the royal society of chemistry1 clickable styryl dyes for fluorescence labeling of pyrrolidinyl pna...
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Clickable Styryl Dyes for Fluorescence Labeling of Pyrrolidinyl PNA Probes for the
Detection of Base Mutations in DNA
Boonsong Ditmangklo,a Jaru Taechalertpaisarn,
a,b Khatcharin Siriwong,
c Tirayut Vilaivan
a,*
aOrganic Synthesis Research Unit, Department of Chemistry, Faculty of Science,
Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand bNational Center for Genetic Engineering and Biotechnology (BIOTEC), National Science
and Technology Development Agency, Pathumthani 12120, Thailand cMaterials Chemistry Research Center, Department of Chemistry and Center of Excellence
for Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002,
Thailand
*e-mail: [email protected]
Table of Content
Item Description Page Experimental
Details general, spectroscopic measurements, structural analysis of
DNA·DNA/PNA·DNA duplexes, docking simulations of styryl dyes on
the DNA·DNA and PNA·DNA hybrids
3
Figure S1 1H (A) and
13C (B) NMR spectra of compound 1 5
Figure S2 1H (A) and
13C (B) NMR spectra of compound 2 6
Figure S3 1H (A) and
13C (B) NMR spectra of compound 3 7
Figure S4 1H (A) and
13C (B) NMR spectra of compound 4 8
Figure S5 1H (A) and
13C (B) NMR spectra of compound 5 9
Figure S6 1H (A) and
13C (B) NMR spectra of compound 6 10
Figure S7 1H (A) and
13C (B) NMR spectra of compound 7 11
Figure S8 1H (A) and
13C (B) NMR spectra of compound 8 12
Figure S9 1H (A) and
13C (B) NMR spectra of compound 9 13
Figure S10 1H (A) and
13C (B) NMR spectra of compound 10 14
Figure S11 1H (A) and
13C (B) NMR spectra of compound 11 15
Figure S12 1H (A) and
13C (B) NMR spectra of compound 12 16
Figure S13 1H (A) and
13C (B) NMR spectra of compound 13 17
Figure S14 1H (A) and
13C (B) NMR spectra of compound 14 18
Figure S15 1H (A) and
13C (B) NMR spectra of compound 15 19
Figure S16 1H (A) and
13C (B) NMR spectra of compound 16 20
Figure S17 1H (A) and
13C (B) NMR spectra of compound 17 21
Figure S18 1H (A) and
13C (B) NMR spectra of compound 18 22
Figure S19 1H (A) and
13C (B) NMR spectra of compound 19 23
Figure S20 HPLC chromatogram and MALDI-TOF mass spectrum of T9-3
24
Figure S21 HPLC chromatogram and MALDI-TOF mass spectrum of T9-5a
25
Figure S22 HPLC chromatogram and MALDI-TOF mass spectrum of T9-6
26
Figure S23 HPLC chromatogram and MALDI-TOF mass spectrum of T9-8
27
Figure S24 HPLC chromatogram and MALDI-TOF mass spectrum of T9-18 28
Figure S25 HPLC chromatogram and MALDI-TOF mass spectrum of T9-19 29
Figure S26 HPLC chromatogram and MALDI-TOF mass spectrum of M10-3 30
Figure S27 HPLC chromatogram and MALDI-TOF mass spectrum of M10-4 31
Figure S28 HPLC chromatogram and MALDI-TOF mass spectrum of M10-5a 32
Figure S29 HPLC chromatogram and MALDI-TOF mass spectrum of M10-6 33
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2019
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Item Description Page
Figure S30 HPLC chromatogram and MALDI-TOF mass spectrum of M10-18 34
Figure S31 HPLC chromatogram and MALDI-TOF mass spectrum of M10-19 35
Figure S32 HPLC chromatogram and MALDI-TOF mass spectrum of M12-3 36
Figure S33 Correlation between (A) absorption and (B) emission energies of dye 3
and solvent parameter ET(30)
37
Figure S34 (A) Absorption and (B) emission spectra of dye 3 in glycerol. 37
Figure S35 (A) Absorption spectra (B) plot of absorption at 532 nm of 3 at different
concentrations in sodium phosphate buffer pH 7.0
38
Figure S36 Fluorescence titration experiments of dsDNA with dye 3 (A) as a
function of dye concentration (B) of dye:DNA (molecules/bp)
38
Figure S37 (A) Absorption and (B) emission spectra of 3 in the presence of various
negatively-charged polymers.
