1

Supporting Information

A rapid and concise setup for the fast screening of FRET pairs

using bioorthogonalized fluorescent dyes

R. Petrovicsa, B. Söveges

a, A. Egyed

a, G. Knorr

a, A. Kormos

a, T. Imre

b, Gy. Török

c, A. Zeke

d,

É. Kocsmáre, G. Lotz

e, P. Kele

a, K. Németh

a*

a Research Centre for Natural Sciences of Hungarian Academy of Sciences, Institute of

Organic Chemistry, “Lendület” Chemical Biology Research Group; H-1117 Budapest,

Magyar tudósok krt. 2. Hungary. Tel.: +36 1 382 6659; E-mail address:

nemeth.krisztina@ttk.mta.hu

b Research Centre for Natural Sciences of Hungarian Academy of Sciences, Instumentation

Center, MS Metabolomics Research Group; H-1117 Budapest, Magyar tudósok krt. 2.

c Research Centre for Natural Sciences of Hungarian Academy of Sciences, Institute of

Enzymology, Molecular Cell Biology Research Group; H-1117 Budapest, Magyar tudósok

krt. 2. Hungary.

d Research Centre for Natural Sciences of Hungarian Academy of Sciences, Institute of

Enzymology, Protein Research Group; H-1117 Budapest, Magyar tudósok krt. 2. Hungary.

e 2

nd Department of Pathology,

Semmelweis University, Budapest H-1091 Üllői str. 93

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2018

2

S1. Spectral characteristics of modified Cy3 and Cy5 dyes

Fluorescent spectra of the cyanine dyes only slightly changed upon modification of the core

framework with - methyltetrazine attached through a vinylene linker - or upon conjugated to

BCN and DNA oligomers.

Fig. S1 Fluorescent excitation and emission spectra of the core scaffold of Cy3 and Cy5 dyes, the tetrazine

harboring Cy3T and Cy5T dyes, BCN conjugated Cy3T and Cy5T and the Clarwt

3’ oligonucleotide modified by

Cy3T and Cy5T

400 500 600 700 800

0

20

40

60

80

100

No

rmaliz

ed

flu

ore

scen

ce in

ten

sity (

%)

Emission wavelength (nm)

Cy3 core ex

Cy3 core em

Cy3T ex

Cy3T em

Cy3T+BCN ex

Cy3T+BCN em

3' Cy3T ex

3' Cy3T em

500 600 700 800

0

20

40

60

80

100

No

rmaliz

ed

flu

ore

scen

ce in

ten

sity (

%)

Emission wavelength (nm)

Cy5 core ex

Cy5 core em

Cy5T ex

Cy5T em

Cy5T+BCN ex

Cy5T+BCN em

3' Cy5T ex

3' Cy5T em

3

S2. Spectral overlap of the selected dyes

Fig. S2 Spectral overlap of the selected dye pairs

400 450 500 550 600 650 700 750 800

0

20

40

60

80

100

No

rmaliz

ed

flu

ore

scen

ce in

ten

sity (

%)

Emission wavelength (nm)

overlap

Cy3T ex

Cy3T em

Cy5T ex

Cy5T em

350 400 450 500 550 600 650 700 750

0

20

40

60

80

100

No

rmaliz

ed

flu

ore

scen

ce in

ten

sity (

%)

Emission wavelength (nm)

overlap

Cy1A ex

Cy1A em

Cy3T ex

Cy3T em

350 400 450 500 550 600 650 700 750

0

20

40

60

80

100

No

rmaliz

ed

flu

ore

scen

ce in

ten

sity (

%)

Emission wavelength (nm)

overlap

Cy1A ex

Cy1A em

CBRD1A ex

CBRD1A em

4

S3. Sequential labeling of the amino modified DNA followed by capillary electrophoresis

Fig. S3 Capillary electrophoretic follow up of the fluorescent labeling of the amino modified DNA oligomer

Conditions 64.5 cm total and 56.5 cm effective length fused silica capillary ID 50 µm,

BGE: 200 mM sodium borate buffer (pH 9.0); injection 50 mbar×6 sec; 30 kV; monitored at 260 nm

Migration time (min)

Heating

60 °C, 5 min.

