Shigeori TakenakaDepartment of Applied Chemistry,

Research Center for Bio-microsensing Technology,

Kyushu Institute of Technology

shige@che.kyutech.ac.jp

International Symposium on Interdisciplinary Education and Research

－Medical Dental and Biotechnological Relationship－

November 11 (Fri), 2016 Kyushu Dental University

Today’s Topics

2

1. Cancer Diagnosis using

Naphthalene diimde

2. Cancer therapy using

Naphthalene diimide

Today’s Topics

3

1. Cancer Diagnosis using

Naphthalene diimde

2. Cancer therapy using

Naphthalene diimide

Voltammogram of ferrocene

Oxi

dat

ion

Curr

ent

Red

uct

ion

Curr

ent

5mA

0 200 400 600

E/mV vs. SCE

1mM Ferrocene

0.1 M LiClO4

in Acetonitrile

- e-+ e-

Fe(II)

Fe(III)

+

N

NO O

O O

N NHN

N N NH

Fe

Fe

O

O

Electrochemical gene detection using FND

DNA probe immobilized electrode

FND

S. Takenaka et al., Anal. Chem., 72, 1334-1341 (2000).

Hybridization with sample DNA

FNDbinding

Current observed according to the amount of hybridized DNA

Citation 435 from Google Scholar at 20016/11/11

Cancer markers

intractable cancer

(pancreatic cancer)

lung cancer, stomach cancer Early diagnosis is

established

Early diagnosis is not

established

Cancer diagnoses 982,100 (2015)

number of death 370,900 (2015)

in Japan

decade National Cancer Center in Japan

Cancer Positiverate

diagnostic accuracy

CEA High Low

PSA Low Low

Ras High Low

P53 Low High

Telomerase High High

Telomerase activity may be used as a good marker for early diagnosis of intractable cancer.

mort

alit

y

6

Telomerase & Telomere DNA

Telomere DNA (Orange parts)

Telomerase : A ribonucleoprotein, synthesizes and directs the telomeric repeats onto the 3’ end. Telomerase activity has been shown to be specifically expressed in cancer cells.

Suggested tetraplex structure

of human telomere

AAUCCCAAUCCCAAUCCC

TTAGGGTTAGGG…

7

TRAP assay as conventional telomerase activity detection

TRAP (Telomeric Repeat Amplification Protocol) assay

Step 1： Addition of Telomeric Repeats by Telomerase

5’-AATCCGTCGAGCAGAGTT ag ggttag ggttag ggttag -3’

5’-AATCCGTCGAGCAGAGTT ag ggttag ggttag ggttag ggttag -3’

TS Primer Telomeric Repeats

TS-Telomerase Product

5’-AATCCGTCGAGCAGAGTT ag ggttag ggttag ggttag ggttag (ggttag)n -3’

Step 2: Amplification of TS-Telomerase Product by PCR

Step 3: PAGE and Data Analysis (6 bp ladder)

Telomerase activity is evaluated from the number of ladders and their intensity

1 h2.5 h

2 h

Detection（5.5 h)

8

Ferrocenylnaphthalene diimide (FND)-based electrochemical telomerase assay

Data analysis

Δi: Current increase ratioI : Before reactioni0 : After reaction

Δi (%) = (i-i0)/i0×100i0

i

This assay enabled simple and quick analysis without PCR and gel electrophoresis

We applied this assay to tongue cancer diagnosis

Detection30 min 1 min

S. Sato et al., Anal. Chem., 77, 7304-7309 (2005).Ferrocenylnaphthalene diimide (FND)

9S. Sato, H. Kondo, T. Nojima, S. Takenaka, Analytical Chemistry, 77, 7304-7309 (2005).