39
Figure S38 MALDI-TOF MS spectra showing the progress of the synthesis of M10-
3 via sequential reductive alkylation-Click chemistry
39
Figure S39 Fluorescence spectra of dye 3 (A) and 18 (B) in the presence of various
PNADNA duplexes
40
Figure S40 (A) Comparison of minor grooves of DNA·DNA and acpcPNA·DNA
duplexes (B) Docking of 3 and 18 into the minor groove of DNA duplex
40
Figure S41 UV-vis spectra and melting temperature of T9-3 in the presence of
various DNA
41
Figure S42 UV-vis spectra (A) and melting temperature (B) of T9-5a in the
presence of various DNA
41
Figure S43 UV-vis spectra (A) and melting temperature (B) of T9-6 in the presence
of various DNA
41
Figure S44 UV-vis spectra (A) and melting temperature (B) of T9-8 in the presence
of various DNA
42
Figure S45 UV-vis spectra (A) and melting temperature (B) of T9-18 in the
presence of various DNA
42
Figure S46 UV-vis spectra (A) and melting temperature (B) of T9-19 in the
presence of various DNA
42
Figure S47 UV-vis spectra (A) and melting temperature (B) of M10-3 in the
presence of various DNA
43
Figure S48 UV-vis spectra (A) and melting temperature (B) of M10-4 in the
presence of various DNA
43
Figure S49 UV-vis spectra (A) and melting temperature (B) of M10-5a in the
presence of various DNA
43
Figure S50 UV-vis spectra (A) and melting temperature (B) of M10-6 in the
presence of various DNA
44
Figure S51 UV-vis spectra (A) and melting temperature (B) of M10-18 in the
presence of various DNA
44
Figure S52 UV-vis spectra (A) and melting temperature (B) of M10-19 in the
presence of various DNA
44
Figure S53 UV-vis spectra (A) and melting temperature (B) of M12-5a in the
presence of various DNA
45
Table S1 Optical properties of styryl dye 3 in various solvents 45
Table S2 Fluorescence and thermal stability data of M10-3 and M10-5a in the
presence of various DNA
45
References 46
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3
Experimental Details
General
All reagent grade chemicals and solvents were purchased from standard suppliers and
were used as received without further purification. Acetonitrile for HPLC experiment was
HPLC grade and was filtered through a membrane filter (13 mm , 0.45 m) before use.
MilliQ water was obtained from an ultrapure water system fitted with a Millipak® 40 filter
unit (0.22 µ). Oligonucleotides were purchased from BioDesign or Pacific Science
(Thailand).
Spectroscopic Measurements
Samples for absorption and fluorescence studies of alkynyl-modified styryl dyes with
or without DNA duplex (dsDNA) were prepared in 10 mM sodium phosphate buffer pH 7.0
at the concentration of dye = 2.0 and dsDNA = 1.0 M. Except otherwise specified, the
samples for absorption and fluorescence studies of PNADNA hybrids were prepared in 10
mM sodium phosphate buffer pH 7.0 at concentration PNA = 1.0 and DNA = 1.2 M. The
concentration of the PNA was determined by UV spectrophotometry using the molar
extinction coefficient at 260 nm (260) as calculated from the base sequence. The thermal
denaturation and absorption spectra were measured on a CARY 100 Bio UV-vis
spectrophotometer (Varian, Australia) and the fluorescence spectra were collected on a Cary
Eclipse Fluorescence Spectrophotometer (Varian/Agilent Technologies) using a quartz
cuvette with a path length of 1.0 cm at 20 °C. The samples for thermal denaturation
experiments were similarly prepared, and the samples were heated from 20 to 90 °C at a
heating rate of 1 °C/min. The melting temperature (Tm) was evaluated by first smoothing of
temperature data and then determined the maximum of the first derivative by using
KaliedaGraph 4.1 (Synergy Software).
The fluorescence quantum yield (ΦF) of the free dyes, single-stranded PNA probes
and PNA·DNA hybrids were calculated using perylene (ΦF = 0.92, ex = 360420),
rhodamine B (ΦF = 0.50, ex = 450540) and nile blue (ΦF = 0.27, ex = 550610) as the
standard.1 The integrated fluorescence intensities and the absorbance values (at ex) of the
standard and the samples were plotted and the slopes were determined to give gradstandard and
gradsample, respectively.
The quantum yield can be calculated according to equation (1):
Φsample = Φstandard (gradsample / gradstandard) (2
sample/ 2
standard) (1)
Where grad is the slope from the plot of integrated fluorescence intensity as a function
of absorbance and η is the refractive index of the solvent used for the fluorescence
measurement.
