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:
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