Supporting Information for Red-White-Blue Emission ... · PRIVILEGED DOCUMENT FOR REVIEW PURPOSES ONLY Table S1. m/z Values of ss-DNA1-4 determined by ESI-MS. DNA Calculated mass

PRIVILEGED DOCUMENT FOR REVIEW PURPOSES ONLY

Supporting Information for

Red-White-Blue Emission Switching Molecular Beacons: Ratiometric Multicolour DNA Hybridization Probes

Reji Varghese and Hans-Achim Wagenknecht*

University of Regensburg Institute for Organic Chemistry

Universitätsstr. 31 D-93053 Regensburg

Germany

Email: [email protected]

Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2009

mailto:[email protected]


1. Experimental Section

General

Chemicals and dry solvents were purchased from commercial suppliers and were

used without further purification unless otherwise mentioned. TLC was performed

on Fluka silica gel 60 F254 coated aluminium foil. Flash chromatography was

carried out with Silica Gel 60 from Aldrich (60-43 μm). Spectroscopic

measurements were recorded in Na-Pi buffer solution (10 mM) using quartz glass

cuvettes (l = 10 mm). ESI mass spectra were measured in the central analytical

facility of the institute on a ThermoQuest Finnigan TSQ 7000 in negative

ionisation mode. NMR spectra were recorded on a Bruker Avance 300

spectrometer in deuterated solvents (1H at 300 MHz, 13C at 75 MHz and 31P at

121.5 MHz). Absorption spectra and the melting temperatures (1 μM DNA, 10-80

°C, 0.7 °C/min, step width 0.5 °C) were recorded on a Varian Cary 100

spectrometer equipped with a 6×6 cell changer unit. Fluorescence was measured

on a Jobin-Yvon Fluoromax 3 fluorimeter with a step width of 1 nm and an

integration time of 0.2 s. All spectra were recorded with an excitation and

emission bandpass of 5 nm and are corrected for Raman emission from the buffer

solution. Phosphoramidite of ethynyl pyrene was commercially available (Glen

research) and the corresponding phosphoramidite of nile red was synthesized

according to our recent publication.1



Synthesis of Molecular Beacons

The oligonucleotides were prepared on an Expedite 8909 DNA synthesizer

(Applied Biosystems) via standard phosphoramidite protocols using CPGs (1

μmol) with a longer coupling time of 15 minutes and a higher concentration of the

phosphoramidite (0.1 M). The chemicals for the DNA synthesis were purchased

from ABI and Glen Research. Unmodified oligonucleotides were purchased from

Metabion. After preparation, the trityl-off oligonucleotide was cleaved off the

resin and was deprotected by treatment with conc. NH4OH at RT for 15 h. The

oligonucleotides were dried and purified by reverse phase HPLC using the

following conditions: A = NH4OAc buffer (50 mM), pH = 6.5; B = MeCN;

gradient = 0-20% B over 50 min for DNA1-3 and 0-30% B over 50 min for

DNA4. The oligonucleotides were lyophilized and quantified by their absorbance

at 260 nm on a Varian Cary Bio 100 spectrometer. Self-hybridized duplexes

(hairpins) were prepared by heating the corresponding DNAs to 90 °C (hold for 10

min.) followed by immediate cooling using an ice bath or liquid nitrogen.

Duplexes with the target of interest were prepared by simply mixing ca. 1

equivalence of the target DNA with hairpin MB at room temperature.



Table S1. m/z Values of ss-DNA1-4 determined by ESI-MS.

DNA Calculated mass (m/z)

Observed mass (m/z)

DNA1 11289.8 11289.0 DNA2 11280.8 11281.2 DNA3 11271.8 11272.2 DNA4 8522.3 8521.0



Figure S1. LC-MS of DNA1.












200 300 400 500 600 700 8000.0

0.3

0.6

0.9

1.2

1.5

ss ds

A

λ / nm200 400 600 800

0.0

0.5

1.0

1.5 ss ds

A

λ / nm

200 400 600 8000.0

0.5

1.0

1.5

2.0 ss ds

A

λ / nm

a) b)

c) d)

200 400 600 8000.0

0.5

1.0

1.5

2.0

ss ds

A

λ / nm

Figure S5: Absorption spectra of ss- and ds-DNAs, a) DNA1, b) DNA2, c) DNA3 and d)

DNA4 (c = 1 μM in Na-Pi buffer, 250 mM NaCl, pH = 7, T = 25 °C).



400 500 600 7000

1

2

3

4

5

60 oC

20 oC

Iint.