Circular dichroism (CD) experiments of PNADNA hybrids were performed on a
JASCO Model J-815 and the samples were prepared in 10 mM sodium phosphate buffer pH
7.0 at concentration PNA = 2.5 and DNA = 3.0 M.
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4
Structural analysis of DNA·DNA/PNA·DNA duplexes: The coordinates of DNA·DNA
duplex retrieving from Protein Databank (PDB code = 4c64) and of acpcPNA·DNA from
literature2 were visualized their structures with Chimera software.
3 Molecular surfaces were
calculated and displayed by the same program, and the minor groove widths were measured
between solvent-excluded molecular surfaces of each strand. Typically, three measurements
of each hybrid were averaged. The minor groove of the acpcPNA·DNA duplex was too
shallow to be determined its width and depth precisely.
Docking simulations of styryl dyes on the DNA·DNA and PNA·DNA hybrids: Structures
of styryl dyes 3 and 18 were built up using Open Babel,4 and their structures were minimized
using Molecular Modelling Toolkit (MMTK) implemented in Chimera. ANTECHAMBER5,6
was used to assign net charges of the molecules. The pre-minimized structures were detected
their rotatable bonds and converted to pdbqt files with AutoDockTools. For the
DNA·DNA/PNA·DNA structures, only polar hydrogen atoms and Kollman charges were
assigned to their structures with AutoDockTools. Grid boxes were set to cover entire
structures, in which DNA·DNA and acpcPNA·DNA duplexes were 80×80×120 and
97×97×97 Å3
respectively. For the docking simulation with Autodock Vina, the
exhaustiveness was set to 64. The results were inspected for their binding interactions
between styryl dyes and DNA·DNA/PNA·DNA hybrids with Chimera.
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5
ab
c
d
f g
h
e
g
hi
a
fb
i j
j
1
e,c
d
i
a
fb je c
d
(A)
(B)
Figure S1. 1
H (400 MHz, CDCl3) (A) and 13
C (100 MHz, CDCl3) (B) NMR spectra of
compound 1
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6
ab
c
d
f g
h
e
g
hi
da,f b
c
i j
k
j
2
k
e
i
d
a,f
b ce
(A)
(B)
Figure S2. 1H (400 MHz, DMSO-d6) (A) and
13C (100 MHz, DMSO-d6) (B) NMR spectra of
compound 2
-
7
ab
c
d
f
g h
e
g hd a,f bc,e
i j3 k
i j
k
d
a,f
b c,ei
(A)
(B)
Figure S3. 1H (400 MHz, DMSO-d6) (A) and
13C (100 MHz, DMSO-d6) (B) NMR spectra of
compound 3
-
8
ab
c
d
f
g
h
e
g hi
d a,f b
c,e
i j k
j
4
k
l
l
i
d a,f b
c,e
(A)
(B)
Figure S4. 1H (400 MHz, DMSO-d6) (A) and
13C (100 MHz, DMSO-d6) (B) NMR spectra of
compound 4
-
9
ab
c
d
f
g h
e
gh
d a,f bc
i
k
l
j5a
l
k
i,e j
d a,f b ci,e j
(A)
(B)
Figure S5. 1H (400 MHz, DMSO-d6) (A) and
13C (100 MHz, DMSO-d6) (B) NMR spectra of
compound 5
-
10
ab
c
d
f
g
h
e
g
hid
f,a bc
i j l m
k
j
6
l
m
n
n
ke
id f,a b c e
(A)
(B)
Figure S6. 1H (400 MHz, DMSO-d6) (A) and
13C (100 MHz, DMSO-d6) (B) NMR spectra of
compound 6
-
11
ab
c
d
f
g h
e
ghi
d a,f b,ec
i jk
l m
l
j
m,k
7
id a,f b,e c
l m,k
(A)
(B)
Figure S7. 1H (400 MHz, DMSO-d6) (A) and
13C (100 MHz, DMSO-d6) (B) NMR spectra of