+ NHS-BCN

5 mM, RT, 20 min

Purification

(G25)

+ fluorescent dye with

bioorthogonal function

CBRD1A

(500 µM, RT, 20 min)

Purification

(G25)

NHS-BCN

unconjugated oligo

DNA oligomer conjugated by BCN

unconjugated oligo

DNA

CBRD1A

DNA oligomer conjugated by BCN

Ab

sorb

ance

@2

60

nm

(m

AU

)

5

Fig. S4 Predicted secondary structure of the single stranded DNA oligomers (Clarwt5’ and Clarwt3’ )

6

S4. Identification of fluorescently labeled DNA oligomers by LC-MS

Fig. S5 Negative ESI-MS spectrum of Clarwt

5’ oligonucleotide (Mw =5338 Da)

Fig. S6 Negative ESI-MS spectrum of Clarwt

3’ oligonucleotide (Mw =5368 Da)

[M-4H]4-

[M-3H]3-

[M-5H]5-

[M-6H]6-

[M-7H]7-

[M-8H]8-

[M-9H]9-

[M-10H]10-

[M-11H]11-

[M-4H]4-

[M-3H]3-

[M-5H]5-

[M-6H]6-

[M-7H]7-

[M-8H]8-

[M-9H]9-

[M-10H]10-

[M-11H]11-

7

Fig. S7 Negative ESI-MS spectrum of Clarwt

K5’ oligonucleotide (Mw =5401 Da)

Fig. S8 Negative ESI-MS spectrum of Clarwt

K5’ oligonucleotide labeled with Cy1A dye (Mw =5958.74 Da)

[M-4H]4-

[M-3H]3-

[M-5H]

5-

[M-6H]6-

[M-7H]

7-

[M-8H]8-

[M-9H]9-

[M-10H]10-

[M-11H]11-

[M-4H]4-

[M-3H]3-

[M-5H]5-

[M-6H]6-

[M-7H]

7-

[M-8H]8-

[M-9H]9-

[M-10H]10-

[M-11H]11-

[M-12H]12-

8

Fig. S9 Negative ESI-MS spectrum of Clarwt

5’ oligonucleotide labeled with Cy3T dye (Mw =6071.96 Da)

Fig. S10 Negative ESI-MS spectrum of Clarwt

3’ oligonucleotide labeled with Cy3T dye (Mw =6101.96 Da)

[M-4H]4-

[M-5H]5-

[M-6H]6-

[M-7H]7-

[M-8H]8-

[M-9H]9-

[M-10H]10-

[M-11H]11-

[M-12H]12-

[M-4H]4-

[M-5H]5-

[M-6H]6-

[M-7H]7-

[M-8H]8-

[M-9H]9-

[M-10H]10-

[M-11H]11-

[M-12H]12-

9

Fig. S11 Negative ESI-MS spectrum of Clarwt

K5’ oligonucleotide labeled with Cy3T dye (Mw =6134.96 Da)

Fig. S12 Negative ESI-MS spectrum of Clarwt

5’ oligonucleotide labeled with Cy5T dye (Mw =6097.99 Da)

[M-4H]4-

[M-5H]5-

[M-6H]6-

[M-7H]7-

[M-8H]8-

[M-9H]9-

[M-10H]10-

[M-11H]11-

[M-12H]12-

[M-4H]4-

[M-5H]5-

[M-6H]6-

[M-7H]

7-

[M-8H]8-

[M-9H]9-

[M-10H]10-

[M-11H]11-

[M-12H]12-

10

Fig. S13 Negative ESI-MS spectrum of Clarwt

3’ oligonucleotide labeled with Cy5T dye (Mw =6127.99 Da)

Fig. S14 Negative ESI-MS spectrum of Clarwt

5’ oligonucleotide labeled with CBRD1A dye

(Mw =5919.69 Da)

[M-4H]4-

[M-5H]5-

[M-6H]6-

[M-7H]

7-

[M-8H]8-

[M-9H]9-

[M-10H]10-

[M-11H]11-

[M-12H]12-

[M-4H]4-

[M-3H]3-

[M-5H]5-

[M-6H]6-

[M-7H]

7-

[M-8H]8-

[M-9H]9-

[M-10H]10-

[M-11H]11-

[M-12H]12-

[M-13H]13-

11

Fig. S15 Negative ESI-MS spectrum of Clarwt

3’ oligonucleotide labeled with CBRD1A dye

(Mw =5949.69 Da)

S5. Purity of fluorescently labeled DNA oligomers measured by CE

The purity of the fluorescently labeled DNA oligomers was analyzed by capillary

electrophoresis. The average purity of the end products is above 90%. In the samples the

initial or BCN conjugated DNA can be found as impurity in negligible quantity. In some of

these samples the DNA can be detected in folded structure (cf. Fig. S4) which resulted in

multiple peaks in the electropherograms but the product peaks can be identified by the

characteristic absorbance peak of the conjugated dyes.