N

N

O O

OO

N

N

Fe

Fe

H

H

Ferrocenylnaphthalene diimide (FND) used in this experiment

N

N

O O

OO

N

NHN

NH

O

O

CH3

CH3

Fe

FeN

N

O O

OO

N

NHN

NH

O

O

CH3

CH3

Fe

Fe N

N

O O

OO

N

NHN

NH

O

O

CH3

CH3

Fe

FeN

N

O O

OO

N

N

CH3

CH3

Fe

Fe

N

N

O O

OO

HN

NH

O

O

Fe

Fe

N N

N N

N

N

O O

OO

HN

NH

O

O

Fe

Fe

N N

N N

N

N

O O

OO

HN

NH

O

O

Fe

Fe

N N

N N

4

N-Methyl-caFc

6

N-Methyl-proFc

3

Piperazine-proFc

5

N-Methyl-acFc

1

Piperazine-caFc

2

Piperazine-acFc

7

N-Methyl-Fc

Different linker chain Different redox potential10

10

Absorption change of FND 7 upon addition of telomere DNA in the presence of Na+

5 mM FND7, 0.1 M AcONa-AcOH(pH5.5), and 0.1 M NaCl at 25 ℃

0

0.05

0.1

0.15

250 300 350 400 450 500

Ab

so

rba

nce

Wavelength/nm

0 mM telomere DNA

15 mM Telomere DNA

Absorption maximum at 384 nm based on FND7 skeleton showed large hypochromic effect and small red shift with isosbestic point upon addition of telomere DNA.

This suggested the single binding mode of FND 7 with telomere DNA.

11

N

N

O O

OO

N

N

Fe

Fe

H

H

Telomere DNA：

5’-aggg ttaggg ttaggg ttaggg-3’11

Scatchard Analysis

3

3.5

4

4.5

5

5.5

6

6.5

7

1.2 1.25 1.3 1.35 1.4 1.45

/L

/ 1

05 M

-1

Fitting equation: /L=K(n-)K: binding constant

n: number of the bound FND

molecules per single telomere DNA

Binding constant

Scatchard analysis was carried out by using absorption change at 384 nm in the FND7 solution upon addition of telomere DNA.

1.5 × 106 M-1

Binding number

1.7

The binding constant of FND with telomere DNA was similar to the ligand reported previously.

12

N

N

O O

OO

N

N

Fe

Fe

H

H

12

Comparison of the binding constant of FND 7 with different DNA structure

0.1 M AcONa-AcOH (pH 5.5) and 0.1 M NaCl at 25 ℃

22-mer oligonucleotide

Binding constant

/10-5M-1

Binding site size

Single stranded DNA5'-CAC CCT ACA CAC CTT CAT CAC C

1.8 2.8

Double stranded DNA5'-CAC CCT ACA CAC CTT CAT CAC C

3'-GTG GGA TGT GTG GAA GTA GTG G

3.1 10.7

Tetraplex DNA5'-AGG GTT AGG GTT AGG GTT AGG G

15.0 1.7

Thermodynamic parameter in the binding of FND with tetraplex DNA

DH=-36.1 kcalmol-1, DS=-92.8 calmol-1K-1

From van’t Hoff plot

10 times

5 times

Entropy control?

13

13

CD spectra

Small increasing peak at 290 nm and large decreasing peak at 260 nm was obtained upon addition of FND7.

Suggestion of the interaction of FND7 with telomere DNA.

0.1 M AcONa-AcOH (pH 5.5) and 0.1 M NaCl

at 25 oC

In Na+

Typical peaks at 240, 260, and 290 nm based on antiparallel tetraplex DNA structure were identified.

-8

-6

-4

-2

0

2

4

6

250 300 350 400

Elli

pticity /

10

6 d

eg

cm

-1 M

-1

Wavelength/nm

0 mM FND

35 mM FND

14

N

N

O O

OO

N

N

Fe

Fe

H

H

14

Binding constant of FNDs with telomere DNA in the presence of Na+ or K+ ion

Buffer FND1 FND2 FND3 FND4 FND5 FND6 FND7

Na+ 10-5K 18.4 15.6 27.9 8.6 10.8 112.6 15.0

n 2.6 3.3 2.5 2.5 2.8 1.9 1.7

K+ 10-5K 7.0 6.6 19.2 8.4 18.6 38.6 5.9

n 3.0 4.1 4.2 1.8 1.8 1.4 2.0

In Na+ ionIn K+ ion

N

N

O O

OO

N

NHN

NH

O

O

CH3

CH3

Fe

Fe

6

All FNDs could bind to telomere DNA with high affinity and their binding number were plurality of FNDs bound to single telomere DNA, especially FND6 showed highest affinity.

15

15

Major?

Telomerase inhibition effect of FND by using TRAP assay

Inhibit conc. FND1 FND2 FND3 FND4 FND5 FND6 FND7

Telomerase/mM 25 25 25 1.0 5.0 5.0 -

PCR/mM - - - 5.0 25 50 -

FND7 does not inhibit the telomerase activity and PCR. FND1-3 only inhibit the telomerase activity. FND4-6 inhibit both reaction.