compound 7
-
12
ab
c
d
lf
g
j
h
e
i
gh
ki
d af ebc
k
jl
8
id a f eb c
(A)
(B)
Figure S8. 1H (400 MHz, DMSO-d6) (A) and
13C (100 MHz, DMSO-d6) (B) NMR spectra of
compound 8
-
13
ab
c
d
mf
g
j
h
e
i
k l
gh
ld af b
ce,i,k
jm
9
l
d af b c
j
m
e,i,k
(A)
(B)
Figure S9. 1H (400 MHz, DMSO-d6) (A) and
13C (100 MHz, DMSO-d6) (B) NMR spectra of
compound 9
-
14
ab
c
d
k
f
g
j
h
e
i
g
h
k
ji
da,f e
bc
10ia,f
d e cb
(A)
(B)
Figure S10. 1H (400 MHz, DMSO-d6) (A) and
13C (100 MHz, DMSO-d6) (B) NMR spectra
of compound 10
-
15
ab
cd
l
f
g
j
h
e
i
k
m
g
hj
ia,f
e
k
bcd
11
i
a,f
eb c
d
l.m
(A)
(B)
Figure S11. 1H (400 MHz, DMSO-d6) (A) and
13C (100 MHz, DMSO-d6) (B) NMR spectra
of compound 11
-
16
ab
c
d
k
f
g
j
h
e
i
d bci
a,e,f,j
gh
12
d b ci
a,e,f,j
(A)
(B)
Figure S12. 1
H (400 MHz, DMSO-d6) (A) and 13
C (100 MHz, DMSO-d6) (B) NMR spectra
of compound 12
-
17
ab
c
d
k
f
g
j
h
e
i
g hji d a
f,e
bc
l
m n
m
n
lk
13
j
i
d a
f,e
b c
(A)
(B)
Figure S13. 1
H (400 MHz, DMSO-d6) (A) and 13
C (100 MHz, DMSO-d6) (B) NMR spectra
of compound 13
-
18
ab
c
d
kf
g
j
h
e i
gh
i-q, e
daf bc
l
m
n
14
q
p
o
i-q, e
b c
(A)
(B)
Figure S14. 1
H (400 MHz, DMSO-d6) (A) and 13
C (100 MHz, DMSO-d6) (B) NMR spectra
of compound 14
-
19
ab
c
d
f
g
j
h
e i
l
g
h
fbc
m
k
n
o15
id,o a e n,mbk jl c
d,o
iaen,m
kjl
(A)
(B)
Figure S15. 1
H (400 MHz, DMSO-d6) (A) and 13
C (100 MHz, DMSO-d6) (B) NMR spectra
of compound 15
-
20
ab
c
d
kf
g
j
h
e
16
i
l
m
m
lg
hjd a b
c,e,k,i
f
mljd a b
c,e,k,i
f
(A)
(B)
Figure S16. 1
H (400 MHz, DMSO-d6) (A) and 13
C (100 MHz, DMSO-d6) (B) NMR spectra
of compound 16
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21
ab
c
d
k
f
gm
ij
ln
o
p
p
h
h
e
k
f
17
ga,j,l
bce mno
di
ppm (f1) 7.508.008.50
f
a,j,l
b ce mnod i
(A)
(B)
Figure S17. 1
H (400 MHz, DMSO-d6) (A) and 13
C (100 MHz, DMSO-d6) (B) NMR spectra
of compound 17
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22
a b
c
d
i
f
l
g
hj
m
e
18
k
m
lji
k
g,e
d,h
c,ba f
i
g e
d,h
c,ba f
(A)
(B)
Figure S18. 1
H (400 MHz, DMSO-d6) (A) and 13
C (100 MHz, DMSO-d6) (B) NMR spectra
of compound 18
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23
a b
c
d
i
f
l
g
hj m
e
k
on
j
i
l
b,d,h
a f
n
o
m
p
p
k
gc
19
e
i
b,d,h
a f gc e
(A)
(B)
Figure S19. 1
H (400 MHz, DMSO-d6) (A) and 13
C (100 MHz, DMSO-d6) (B) NMR spectra
of compound 19
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24
(A)
(B)
Figure S20. HPLC chromatogram (A) and MALDI-TOF mass spectrum (B) of T9-3 [Ac-
TTTT(3)TTTTT-LysNH2] (CCA matrix) (calcd. for [M+H]+: m/z = 3598.0).
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25
(A)
(B)
Figure S21. HPLC chromatogram (A) and MALDI-TOF mass spectrum (B) of T9-5a [Ac-
TTTT(5a)TTTTT-LysNH2] (CCA matrix) (calcd. for [M+H]+: m/z = 3569.9).
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26
(A)
(B)
Figure S22. HPLC chromatogram (A) and MALDI-TOF mass spectrum (B) of T9-6 [Ac-
TTTT(6)TTTTT-LysNH2] (CCA matrix) (calcd. for [M+H]+: m/z = 3656.1).
