J- vs. H-type assembly: pentamethine cyanine (Cy5) as near ... · measured against Cy5 (quantum...

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Supporting Information:

J- vs. H-type assembly: pentamethine cyanine (Cy5) as near IR chiroptical

reporter

Larysa I. Markova, a,b Vladimir L. Malinovskii, a Leonid D. Patsenkerb and Robert Häner*a

a Department of Chemistry and Biochemistry, University of Bern,Freiestrasse 3, CH-3012 Bern, Switzerland

b Department of Organic Luminophores and Dyes, State Scientific Institution "Institute for Single Crystals"of the NAS of Ukraine, 60, Lenin Ave., Kharkiv 61001, Ukraine.

TABLE OF CONTENTS

1. General S2

2. Experimental procedures S2

3. Mass-spectrometry and HPLC data of the cyanine (Cy5) modified ON1 and ON2 and squaraine (Sq) modified ON5

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4. Excitation spectra S6

5. Influence of the temperature on the absorption and emission spectra of Cy5 containing oligonucleotide ON2 and duplex ON1*ON2

S7

6. Tm experiments S8

7. Quantitative analysis of asymmetry of exciton couplet S9

7.1 Fitting of absorbance and CD bands shape. S9

7.2 Integration of signal intensity and ratio of g-factors S10

8. Titration experiment S12

9. CD spectra of the oligonucleotides ON2 and ON5 and hybrids ON3*ON5 and ON3*ON4 S13

Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2013

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1. General Absorption spectra were recorded in 1-cm quartz cells at 20 °C on Varian Cary-100 Bio-UV/VIS spectrophotometer equipped with a Varian Cary-block temperature controller. Emission spectra and quantum yields (ΦF) were measured in 1-cm quartz cells at 20 °C on Varian Cary Eclipse fluorescence spectrophotometer equipped with a Varian Cary-block temperature controller. CD spectra were recorded on a JASCO J-715 spectropolarimeter using quartz cuvettes with an optical path of 1 cm. HPLC purity determination was performed with a Shimadzu LC system equipped with a Shimadzu-block temperature controller. Oligonucleotides ON1 and ON2 containing Cy5 molecule in the backbone were from Microsynth (Balgach, Switzerland) and BaseClick (Tutzing, Germany). Not modified oligonucleotides ON3 and ON4 were from Microsynth (Balgach, Switzerland). Oligonucleotide ON5 containing squaraine (Sq) molecule in the backbone was synthesized according to [1]. 2. Experimental procedures The purity of the oligonucleotides was determined by reverse phase HPLC: column LiChrospher® 100 RP-18, 250 mm × 4 mm, Merck; mobile phase A = (Et3NH)OAc (0.1 M, pH 7.4); mobile phase B = MeCN; elution at 20 °C; gradient 0 – 40% B over 22 min, then 40-100% B over 5 min. Measurements of spectra in aqueous solutions (except quantum yields determination) were performed at a concentration of 1.5 µM (10 mM PB, pH = 7.4, 100 mM NaCl) for oligonucleotides and at a concentration of 1.5 µM + 1.5 µM of each strand (10 mM PB, pH = 7.4, 100 mM NaCl) for duplexes. Concentration of oligonucleotides was determined using molar absorbtivities of ε260(ON1) = 227 400 M-1 cm-1, ε260(ON2) = 206 200 M-1 cm-1, ε260(ON3) = 231 400 M-1 cm-1, ε260(ON4) = 216 500 M-1 cm-1 and ε260(ON5) = 212 200 M-1 cm-1; a value of ε260(Cy5) = 5 000 M-1 cm-1 was applied for calculation of ε260(ON1) and ε260(ON2) and a value of ε260(Sq) = 11 000 M-1 cm-1 was applied for calculation of ε260(ON5). Thermal denaturation experiments were carried out on a Varian Cary-100 Bio-UV/VIS spectrophotometer and data were collected at 260, 590 and 645 nm for duplex ON1*ON2 with internal Cy5 modifications in the backbone and at 260 nm for non-modified duplex ON3*ON4 (cooling-heating cycles in the temperature range of 20-90 °C, temperature gradient of 0.5 °C/min). Melting temperature (Tm) values were determined as the maximum of the first derivative of the melting curve. For the determination of the quantum yields, the integrated relative intensities of the samples were measured against Cy5 (quantum yield, QYCy5 = 27% in water [2]) as the reference. Absorbance of the solutions at the excitation wavelength (600 nm for the ON1, ON2 and ON5, and 625 nm for the

1 L.I. Markova, V.L. Malinovskii, L.D. Patsenker, R.Häner, Synthesis and properties of squaraine-modified DNA. Org. Biomol. Chem. 2012, 10, 8944-8947. 2 R.B. Mujumdar, L.A. Ernst, S.R. Mujumdar, C.J. Lewis, A.S. Waggoner, Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug. Chem. 1993, 4, 105–111.