[M-4H]4-

[M-3H]3-

[M-5H]5-

[M-6H]6-

[M-7H]7-

[M-8H]8-

[M-9H]9-

[M-10H]10-

[M-11H]11-

[M-12H]12-

12

Fig. S16 Electropherogram of Clarwt

K5’ oligonucleotide

Fig. S17 Electropherogram of Clarwt

3’ oligonucleotide

min5 6 7 8 9 10 11

mAU

0

20

40

60

80

100

120

140

DAD1 B, Sig=214,20 Ref=off (D:\DOCUMENTS\RESEARCH\KELE GROUP\RÉKA\CE\170220000012.D)

Area: 301.026

8.846

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

1 4 0

D A D 1 , 8 . 8 2 3 ( 1 4 6 m A U , - ) o f 1 7 0 2 2 0 0 0 0 0 1 2 . D

min5 6 7 8 9 10 11

mAU

0

20

40

60

80

100

DAD1 B, Sig=214,20 Ref=off (D:\DOCUMENTS\RESEARCH\KELE GROUP\RÉKA\CE\170206000003.D)

Area: 203.99

8.802

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

2 5

5 0

7 5

1 0 0

1 2 5

1 5 0

1 7 5

D A D 1 , 8 . 8 0 3 ( 1 9 9 m A U , A p x ) o f 1 7 0 2 0 6 0 0 0 0 0 3 . D

13

Fig. S18 Electropherogram of Clarwt

5’ oligonucleotide

Fig. S19 Electropherogram of Clarwt

K5’ oligonucleotide labeled with Cy1A dye

min5 6 7 8 9 10 11

mAU

0

20

40

60

80

100

120

140

DAD1 B, Sig=214,20 Ref=off (D:\DOCUMENTS\RESEARCH\KELE GROUP\RÉKA\CE\170206000004.D)

Area: 295.572

8.806

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

D A D 1 , 8 . 8 0 3 ( 2 8 7 m A U , - ) o f 1 7 0 2 0 6 0 0 0 0 0 4 . D

min5 6 7 8 9 10 11

mAU

0

10

20

30

40

DAD1 C, Sig=260,40 Ref=off (D:\DOCUMENTS\RESEARCH\KELE GROUP\RÉKA\CE\170308000004.D)

Area: 5.80

412

9.103

Area: 36.6

973

9.682

Area: 54.0

475

10.26

9

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

- 4

- 2

0

2

4

6

8

1 0

D A D 1 , 9 . 0 9 3 ( 1 5 . 0 m A U , - ) o f 1 7 0 3 0 8 0 0 0 0 0 4 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

- 4

- 2

0

2

4

6

8

1 0

D A D 1 , 9 . 7 4 7 ( 1 6 . 6 m A U , - ) o f 1 7 0 3 0 8 0 0 0 0 0 4 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

D A D 1 , 1 0 . 2 7 0 ( 8 1 . 7 m A U , - ) o f 1 7 0 3 0 8 0 0 0 0 0 4 . D

14

Fig. S20 Electropherogram of Clarwt

5’ oligonucleotide labeled with Cy3T dye

Fig. S21 Electropherogram of Clarwt

3’ oligonucleotide labeled with Cy3T dye

min5 6 7 8 9 10 11

mAU

0

10

20

30

40

DAD1 C, Sig=260,40 Ref=off (D:\DOCUMENTS\RESEARCH\KELE GROUP\RÉKA\CE\170316000024.D)

Area: 3.63

329

9.340

Area: 93.7

904

9.673

Area: 5.32

871

9.933

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

- 2

- 1

0

1

2

3

4

5

6

D A D 1 , 9 . 3 3 3 ( 8 . 8 m A U , - ) o f 1 7 0 3 1 6 0 0 0 0 2 4 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

2 0

4 0

6 0

8 0

D A D 1 , 9 . 6 7 3 ( 1 0 0 m A U , - ) o f 1 7 0 3 1 6 0 0 0 0 2 4 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

2

4

6

8

1 0

D A D 1 , 9 . 9 3 3 ( 1 2 . 1 m A U , - ) o f 1 7 0 3 1 6 0 0 0 0 2 4 . D

min5 6 7 8 9 10 11

mAU

0

10

20

30

40

DAD1 C, Sig=260,40 Ref=off (D:\DOCUMENTS\RESEARCH\KELE GROUP\RÉKA\CE\170316000027.D)