FND7 was suitable for telomerase assay. FND1-6 might have effective telomerase inhibitor.

16

FND70.5

mM

1.0

mM

5.0

mM

10 m

M

25 m

M

50 m

M

FND1

0.5

mM

1.0

mM

5.0

mM

10 m

M

25 m

M

50 m

M

FND4

0.5

mM

1.0

mM

5.0

mM

10 m

M

25 m

M

50 m

M

16

*TRAP Assay Reaction solution(TRAPeze by Millipore)20 mM Tris-HCl, pH8.3, 1.5 mM MgCl2, 63 mM KCl, 0.05% Tween 20, 1 mM EGTA, 50 μM each dNTP mixture, 1×TS Primer, 1×Primer Mix, 2 Units Taq polymerase, 250 cells Telomerase, 0.5-50 μM FND 1-7

36 bpInternal Control(PCR ascertainment)

Ferrocenylnaphthalene diimide (FND)-based electrochemical telomerase assay (ECTA)

Data analysis

Δi: Current increase ratioI : Before reactioni0 : After reaction

Δi (%) = (i-i0)/i0×100i0

i

This assay enabled simple and quick analysis without PCR and gel electrophoresis

We applied this assay to tongue cancer diagnosis

Detection30 min 1 min

S. Sato et al., Anal. Chem., 77, 7304-7309 (2005).

Ferrocenylnaphthalene

diimide (FND)

17K. Mori et al., Clin. Chem., 59, 289 (2013).

Initial oral cancer

After surgery (resection alone)before surgery

Advancing oral cancer

After surgery (resection ＋ repair operation) QOL decaybefore surgery

hospital charges: ca.

500,000 yen

（surgical cost: ca.

180,000 yen)

length of hospital

stay: ca. 10 days

Five year survival rate

９０％

３０％

Importance of early diagnosis of oral cancer

hospital charges: ca.

3,000,000 yen

（surgical cost: ca.

2,000,000 yen)

length of hospital

stay: over1.5 months

It is very

important to

develop early oral

cancer diagnosis.

① Increasing patient under super-aging society

4992

5939

6873

7842

8678

0

2500

5000

7500

10000

1995 2000 2005 2010 2015 2020

Nu

mb

er

of

pat

ien

t

(年)

男 女 計

Future forecast of oral cancer patient

(出所)新図説耳鼻咽喉科・頭頸部外科講座 第5巻 頭頸部腫瘍（2001年、7頁）より

1.7

tim

es

げ

Present oral caner diagnosis

Inefficient

method

② Early diagnosis required medical specialist

Initial Oral Cancer

Hard to discriminate

Stomatitis

(出所) 厚生労働省：統計情報・白書（2010）

③ Limited number of medical specialist (Doctor of dental surgery)

Limited number

Dental surgery

(4.0%)

Dentistry

(87.6%)

Orthodontic

dentistry (3.5%)Child dentistry

(2.0%)

Clinical training

dentistry (2.1%)

Others

(0.7%)

Present state of oral cancer in Japan

TERT expression in oral cancer cell lines

HSC-2 cells, human oral squamous cell carcinoma cell lines

HSC-3 cells, human tongue squamous cell carcinoma cell lines

Ca9-22 cells, human gingival squamous cell carcinoma cell lines

SAS cells, and human tongue squamous cell carcinoma cells

20

EXPERIMENTAL

1 ) Dissolve samples in 500 μL of lysis buffer, centrifuge.

2) Obtain supernatant of cells.

3) Reverse transcription by Super-Script Reverse TranscriptaseII

(Invitrogen)

4) RT-PCR by FAST SYBR® Green Master Mix

＊Internal control: GAPDH gene

oral cancer cell lines

Higher and lower mRNA concentration for the

hTERT, the catalytic subunit gene of telomerase, is

SAS and Ca9-22 cells.