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duplexes ON1*ON2 and ON1*ON5) was between 0.04–0.06 measured in a 1-cm cell. The emission spectra of the solutions were recorded and the quantum yields of the samples were determined as described in [3] according to formula:

QY = QYCy5 × (F/F Cy5) × (ACy5/A) × (n2/n2Cy5),

where F and FCy5 are the integrated areas of the fluorescence spectra, A and ACy5 are the absorbance at the excitation wavelength and n and nCy5 are the refraction indices of solvents used for the sample under examination and Cy5, respectively. 3. Mass-spectrometry and HPLC data of the cyanine (Cy5) modified ON1 and ON2 and squaraine (Sq) modified ON5 Table S1. Mass spectrometry data of the ON1, ON2 (Qualitative analysis report) and ON5 (ESI-MS, negative mode, MeCN/H2O/0.5%, Et3N) Oligo-mer

Sequence Molecular formula

Mol Weight

Found mass

ON1 5’ AGCTCGGTCACy5CGAGAGTGCA C226H282N83O120P20- 6700.62 6700.24

ON2 3’ TCGAGCCAGTCy5GCTCTCACGT C224H284N73O124P20- 6602.54 6602.17

ON5 3’ TCGAGCCAGTSqGCTCTCACGT C231H293N73O126P20 6724.35 6724.34

Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

24001: 260 nm, 4 nm 2: 650 nm, 4 nm

Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

24001: 260 nm, 4 nm 2: 650 nm, 4 nm

Figure S1. HPLC data of the oligonucleotides ON1 (left) and ON2 (right) recorded at 260 and 650 nm.

Minutes2 4 6 8 10 12 14 16 18 20 22 24 26

mAu

0

100

200

300

400

500

600

700

800

900

1000

1100

mAu

0

100

200

300

400

500

600

700

800

900

1000

11001: 260 nm, 4 nm 2: 630 nm, 4 nm

Figure S2. HPLC data of the oligonucleotide ON5 recorded at 260 and 630 nm.

3 C.A. Parker, Photoluminescence of Solutions, Elsevier Publishing Company, Amsterdam, 1968.


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S5


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Figure S3. HR-MS of the ON5. 4. Excitation spectra

300 400 500 600 700 8000

100

200

300

400

500

600

700ON2

Flu

ores

cenc

e

Wavelength [nm]300 400 500 600 700 800

0

50

100

150

200

Hybrid ON1*ON2

Flu

ores

cenc

e

Wavelength [nm]

Figure S4. Excitation spectra of the oligonucleotide ON2 (left) and duplex ON1*ON2 (right). λem = 670 nm.


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5. Influence of the temperature on the absorption and emission spectra of Cy5 containing oligonucleotide ON2 and duplex ON1*ON2

200 300 400 500 600 7000.0

0.1

0.2

0.3

0.4

80 0C70 0C60 0C50 0C40 0C30 0C

t = 20 0C

Abs

orba

nce

Wavelength [nm]550 600 650 700 750 8000

100

200

300

400

500

600

80 0C70 0C60 0C50 0C40 0C30 0C

t = 20 0C

Em

issi

on

Wavelength [nm] Figure S5. Influence of the temperature on the absorption and emission spectrum of the Cy5 containing oligonucleotide ON2 in the temperature range 20-80 ºC.

500 550 600 650 7000.0

0.1

0.2

0.3

0.4

0.5

80 0C70 0C60 0C50 0C40 0C30 0C

t = 20 0C

Em

issi

on

Wavelength [nm] Figure S6. Influence of the temperature on the absorption spectrum of the Cy5 containing duplex ON1*ON2 in the temperature range 20-80 ºC.


