 4.6

Created Wed Sep 18 02:20:51 2013

dG = -243.25 [Initially -260.90] GFP mRNA

AU

GG

UG

A

GC

A

AG

GG

CG

AG

GAGC

UG

UU

CAC

CG

G

GG

UG

GUG

CC

CA

UC

CU

GG

UC

GA

GC

UG

GA

C

GG

CG

AC

G

UAAA

CG

GC

CAC

A AG

UU

CA

GC

GU

GU

CC

GGC G

A

GG

GC

GAG

GG

CGA

UG

CC

ACCUA

C GG

CA

AGC

UG

A C C C UG

AA

GU

UC

AU

CUGC

ACCA

CC

GGC

AAG

CU

GC

C

C

GU

GC

CC

UG

GC

CCA

C

CC

UC

G

UG

ACC

A

CC

CUG

AC

CUA

CG

GC

GU

G

CA

G

UGCUUCAGCCGC

UA

CC

CC

G A C CA

CA

U G A A G C AGC

A CG

AC

UUC

UU C

AA

GU

CC

GC

CAU

GC

CCG

A A

GG

CU

AC

GU

CC

AG

GA

GCG

CA

C CA

U

CU

UC

UU C

AA

GG

AC

G

A

C

GG

CA A C U A

CA

AGA

C

C

CG

CG

CC

G

AG

GU G

A AGUUCGA

GG

GCG

A

CAC

CC U

G

GUG

AAC

CGC

A

UCGAGCU

G

A

AG

GG C A U C

GACU

UC

A AGG

AG

G AC

GG

C

AA C

AU

CCU

GG

GG

CA

C A A GC

UG

GA

GU

A

C AAC

UA

CA

AC

AG

CCA C

AA

CG

UC

UA

UA U

CA

UG

GC

CG

ACAAG

CA

G AA

GAA

CG

G CA

UC

AA

GG

UG

AA

CU

UC

A AG

A

U

C

CG

CC A C A A

CA

U C G AG G A

C G G C A G C G U G C A G C U C G C C G A

CC

A C U A CC

AGCA

GA

ACACCC

CC

A

UCGGCGA

CG

GCCCCGUGCUGCUGCCCGAC

AA

CC

AC

UAC

C

UG

AG

CA C C

CAG

UC

CG

CC

CU

GA

GC A

AAG

AC

C C CA

AC

G

A

G

A

AG

CG

CG

AU

CAC

AU

GG

UC

CU

GC

UGGA

GUUCGU G

ACCG

CC

GCCGGGA

UCA

C

UC

UC

GG

CA

UG

G

AC

GA

GC

UGUAC

AA

GUAA

5’

3’

40

80

120

160

200

240

280

320

360

400

440

480

520

560

600

640

680

3’

AS-a�K-AS-a1�K-AS-a2�K-AS-a3�

AS-b�K-AS-b1�K-AS-b2�

AS-c�K-AS-c1�

AS-d�K-AS-d1�K-AS-d2�

S8

Figure S3. Relative amount of β-actin (a) and SIN3B (b) mRNA in GFP-HeLa cells before and

after the photoirradiation with K-AS-a1 treatment ([antisense] = 100 nM, photoirradiation: 366 nm,

10 sec)

0 20 40 60 80

100 120 140 160

0 20 40 60 80

100 120 140 160

K-AS-a1�

Rel

ativ

e am

ount

of

β-ac

tin m

RN

A / %�

Without AS�

: Without irradiation�

: With 10 sec of 366 nm irradiation�

K-AS-a1�

Rel

ativ

e am

ount

of

SIN

3B m

RN

A / %�

Without AS�

: Without irradiation�

: With 10 sec of 366 nm irradiation�a)� b)�

S9

Figure S4. Cell viability (a) and relative amount of pyrimidine (6-4) pyrimidone photoproducts (6-4

PP) (b) in GFP-HeLa cells as a function of irradiation time. Cell viability was estimated by WST-1

assay 48 h after the photoirradiation. The amount of 6-4 PP was measured by ELISA kit (Cell

Biolabs, CA) 48 h after the photoirradiation according to a manufacture’s protocol.

0

50

100

150

0.1 1 10 100

B

Cel

l via

bilit

y / %

Irradiation time / sec

0

100

200

300

0.1 1 10 100

B

Rel

ativ

e am

ount

of 6

-4 P

P (%

)

Irradiation time / secIrradiation time / sec�

Cel

l via

bilit

y / %�

0�Irradiation time / sec�

Rel

ativ

e am

ount

of

6-4

PP

/ %�

0�

a)� b)�

S10

Figure S5. Relative amount of GFP mRNA in GFP-HeLa cells after the treatment of siRNA

targeting GFP mRNA ([siRNA] = 100 nM, siRNA (sense); 5’-GCAAGCUGACCCUGAAGUUCA

U-3’, siRNA (antisense); 5’-GAACUUCAGGGUCAGCUUGCCG-3’)

References

1. Y. Yoshimura, K. Fujimoto, Org. Lett., 2008, 10, 3227.

2. M. Zuker, Nucleic Acids Res., 2003, 31, 3406.

0

20

40

60

80

100

120

50 h�

Rel

ativ

e am

ount

of

GFP

mR

NA

/ %�

2 h�

: Without siRNA�

: With siRNA�

26 h