The Royal Society of Chemistry1 Clickable Styryl Dyes for Fluorescence Labeling of Pyrrolidinyl PNA...

1

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 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


26


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 S28 HPLC chromatogram and MALDI-TOF mass spectrum of M10-5a 32


Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2019

2

Item Description Page




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


42

Figure S46 UV-vis spectra (A) and melting temperature (B) of T9-19 in the


42

Figure S47 UV-vis spectra (A) and melting temperature (B) of M10-3 in the


43



43

Figure S49 UV-vis spectra (A) and melting temperature (B) of M10-5a in the


43



44



44



44

Figure S53 UV-vis spectra (A) and melting temperature (B) of M12-5a in the


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


45

References 46

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.

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.

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

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)



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)



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)



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)



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)



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)



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)



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)


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)


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



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



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



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



of compound 16

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



of compound 17

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



of compound 18

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



of compound 19

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).

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).

26

(A)

(B)



27

(A)

(B)



28

(A)

(B)



29

(A)

(B)



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).

31

(A)

(B)



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).

33

(A)

(B)



34

(A)

(B)



35

(A)

(B)



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).

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

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

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

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 + mismatched


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 + mismatched


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 + mismatched


T9-6 + abasic

(B)


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 + mismatched


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 + mismatched


T9-8 + abasic

(B)


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 + mismatched


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 + mismatched


T9-18 + abasic

(B)


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 + mismatched


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 + abasic

(B)


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 + mismatched


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 + mismatched


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 + mismatched


M10-4 + abasic

(B)


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 + mismatched


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 + mismatched


M10-6 + abasic

(B)


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 + mismatched


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 + mismatched


M10-18 + abasic

(B)


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 + mismatched


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 + mismatched


M10-19 + abasic

(B)


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

References:

1. A. M. Brouwer, Pure Appl. Chem., 2011, 83, 2213.

2. N. Poomsook, T. Vilaivan and K. Siriwong, J. Mol. Graph. Modell., 2018, 84, 36.

3. E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. E. C. Meng

and T. E. Ferrin, J Comput. Chem., 2004, 25, 1605.

4. N. M. O'Boyle, M. Banck, C. A. James, C. Morley, T. Vandermeersch and G. R.

Hutchison, J. Cheminf., 2011, 3, 33.

5. J. Wang, W. Wang, P. A. Kollman, D. A. Case, J. Mol. Graph. Modell., 2006, 25, 247.

6. J. Wang, R. M. Wolf, J. W. Caldwell, P. A. Kollman and D. A. Case, J. Comput. Chem.,

2004, 25, 1157.

7. J. P. Cerón-Carrasco, D. Jacquemin, C. Laurence, A. Planchata, C. Reichardt and K.

Sraïdi, J. Phys. Org. Chem., 2014, 27, 512.

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