Area: 10.1

429

9.175

Area: 101.018

9.758

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

- 2

0

2

4

6

8

1 0

D A D 1 , 9 . 1 7 7 ( 1 3 . 2 m A U , - ) o f 1 7 0 3 1 6 0 0 0 0 2 7 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

D A D 1 , 9 . 7 5 3 ( 8 0 . 6 m A U , - ) o f 1 7 0 3 1 6 0 0 0 0 2 7 . D

15

Fig. S22 Electropherogram of Clarwt

K5’ oligonucleotide labeled with Cy3T dye

Fig. S23 Electropherogram of Clarwt

5’ oligonucleotide labeled with Cy5T dye

min5 6 7 8 9 10 11

mAU

0

5

10

15

20

25

DAD1 C, Sig=260,30 Ref=off (D:\DOCUMENTS\RESEARCH\KELE GROUP\RÉKA\CE\170216000023.D)

Area: 5.23

525

9.361

Area: 1.88

759

9.527

Area: 84.4

255

9.737

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

2

4

6

8

1 0

D A D 1 , 9 . 3 5 7 ( 1 3 . 0 m A U , - ) o f 1 7 0 2 1 6 0 0 0 0 2 3 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

- 1

0

1

2

3

4

5

6

7

D A D 1 , 9 . 5 3 0 ( 9 . 0 m A U , - ) o f 1 7 0 2 1 6 0 0 0 0 2 3 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

1 0

2 0

3 0

4 0

5 0

D A D 1 , 9 . 6 7 7 ( 5 3 . 9 m A U , - ) o f 1 7 0 2 1 6 0 0 0 0 2 3 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

1 0

2 0

3 0

4 0

5 0

D A D 1 , 9 . 7 3 7 ( 5 6 . 9 m A U , - ) o f 1 7 0 2 1 6 0 0 0 0 2 3 . D

min5 6 7 8 9 10 11

mAU

0

10

20

30

40

DAD1 C, Sig=260,40 Ref=off (D:\DOCUMENTS\RESEARCH\KELE GROUP\RÉKA\CE\170316000025.D)

Area: 4.75

374

9.358

Area: 102.607

9.634

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

2

4

6

8

D A D 1 , 9 . 3 5 0 ( 1 0 . 0 m A U , - ) o f 1 7 0 3 1 6 0 0 0 0 2 5 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

D A D 1 , 9 . 6 3 3 ( 8 2 . 7 m A U , - ) o f 1 7 0 3 1 6 0 0 0 0 2 5 . D

16

Fig. S24 Electropherogram of Clarwt

3’ oligonucleotide labeled with Cy5T dye

Fig. S25 Electropherogram of Clarwt

5’ oligonucleotide labeled with CBRD1A dye

min5 6 7 8 9 10 11

mAU

0

10

20

30

40

DAD1 C, Sig=260,40 Ref=off (D:\DOCUMENTS\RESEARCH\KELE GROUP\RÉKA\CE\170316000031.D)

Area: 8.51

546

9.014

Area: 81.8

267

9.483

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

2

4

6

8

1 0

1 2

1 4

D A D 1 , 9 . 0 1 3 ( 1 6 . 6 m A U , - ) o f 1 7 0 3 1 6 0 0 0 0 3 1 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

D A D 1 , 9 . 4 8 3 ( 8 3 . 0 m A U , - ) o f 1 7 0 3 1 6 0 0 0 0 3 1 . D

min5 6 7 8 9 10 11

mAU

0

5

10

15

20

25

DAD1 C, Sig=260,40 Ref=off (D:\DOCUMENTS\RESEARCH\KELE GROUP\RÉKA\CE\170308000001.D)

Area: 109.992

9.881

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

1 0

2 0

3 0

4 0

D A D 1 , 9 . 8 3 0 ( 5 2 . 1 m A U , - ) o f 1 7 0 3 0 8 0 0 0 0 0 1 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

1 0

2 0

3 0

4 0

5 0

6 0

D A D 1 , 9 . 8 8 0 ( 6 8 . 1 m A U , - ) o f 1 7 0 3 0 8 0 0 0 0 0 1 . D

17

Fig. S26 Electropherogram of Clarwt

3’ oligonucleotide labeled with CBRD1A dye

min5 6 7 8 9 10 11

mAU

0

10

20

30

40

50

DAD1 B, Sig=214,20 Ref=off (D:\DOCUMENTS\RESEARCH\KELE GROUP\RÉKA\CE\170215000005.D)