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(A)
(B)
Figure S23. HPLC chromatogram (A) and MALDI-TOF mass spectrum (B) of T9-8 [Ac-
TTTT(8)TTTTT-LysNH2] (CCA matrix) (calcd. for [M+H]+: m/z = 3650.1).
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(A)
(B)
Figure S24. HPLC chromatogram (A) and MALDI-TOF mass spectrum (B) of T9-18 [Ac-
TTTT(18)TTTTT-LysNH2] (CCA matrix) (calcd. for [M+H]+: m/z = 3592.0).
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29
(A)
(B)
Figure S25. HPLC chromatogram (A) and MALDI-TOF mass spectrum (B) of T9-19 [Ac-
TTTT(19)TTTTT-LysNH2] (CCA matrix) (calcd. for [M+H]+: m/z = 3650.0).
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30
(A)
(B)
Figure S26. HPLC chromatogram (A) and MALDI-TOF mass spectrum (B) of M10-3 [Ac-
GTAGA(3)TCACT-LysNH2] (CCA matrix) (calcd. for [M+H]+: m/z = 3977.4).
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31
(A)
(B)
Figure S27. HPLC chromatogram (A) and MALDI-TOF mass spectrum (B) of M10-4 [Ac-
GTAGA(4)TCACT-LysNH2] (CCA matrix) (calcd. for [M+H]+: m/z = 3977.4).
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32
(A)
(B)
Figure S28. HPLC chromatogram (A) and MALDI-TOF mass spectrum (B) of M10-5a [Ac-
GTAGA(5a)TCACT-LysNH2] (CCA matrix) (calcd. for [M+H]+: m/z = 3949.3).
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33
(A)
(B)
Figure S29. HPLC chromatogram (A) and MALDI-TOF mass spectrum (B) of M10-6 [Ac-
GTAGA(6)TCACT-LysNH2] (CCA matrix) (calcd. for [M+H]+: m/z = 4035.5).
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34
(A)
(B)
Figure S30. HPLC chromatogram (A) and MALDI-TOF mass spectrum (B) of M10-18 [Ac-
GTAGA(18)TCACT-LysNH2] (CCA matrix) (calcd. for [M+H]+: m/z = 3971.4).
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35
(A)
(B)
Figure S31. HPLC chromatogram (A) and MALDI-TOF mass spectrum (B) of M10-19 [Ac-
GTAGA(19)TCACT-LysNH2] (CCA matrix) (calcd. for [M+H]+: m/z = 4029.4).
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36
(A)
(B)
Figure S32. HPLC chromatogram (A) and MALDI-TOF mass spectrum (B) of M12-5a [Ac-
CCCAGT(5a)GTTGGG-LysNH2] (CCA matrix) (calcd. for [M+H]+: m/z = 4656.0).
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37
1.84x104
1.85x104
1.86x104
1.87x104
1.88x104
1.89x104
35 40 45 50 55 60 65
1/
ma
x (
cm
-1)
ET(30)
1.63x104
1.64x104
1.65x104
1.66x104
1.67x104
1.68x104
35 40 45 50 55 60 65
1/
ma
x (
cm
-1)
ET(30)
A B
Figure S33. Correlation between (A) absorption and (B) emission energies of dye 3 and
solvent parameter ET(30)
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
200 300 400 500 600 700 800
Absorb
an
ce (
A.U
.)
Wavelength (nm)
0%
80%
glycerol
0
50
100
150
200
250
300
350
400
550 600 650 700 750 800
Flu
ore
scence
(arb
itra
ry u
nit)
Wavelength (nm)
0%
80%
glycerol
A B
Figure S34. (A) Absorption and (B) emission spectra of dye 3 in the presence of glycerol.
Conditions: 0, 20, 40, 60 and 80% aqueous glycerol containing 10 mM sodium phosphate
buffer pH 7.0, [3] = 2 M, ex = 525 nm
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38
A B
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
200 300 400 500 600 700 800
Absorb
an
ce (
A.U
.)
Wavelength (nm)
5 M
0.1 M
0.00
0.05
0.10
0.15
0.20
0 1x10-6
2x10-6
3x10-6
4x10-6
5x10-6
6x10-6
Absorb
ance
at 53
2 n
m
[Dye] (M)
Figure S35. (A) Absorption spectra (B) plot of absorption at 532 nm of 3 at concentrations of
0.1, 0.5, 1, 1.5, 2, 3, 4 and 5 µM in sodium phosphate buffer pH 7.0
0
50
100
150
200
250
0 1x10-6
2x10-6
3x10-6
4x10-6
Free dyeDye+dsDNA
Flu
ore
scence
at 605
nm
(A
.U.)