λ / nm

a) b)

25 30 35 40 45 50 551.0

1.5

2.0

2.5

3.0

3.5

I435

T / oC

TT

AT

AG

TA G A A A

CC

ACA

C

AA

GTATCCA

dUG

AGGdUAC

PyNr

GdU A CCT CTT ATA GTA GAA ACC ACA A AG TAA GGdU AC

Nr PyΔ

c)

Figure S6: a) Temperature dependent fluorescence spectra of DNA1 and b) plot of I435 of

DNA1 against temperature. (c = 1 μM in Na-Pi buffer, 250 mM NaCl, pH = 7, λexc = 380

nm, T = 25 °C). A melting temperature of ≈ 40 °C is observed fort the melting of the

hairpins of DNA1. c) A pictorial representation of the temperature dependent association

and dissociation of the hairpin.



20 40 60

1.05

1.10

1.15

1.20

Tm = 38.0 oCHyp

erch

rom

icity

T / oC20 40 60 80

1.9

2.0

2.1

2.2

2.3

Tm = 59.1 oC

Hyp

erch

rom

icity

T / oC

20 40 60 80

0.80

0.84

0.88

0.92

Tm = 34.2 oC

Hyp

erch

rom

icity

T / oC20 40 60 80

1.8

1.9

2.0

2.1

2.2

Tm = 58.1 oC

Hyp

erch

rom

icity

T / oC

20 40 60 80

2.10

2.15

2.20

2.25

2.30

Tm = 58.4 oC

Hyp

erch

rom

icity

T / oC

20 40 60 800.72

0.74

0.76

0.78

0.80

Tm = 39.3 0C

Hyp

erch

rom

icity

T / oC

20 40 60 800.88

0.92

0.96

1.00

1.04

Tm = 31.2 0C

hype

rchr

omoc

ity

T / oC

20 40 60 801.50

1.55

1.60

1.65

1.70

1.75

1.80

Tm = 45.4 oC

Hyp

erch

rom

icity

T / oC

a)

b)

c)

d)

TT

AT

AG

TA G A A A

CC

ACA

C

AA

GTA

TCCA

dUG

AGGdUAC

PyNr

TT

AT

AG

TA G A A A

CC

ACA

C

AA

GTA

TCGA

dUC

AGC

dUG

PyNrT

TT

AT

AG

TA G A A A

CC

ACA

C

AA

GTA

TCGT

dUC

AGC

TG

PyNr

dU

TCGA

dUC

AGCdUAG

PyNr

TA

ATA

G A AC

GT

A

AC

GdUA CCT CTT ATA GTA GAA ACC ACA AAG TAA GGdU AC

GAA TAT CAT CTT TGG TGT TTC AT

Nr Py

CdUA GCT CTT ATA GTA GAA ACC ACA AAG TAA GCT dUG

GAA TAT CA T CTT TGG TGT TTC AT

Nr Py

CdUT GCT CTT ATA GTA GAA ACC ACA AAG TAA GCdU TG

GAA TAT CAT CTT TGG TGT TTC AT

Nr Py

CdUA GCT TAA TAG AAC ACA GTA GC dUAG

ATT ATC TTG TGT CA

Nr Py

Figure S7. Melting curves for hairpins (left) and duplexes (right) monitored at 260 nm

(heating cycle); a) DNA1, b) DNA2, c) DNA3, d) DNA4 (c = 1 μM in Na-Pi buffer, 250



mM NaCl, pH= 7). The weak transition observed in the temperature range of 20-40 °C in

the duplexes of DNA1-4 may be attributed to the melting of the duplex formed through

the “sticky end” association2 (c = 1 μM in Na-Pi buffer, 250 mM NaCl, pH = 7).

a) b)

20 40 60 80 1001.6

1.7

1.8

1.9

2.0

Hyp

erch

rom

icity

T / oC

Tm = 74.5 oC

GdUA CCT CTT ATA GTA GAA ACC ACA AAG TAA GGdU AC

CA T GGA GAA TAT CAT CTT TGG TGT TTC ATT CCA TG

1.5

1.6

1.7

1.8

1.9

Nr Py

20 40 60 80 100

Hyp

erch

rom

icity

T / oC

Tm = 74.8 oC


GA A CGA GAA TAT CAT CTT TGG TGT TTC ATT CGA AC

Nr Py

Figure S8. Melting curves for the duplexes of a) DNA1 and b) DNA3 with full length

complementary strands (5′-CATGGAGAATATCATCTTTGGTGTTTCATTCCATG-3′

and 5′-GAACGAGAATATCATCTTTGGTGTTTCATTCGAAC-3′ for DNA1 and

DNA3, respectively). Absorbance changes are monitored at 260 nm (c = 1 μM in Na-Pi

buffer, 250 mM NaCl, pH= 7). This experiment is suggested by one of the reviewer

during the revision of the paper in order to prove the weak transition appeared in the

melting of the duplex in Figure S7 corresponds to the melting of the duplex formed

through the “stick end” association. The disappearance of the weak transition in this case

clearly supports the proposed hypothesis.