Area: 9.03

181

9.179

Area: 4.27

87

9.617

Area: 9.23

248

9.798

Area: 97.3

705

9.879

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

2

4

6

8

1 0

D A D 1 , 9 . 1 7 7 ( 1 1 . 9 m A U , - ) o f 1 7 0 2 1 5 0 0 0 0 0 5 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

- 1

0

1

2

3

4

5

6

D A D 1 , 9 . 6 2 7 ( 7 . 7 m A U , - ) o f 1 7 0 2 1 5 0 0 0 0 0 5 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

1

2

3

4

5

6

7

8

D A D 1 , 9 . 7 8 7 ( 8 . 8 m A U , - ) o f 1 7 0 2 1 5 0 0 0 0 0 5 . D

n m2 0 0 3 0 0 4 0 0 5 0 0

m A U

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

D A D 1 , 9 . 8 8 0 ( 9 0 . 5 m A U , - ) o f 1 7 0 2 1 5 0 0 0 0 0 5 . D

18

S6. Estimation of distances of 17-mer double stranded DNA oligomer

Table S1 Calculated distances (5'-phosphate-P to 5'-phosphate-P in trans position) of different

DNA structures from PDB database

4Q45 (damaged DNA) Chain:B Chain:C 59.4 Å

1D66 (bound to TFs) Chain:D Chain:E 51.9 Å

1W0T (telomeric DNA) Chain:C Chain:D 52.2 Å

1W0U (telomeric DNA) Chain:C Chain:D 50.2 Å

5CDP (broken DNA) Chain:F Chain:H 42.1 Å

1O3R (deformed DNA) Chain:B Chain:C 40.0 Å

3L2P (nicked DNA) Chain:B Chain:D 57.5 Å

4E54 (damaged DNA) Chain:F Chain:G 50.8 Å

4E5Z (damaged DNA) Chain:F Chain:G 49.0 Å

1A02 (bound to TFs) Chain:A Chain:B 49.2 Å

1DCT (crosslinked DNA) Chain:F Chain:M 56.4 Å

1HF0 (bound to TFs) Chain:M Chain:N 50.4 Å

1IGN (telomeric DNA) Chain:C Chain:D 50.1 Å

1T2K (bound to TFs) Chain:E Chain:F 50.9 Å

1U78 (bound to TFs) Chain:B Chain:C 52.7 Å

2AS5 (bound to TFs) Chain:A Chain:B 45.1 Å

2Q2U (damaged DNA) Chain:I Chain:J 55.3 Å

2RBA (damaged DNA) Chain:C Chain:D 58.4 Å

4FTH (bound to TFs) Chain:C Chain:D 49.7 Å

5CQQ (bound to TFs) Chain:C Chain:D 44.5 Å

Average 50.8 Å

SE 5.2 Å

19

S7. Temperature controlled denaturation of fluorescently labeled DNA oligomer pairs

Fig. S27 Changes in FRET efficiencies as a function of temperature in case of the oligonucleotide pairs

labeled at proximal (black squares) and distant termini (red circles);

A: Cy3T/Cy5T; B: Cy1A/Cy3T; C: Cy1A/CBRD1A;

50 60 70

0

20

40

60

80

100

FR

ET

effic

iency (

%)

Temperature(oC)

A

50 60 70

0

10

20

30

40

50

60

70

80

90

100

FR

ET

effic

iency (

%)

Temperature(oC)

B

50 60 70

0

10

20

30

40

50

60

70

80

90

100

FR

ET

effic

iency (

%)

Temperature(oC)

C

20

S8. Flow cytometric analysis

For flow cytometric analysis HEK293T cells were transferred into 24-well plate (Greiner-bio-

one 662160) (40,000 cell/well) and incubated for 48 h at 37°C in a 5% CO2 atmosphere. Cells

were transfected with 25 pmol fluorescently labeled DNA using Lipofectamine-3000

(Invitrogene L3000-001) and Optimem medium (Gibco 70011-044) and incubated for 4 h at

37°C in a 5% CO2 atmosphere. Before measurements cells were harvested using 10%

Trypsin-EDTA solution (Sigma T3924) and complete DMEM. Cells were washed twice with

PBS solution (400 g, 5 min). Flow cytometric analysis were carried out with BD FACSCanto

II system (Beckton Dickinson) equipped with 405, 488, 532 and 633 nm lasers with AmCyan

(510 50nm), PE (585 42 nm) and PerCP (LP 670) filters.

Fig. S28 Flow cytometric analysis of HEK293T cells labelled by fluorescent DNA oligomers

21

S9. NMR spectra of Cy1A dye

22