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6. Tm experiments

20 30 40 50 60 70 80 900.65

0.70

0.75

0.80

0.85260 nm

Abs

orba

nce

Temperature [0C]20 30 40 50 60 70 80 90

0.000

0.004

0.008

0.012

0.016

Temperature [0C] Figure S7. Example of Tm determination procedure: duplex ON3*ON4, melting curves at 260 nm (left) and first derivative curve (right), Tm = 70.7 °C (right). Table S2. Hybridization data of the cyanine containing duplex ON1*ON2 and its non-modified analog ON3*ON4. (Cy5-Cy5 vs A-T base pair)*. Oligomer Duplex Tm, °C ∆Tm, °C

ON3 5’ AGCTCGGTCATCGAGAGTGCA 3’ TCGAGCCAGTAGCTCTCACGT 70.7 –

ON4

ON1 5’ AGCTCGGTCACy5CGAGAGTGCA 3’ TCGAGCCAGTCy5GCTCTCACGT

66.7 - 4.0 ON2

* from the first derivative of the cooling (90 °C – 20 ºC) curve


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7. Quantitative analysis of asymmetry of exciton couplet: 7.1 Fitting of absorbance and CD bands shape.

200 300 400 500 600 700 800

0.0

0.2

0.4

0.6

554.

0590

9

601.

3224

1

637.

1984

566

6.58

497

Figure S8. Fitting of absorbance band shape of Cy-Cy containing hybrid.

200 300 400 500 600 700 800

-10

-5

0

5

10

15

20

25

555.

8380

8

601.

2025

633.

2017

6

668.

5423

2


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2 0 0 4 0 0 6 0 0 8 0 0

-1 x 1 0 1

0

1 x 1 0 1

2 x 1 0 1

3 x 1 0 1

B

A

P e a k A n a ly s is

A d j . R - S q u a r e = 9 .3 1 5 5 7 E - 0 0 1 # o f D a t a P o in t s = 1 1 6 1 .

D e g r e e o f F r e e d o m = 1 1 4 9 .S S = 1 . 8 1 0 7 6 E + 0 0 3

C h i^2 = 1 . 5 7 5 9 5 E + 0 0 0

D a t e : 1 7 . 0 3 . 2 0 1 3D a t a S e t : [ D U P L 2 0 C C O R ] S h e e t 1 ! B

F it t in g R e s u l t s

M a x H e ig h t- 1 .7 4 6 7 3- 8 .7 5 7 7 2- 5 .5 3 4 5 92 3 .0 2 2 3 1

A r e a In tg P-8 .6 0 0 8 2-2 4 .6 4 4 1 5-1 1 .9 3 6 1 75 9 .0 4 0 2 3

F W H M4 8 .7 7 9 8 92 7 .8 7 7 1 62 1 .3 6 5 12 5 .4 0 5 3 4

C e n te r G r v ty5 6 4 .5 4 7 7 76 0 0 .5 5 9 6 36 3 3 .3 4 6 8 56 6 8 .2 9 9 4

A r e a In tg- 9 0 .6 9 8 0 6- 2 5 9 .8 7 9 3 3- 1 2 5 .8 7 0 1 76 2 2 .5 9 5 5 4

P e a k T yp eG a u s s ia nG a u s s ia nG a u s s ia nG a u s s ia n

P e a k In d e x1 .2 .3 .4 .

Figure S9. Fitting of CD shape of Cy-Cy containing hybrid. 7.2 Integration of CD signals.

200 300 400 500 600 700 800

-10

-5

0

5

10

15

20

25

280

601

633

667.

5

1 2 3 4

2 3 4

-400

-200

0

200

400

600

Are

a

Index

Integral Result of B










Index Area AreaIntgP (%)

Row Index

Beginning X

Ending X

FWHM Center Height

1 45.8718 3.42315 120 228 309.5 23.70212 280 5.7309 2 -354.14384 -26.42773 762 521 622 31.77151 601 -9.12614 3 -99.23928 -7.40566 826 622 645 19.21005 633 -5.62839 4 607.59975 45.3417 895 645 710.5 24.58379 667.5 23.32792


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Ratio of g-factors Circular dichroism is defined as difference in absorbance of left and right circularly polarized light beams, CD = AL – AR g-factor, is defined as [4]:

Where AL and AR are the absorptions of left and right circularly polarized light; and A represents the absorbance of nonpolarized light. Output of CD instrument is usually presenting in ellipticity in mdeg, Ѳ (mdeg): Ѳ (mdeg) = ~33000 CD = ~33000(AL – AR) Therefore value of CD, or (AL – AR) can be obtained from experimental data as Ѳ (mdeg) / 33000 Importantly, g-value calculation as g = (AL – AR) /A does not required concentration or extinction

coefficient (ǫ), when CD and absorbance are measured for the same sample. This is very valuable,

because concentration and/or exact ǫǫǫǫ are often barely defined, especially for aggregated materials, polymers, etc. g-factor is also called as anisotropy or dissymetry factor, and was applied in our work to characterize an asymmetry of exciton couplet.