a)

20 40 60 801.5

1.6

1.7

1.8

1.9Tm = 54.6 oC

Hyp

erch

rom

icity

T / oC20 40 60 80

1.6

1.7

1.8

1.9

2.0T

m = 53.2 oC

Hyp

erch

rom

icity

T / oC

20 40 60 801.7

1.8

1.9

2.0T

m = 53.5 oC

Hyp

erch

rom

icity

T / oC

b)

c) d)

20 40 60 801.40

1.45

1.50

1.55

1.60

1.65

1.70

Hyp

erch

rom

icity

T / oC

Tm = 33.0 oC


GAA TAT CAT CTA TGG TGT TTC AT

Nr Py

mismatch



Nr Py

mismatch



Nr Py

mismatch

CdUA GCT TAA TAG AAC ACA GTA GC dUAG

ATT ATC TAG TGT CA

Nr Py

mismatch

Figure S9. Melting curves of the duplex of DNA1-4 with target DNAs contains SNP monitored at 260 nm (heating cycle); a) DNA1, b) DNA2, c) DNA3, d) DNA4. Table S2. Melting temperatures of duplex DNA1-4 with matched targets and targets with SNP. ΔT represents the difference in melting temperatures between duplex and hairpin.

DNA Matched target Mismatch target

Tm (hairpin) (°C)

Tm (duplex) (°C)

ΔT (°C)

Tm (hairpin) (°C)

ΔT (°C)

DNA1 38.0 59.1 21.1 54.6 16.6

DNA2 34.2 58.1 23.9 53.2 19.0

DNA3 31.2 58.4 27.2 53.5 22.3

DNA4 39.3 45.4 6.1 33.0 -6.3



0,0 0,2 0,4 0,6 0,8 1,0 1,20

1

2

3

4

I 435n

m /

I 660n

m

[DNA1] / μM

Figure S10. Plot of I435/I660 of DNA1 against the concentration of DNA5 (c = 1 μM in

Na-Pi buffer, 250 mM NaCl, pH = 7).

b)a)

c)

400 500 600 7000

1

2

3

4

5

6

Fint.

λ / nm0.0 0.2 0.4 0.6 0.8 1.0 1.2

3

4

5

6

I435

[DNA5] / μM

0.0 0.2 0.4 0.6 0.8 1.0

2

3

4

5

6

I435

[DNA] / μM

d)

400 500 600 7000

2

4

6

Iint.

λ / nm

Figure S11. Fluorescence titration spectra of DNA2 with a) DNA5 and c) DNA6. Plot of

I435 against the concentration of b) DNA5 and d) DNA6 (c = 1 μM in Na-Pi buffer, 250

mM NaCl, pH = 7).



0,0 0,2 0,4 0,6 0,8 1,0 1,25

6

7

8

9

I435

[DNA5] / μM

a) b)

400 500 600 7000

3

6

9

Fint.

λ / nm

400 500 600 7000

2

4

6

8

Iint.

λ / nm

c) d)

0.0 0.2 0.4 0.6 0.8 1.0

3

4

5

6

7

8

I435

[DNA6] / μM



mM NaCl, pH = 7).



0,0 0,4 0,8 1,21

2

3

4

5

I435

[DNA7] / μM

0,0 0,5 1,0 1,5 2,0 2,5 3,0

1

2

3

4

I435

[DNA8] / μM

b)a)

400 500 600 7000

1

2

3

4

5

6

Fint.

λ / nm

400 500 600 7000

1

2

3

4

5

Iint.

λ / nm

c) d)



mM NaCl, pH = 7). DNA8 = ACTGTGATCTATTA (5′→3′) (underline indicates a

mismatch).

References: 1. R. Varghese and H.-A. Wagenknecht, Chem. Eur. J., 2009, 15, 9307. 2. I. V. Nesterova, S. S. Erdem, S. Pakhomov, R. P. Hammer and S. A. Soper, J.

Am. Chem. Soc., 2009, 131, 2432.


Documents

Supporting Information for Red-White-Blue Emission ... · PRIVILEGED DOCUMENT FOR REVIEW PURPOSES ONLY Table S1. m/z Values of ss-DNA1-4 determined by ESI-MS. DNA Calculated mass