[Dye] (M)
0
50
100
150
200
250
0 0.05 0.1 0.15
Flu
ore
scence
at 6
05
nm
(A
.U.)
Dye:DNA (molecules/bp)
A B
Figure S36. Fluorescence titration experiments of dsDNA with dye 3 (A) as a function of dye
concentration (B) as a function of dye:DNA (molecules/bp); Conditions: phosphate buffer pH
7.0, [3] = 2 M, ex = 525 nm
dsDNA = 5-CGCGGCGTACAGTGATCTACCATGCCCTGG-3 +
3-GCGCCGCATGTCACTAGATGGTACGGGACC-5
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39
0.0
0.2
0.4
0.6
0.8
1.0
200 300 400 500 600 700 800
DyedsDNAPSSHeparin
Absorb
an
ce (
A.U
.)
Wavelength (nm)
0
20
40
60
80
100
120
140
160
550 600 650 700 750 800
DyedsDNAPSSHeparin
Flu
ore
scence
(A
.U.)
Wavelength (nm)
A B
Figure S37. (A) Absorption and (B) emission spectra of 3 in the presence of various
negatively-charged polymers. PSS = sodium poly(styrenesulfonate). Conditions: sodium
phosphate buffer pH 7.0, [3] = 2 M, polymer/dye ratio (expressed as number of negative
charges/dye molecule) = 30, ex = 525 nm
Figure S38. MALDI-TOF MS spectra showing the progress of the synthesis of styryl-dye-
labeled acpcPNA (M10-3) via sequential reductive alkylation-Click chemistry
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40
A B
0
20
40
60
80
100
120
140
160
550 600 650 700 750 800
Dye 3complementarymismatchedbase insertedabasic
Flu
ore
sce
nce (
arb
itra
ry u
nit)
Wavelength (nm)
0
5
10
15
20
25
30
35
40
600 650 700 750 800
Dye 18complementarymismatchedbase insertedabasic
Flu
ore
sce
nce (
arb
itra
ry u
nit)
Wavelength (nm)
Figure S39. Fluorescence spectra of dye 3 (A) and 18 (B) in the presence of various
PNADNA duplexes in 10 mM sodium phosphate buffer pH 7.0; complementary = M10 +
dAGTGATCTAC, mismatched = M10 + dAGTGCTCTAC, base-inserted = M10 +
dAGTGACTCTAC, abasic = M10 + dAGTGXTCTAC; [PNADNA] = 1.0 M, [dye] = 1.0
M, ex = 525 nm for 3 and ex = 560 nm for 18, See Table 3 for the DNA sequence.
A B
Figure S40. (A) Comparison of minor grooves of DNA·DNA (left) and acpcPNA·DNA
(right) duplexes (B) Docking of 3 and 18 into the minor groove of DNA duplex.
-
41
0
0.1
0.2
0.3
0.4
0.5
0.6
200 300 400 500 600 700 800
Ab
s
Wavelength (nm)
T9-3
T9-3 + complementary
T9-3 + mismatched
T9-3 + base inserted
T9-3 + abasic
0.00
0.02
0.04
0.06
450 500 550 600
(A)
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
20 30 40 50 60 70 80 90
No
rma
lize
d A
26
0
Temperature (C)
T9-3 + complementary
T9-3 + mismatched
T9-3 + base inserted
T9-3 + abasic
(B)
Figure S41. UV-vis spectra (A) and melting temperature (B) of T9-3 in the presence of
various DNA
0
0.1
0.2
0.3
0.4
0.5
0.6
200 300 400 500 600 700 800
Ab
s
Wavelength (nm)
T9-5a
T9-5a + complementary
T9-5a + mismatched
T9-5a + base inserted
T9-5a + abasic
0
0.02
0.04
0.06
400 450 500 550 600
(A)
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
20 30 40 50 60 70 80 90
Norm
ali
zed
A260
Temperature (C)
T9-5a + complementary
T9-5a + mismatched
T9-5a + base inserted
T9-5a + abasic
(B)
Figure S42. UV-vis spectra (A) and melting temperature (B) of T9-5a in the presence of
various DNA
0
0.1
0.2
0.3
0.4
0.5
0.6
200 300 400 500 600 700 800
Ab
s
Wavelength (nm)
T9-6
T9-6 + complementary
T9-6 + mismatched
T9-6 + base inserted
T9-6 + abasic
0.00
0.05
0.10
400 450 500 550 600
(A)