Experimental data for ON1-ON2 hybrid at 20 ºC:

A(668)= 0.59678 ; A(633)= 0.34578 A(601)= 0.32854

CD(668)= 23.3657 ;CD(633)= -5.53441 ; CD(601)= -9.03216

g(668)= 7.01 x 10-4 ; g(633)= -1.68 x 10-4 ; g(601)= -2.74 x 10-4 that gives g(668nm)/g(633nm)= 4.17 and g(668nm)/g(601nm) = 2.56

4 N. Berova, L. Di Bari, G. Pescitelli, Chem.Soc.Rev., 2007, 36, 914-931.


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8. Titration experiment. Titration of the oligonucleotide ON1 (starting conc. 1.5 µM, 10 mM phosphate, pH = 7.4, 100 mM NaCl, 20 ºC, V = 1000 µL) by addition of stock solution of oligonucleotide ON2 (C stock sol. = 7.5×10-5 M) in 40 µL steps. At first step, 20 µL of ON2 stock solution was used. First step corresponds to the 7.4×10-8 M of duplex formed, assuming a total shift of equilibrium to the hybrid at 20 ºC. Linear response of CD signal intensity on ON2 concentration reveals a fast kinetic of hybridization and correlates with formation of one chiral product upon Cy-Cy assembly within hybrid ON1-ON2.

500 550 600 650 700 750-10

-5

0

5

10

15

20

25

CD

[mde

g]

Wavelength [nm]

7.35294E-08 1.318126

2.12264E-07 4.140826

3.40909E-07 7.066873

4.60526E-07 9.312865

5.72034E-07 12.10704

6.7623E-07 14.44468

7.7381E-07 16.76052

8.65385E-07 18.14449

9.51493E-07 20.48726

1.03261E-06 22.40637

0.0 2.0x10-7 4.0x10-7 6.0x10-7 8.0x10-7 1.0x10-6 1.2x10-6

0

5

10

15

20

25

CD

inte

nsity

at 6

68 n

m (

mde

g)

C (ON2), mol/dm3

Equation y = a + b*x

Weight No Weighting

Residual Sum of Squares

0.4005

Pearson's r 0.99956

Adj. R-Square 0.999

Value Standard Erro

?$OP:A=1 Intercept -0.47458 0.15518

?$OP:A=1 Slope 2.19734E7 231783.2041


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9. CD spectra of the oligonucleotides ON2 and ON5 and hybrids ON3*ON5 and ON3*ON4

300 400 500 600 700

-4

-2

0

2

4

6

8ON2

CD

[mde

g]

Wavelength [nm] 300 400 500 600 700

-40

-20

0

20

40

60

80ON2

∆ε

Wavelength [nm]

300 400 500 600 700

-4

-2

0

2

4

6

8ON5

CD

[mde

g]

Wavelength [nm] 300 400 500 600 700

-40

-20

0

20

40

60

80ON5

∆ε

Wavelength [nm] Figure S10. The CD spectra of the oligonucleotides ON2 and ON5 (conditions: 1.5 µM single strand conc., 10 mM phosphate, pH = 7.4, 100 mM NaCl).


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300 400 500 600 700-6

-4

-2

0

2

4

6

8ON3*ON5

CD

[mde

g]

Wavelength [nm] 300 400 500 600 700

-60

-40

-20

0

20

40

60

80ON3*ON5

∆ε

Wavelength [nm]

300 400 500 600 700

-4

-2

0

2

4

6

8 ON3*ON4

CD

[mde

g]

Wavelength [nm] 300 400 500 600 700

-40

-20

0

20

40

60

80 ON3*ON4

∆ε

Wavelength [nm] Figure S11. The CD spectra of the hybrids ON3*ON5 and ON3*ON4 (conditions: 1.5 µM each strand conc., 10 mM phosphate, pH = 7.4, 100 mM NaCl).


J- vs. H-type assembly: pentamethine cyanine (Cy5) as near ... · measured against Cy5 (quantum...

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