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
20 30 40 50 60 70 80 90
No
rma
lize
d A
26
0
Temperature (C)
T9-6 + complementary
T9-6 + mismatched
T9-6 + base inserted
T9-6 + abasic
(B)
Figure S43. UV-vis spectra (A) and melting temperature (B) of T9-6 in the presence of
various DNA
-
42
0
0.1
0.2
0.3
0.4
0.5
200 300 400 500 600 700 800
Ab
s
Wavelength (nm)
T9-8
T9-8 + complementary
T9-8 + mismatched
T9-8 + base inserted
T9-8 + abasic
0.00
0.02
0.04
500 550 600 650
(A)
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
20 30 40 50 60 70 80 90
No
rma
lize
d A
26
0
Temperature (C)
T9-8 + complementary
T9-8 + mismatched
T9-8 + base inserted
T9-8 + abasic
(B)
Figure S44. UV-vis spectra (A) and melting temperature (B) of T9-8 in the presence of
various DNA
0
0.1
0.2
0.3
0.4
0.5
0.6
200 300 400 500 600 700 800
Ab
s
Wavelength (nm)
T9-18
T9-18 + complementary
T9-18 + mismatched
T9-18 + base inserted
T9-18 + abasic
0.00
0.02
0.04
450 500 550 600 650 700
(A)
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
20 30 40 50 60 70 80 90
Norm
alize
d A
260
Temperature (C)
T9-18 + complementary
T9-18 + mismatched
T9-18 + base inserted
T9-18 + abasic
(B)
Figure S45. UV-vis spectra (A) and melting temperature (B) of T9-18 in the presence of
various DNA
0
0.1
0.2
0.3
0.4
0.5
0.6
200 300 400 500 600 700 800
Ab
s
Wavelength (nm)
T9-19
T9-19 + complementary
T9-19 + mismatched
T9-19 + base inserted
T9-19 + abasic
0.00
0.02
0.04
0.06
450 500 550 600 650 700
(A)
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
20 30 40 50 60 70 80 90
Norm
alize
d A
260
Temperature (C)
T9-19+ complementary
T9-19 + mismatched
T9-19 + base inserted
T9-19 + abasic
(B)
Figure S46. UV-vis spectra (A) and melting temperature (B) of T9-19 in the presence of
various DNA
-
43
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
200 300 400 500 600 700 800
Ab
s
Wavelength (nm)
M10-3
M10-3 + complementary
M10-3 + mismatched
M10-3 + base inserted
M10-3 + abasic
0
0.01
0.02
0.03
0.04
0.05
450 500 550 600 650
(A)
0.95
1.00
1.05
1.10
1.15
1.20
20 30 40 50 60 70 80 90
No
rmali
zed
A2
60
Temperature (oC)
M10-3 + complementary
M10-3 + mismatched
M10-3 + base inserted
M10-3 + abasic
(B)
Figure S47. UV-vis spectra (A) and melting temperature (B) of M10-3 in the presence of
various DNA
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
200 300 400 500 600 700 800
Ab
s
Wavelength (nm)
M10-4
M10-4 + complementary
M10-4 + mismatched
M10-4 + base inserted
M10-4 + abasic
0.00
0.02
0.04
380 430 480 530 580
(A)
0.95
1.00
1.05
1.10
1.15
1.20
20 30 40 50 60 70 80 90
Norm
ali
zed
A2
60
Temperature (C)
M10-4 + complementary
M10-4 + mismatched
M10-4 + base inserted
M10-4 + abasic
(B)
Figure S48. UV-vis spectra (A) and melting temperature (B) of M10-4 in the presence of
various DNA
0
0.1
0.2
0.3
0.4
0.5
0.6
200 300 400 500 600 700 800
Ab
s
Wavelength (nm)
M10-5a
M10-5a + complementary
M10-5a + mismatched
M10-5a + base inserted
M10-5a + abasic
0.00
0.02
0.04
380 430 480 530 580
(A)
0.95
1.00
1.05
1.10
1.15
1.20
20 30 40 50 60 70 80 90
No
rma
lize
d A
26
0
Temperature (C)
M10-5a + complementary
M10-5a + mismatched
M10-5a + base inserted
M10-5a + abasic
(B)
Figure S49. UV-vis spectra (A) and melting temperature (B) of M10-5a in the presence of
various DNA
-
44
0
0.1
0.2
0.3
0.4
0.5
0.6
200 300 400 500 600 700 800
Ab
s
Wavelength (nm)
M10-6
M10-6 + complementary
M10-6 + mismatched
M10-6 + base inserted
M10-6 + abasic
0.00
0.03
0.06
400 450 500 550 600 650
(A)
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
20 30 40 50 60 70 80 90
Norm
alize
d A
260
Temperature (C)
M10-6 + complementary
M10-6 + mismatched
M10-6 + base inserted
M10-6 + abasic
(B)
Figure S50. UV-vis spectra (A) and melting temperature (B) of M10-6 in the presence of
various DNA
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
200 300 400 500 600 700 800
Abs
Wavelength (nm)
M10-18
M10-18 + complementary
M10-18 + mismatched
M10-18 + base inserted
M10-18 + abasic
0.00
0.02
0.04
0.06
500 570 640 710
(A)
0.95
1.00
1.05
1.10
1.15
1.20
20 30 40 50 60 70 80 90
Norm
ali
zed
A2
60
Temperature (C)
M10-18 + complementary
M10-18 + mismatched
M10-18 + base inserted
M10-18 + abasic
(B)
Figure S51. UV-vis spectra (A) and melting temperature (B) of M10-18 in the presence of
various DNA
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
200 300 400 500 600 700 800
Ab
s
Wavelength (nm)
M10-19
M10-19 + complementary
M10-19 + mismatched
M10-19 + base inserted
M10-19 + abasic
0.00
0.02
0.04
0.06
500 570 640
(A)
0.95
1.00
1.05
1.10
1.15
1.20
20 30 40 50 60 70 80 90
No
rma
lize
d A
26
0
Temperature (C)
M10-19 + complementary
M10-19 + mismatched
M10-19 + base inserted
M10-19 + abasic
(B)
Figure S52. UV-vis spectra (A) and melting temperature (B) of M10-19 in the presence of
various DNA
-
45
0
0.1
0.2
0.3
0.4
0.5
0.6
200 300 400 500 600 700 800
Ab
s
Wavelength (nm)
M12-5a
M12-5a + Wild type
M12-5a + Liddle's syndrome
(A)
0.95
1.00
1.05
1.10
1.15
1.20
20 30 40 50 60 70 80 90
Norm
ali
zed
A260
Temperature (C)
M12-5a + Wild type
M12-5a + Liddle's syndrome
(B)
Figure S53. UV-vis spectra (A) and melting temperature (B) of M12-5a in the presence of
various DNA
Table S1. Optical properties of the representative styryl dye 3 in various solvents
Solvent ET(30)
a viscosity abs (nm) em (nm) F
THF 37.5 0.55 543 601 0.016
EtOAc 38.0 0.46 540 601 0.019
DMSO 45.1 2.24 540 609 0.011
MeCN 45.6 0.37 539 600 0.002
MeOH 55.4 0.55 538 594 0.003
H2O 63.1 1.00 532 601 0.001 aET(30) values were obtained from literature data.
7
Table S2. Fluorescence and thermal stability data of M10-3 and M10-5a in the presence of
various DNA (complementary, single mismatch, base insertion, abasic and non-
complementary)
PNA DNA sequence
( 53)a,b
Tm (Tm)
ºC
abs (nm)
F/F0 F F(ds)
F(ss)
Notes
M10-3 (ex = 525 nm
em = 602 nm)
none - 542 - 0.019 - single stranded probe
AGTGATCTAC 52 543 2.5 0.059 3.1 complementary
AGTGCTCTAC 35 (–17) 545 3.8 0.077 4.1 single base mismatched
AGTGACTCTAC 33 (–19) 564 10.4 0.222 11.7 base insertion (C)
AGTGAATCTAC 40 (–12) 559 6.6 0.133 7.0 base insertion (A)
AGTGATTCTAC 42 (–10) 563 8.2 0.175 9.2 base insertion (T)
AGTGAGTCTAC 38 (–14) 562 7.4 0.135 7.1 base insertion (G)
AGTCGATCTAC 38 (–14) 542 2.2 0.047 2.5 indirect base insertion
AGTGATCCTAC 41 (–11) 558 2.6 0.064 3.4 indirect base insertion
AGTGXTCTAC 36 (–16) 555 5.1 0.066 3.5 abasic site
AGTGATCTXC 39 (–13) 548 2.0 0.030 1.6 indirect abasic site
TCTGCATTTAG
-
46
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