TRAP assay in oral cancer cell lines

21

Number of

cells SAS Ca9-22 HSC-2 HSC-3

0 - - - -

10 - - - -

50 ＋ - - -

75 ＋ ± ± ±

100 ＋ ± ＋ ＋

200 ＋ ＋ ＋ ＋

SAS

ladder=0, -; 0<ladder<5, ±; ladder≧5, +

Detection limit is over 200 cells

TRAP Assay Reaction solution(TRAPeze by Millipore)

20 mM Tris-HCl, pH8.3, 1.5 mM MgCl2, 63 mM KCl, 0.05%

Tween 20, 1 mM EGTA, 50 μM each dNTP mixture, 1×TS

Primer, 1×Primer Mix, 2 Units Taq polymerase, 0, 10, 50, 75, 200 cells

Telomerase reaction, 37 oC, 60 min

40

60

80

100

140

200

I.S.

M

ECTA in oral cancer cell lines

-S-5’-TTTTTTTTTT AATCCGTCGAGCAGAGTTAGGGTS primer sequenece

Pretreatment of gold electrode: plasma treatment for 30sec

↓

5 nM SS-T8TS1 in 1 mM MCH, 0.1 M NaCl 300 μL, 37 °C, 2 h

↓

i0 measurement (SWV)

0.1 M AcOH-AcOK, 0.1 M KCl (pH 5.5), 20 μM FND

↓

Reaction with cell extract, sample solution 20 μL

[10 ng/μL, 50 mM Tris-HCl (pH 8.0), 1.0 mM MgCl2, 50 mM KCl, 0.10 mM 2-mercaptoethanol,

0.10 M spermidine, 20 μM dNTP mixture]

37 °C, 30 min

↓

i measurement (SWV)

1 min

2 h

1 min

30 min

1 min

Δi (%) = 100×(i – i0)/ i0

SWV: square wave voltammetry 22

ECTA in oral cancer cell lines

23

SAS ○, Ca9-22 ●HSC-2 ×, HSC-3 □100 Ca9-22 cells

Detection limit is 10 cells (P<0.001).

The detection limit of ECTA was five times lower than that

of TRAP.

0

2

4

6

8

10

0 0.1 0.2 0.3 0.4

Curr

ent/

µA

Potential/V vs. Ag/AgCl

(A) (B)

0

10

20

30

40

50

0 50 100 150 200 250

∆i/

%

Number of cells

Correlation of the current increase in ECTA with mRNA for correlation

24

The current increase was found to be proportional to the amount of

mRNA for hTERT in the 10-cell lysate quantified by RT-PCR.

Preparation of clinical samples

Samplesexfoliated buccal cells (B)・・・total oral diagnosis local exfoliated cells (L)・・・local diagnosis

cancer tissue (T)

① Obtain informed consent from all study subjects.

② Collect tissue samples (B, L, T).

③ Dissolve samples in 500 μL of lysis buffer, centrifuge.

④ Obtain supernatant of cells.

⑤ Measure protein concentration in these samples.

⑥ Adjust concentration （TRAP 2000 ng, Electrochemical method 200 ng)．

⑦ Detect telomerase activity by TRAP assay or electrochemical assay.

oral squamous cell cancer：24 sampleshealthy individuals：2 samples

Sample preparation

Number of cells：1×107 cells/500 μL

Medical checkup

Cancer suspect patient

25

Collecting method of exfoliated buccal cells (B)

26

Collecting method of local exfoliated cells (L)

27

0

1

2

3

4

5

6

7

0 0.1 0.2 0.3 0.4 0.5

Cu

rren

t/m

A

Potential/V vs. Ag/AgCl

0

1

2

3

4

5

6

7

0 0.1 0.2 0.3 0.4 0.5

Cu

rren

t/m

A

Potential/V vs. Ag/AgCl

SWV results on local exfoliated cells from cancer patient and healthy volunteer

cancer patient healthy volunteer

Cancer patient: current increased after treatmentHealthy volunteer: current unchanged before and after treatment

Telomerase extension with clinical samples was successful on the electrode by FND-based electrochemical assay

Cu

rren

t/μ

A

6

5

4

3

2

1

0

cancer patientheated sample

0 0.1 0.2 0.3 0.4 0.5

Potential/V vs. Ag/AgCl0 0.1 0.2 0.3 0.4 0.5

Potential/V vs. Ag/AgCl

0 0.1 0.2 0.3 0.4 0.5

Potential/V vs. Ag/AgCl

SWV: square wave voltammetry28

The calculation of the positive rate in ECTA and TRAP

29

0

1

2

3

4

5

6

7

0 0.1 0.2 0.3 0.4 0.5

Cu

rren

t/m

A

Potential/V vs. Ag/AgCl

Classified by Δi

Δi/%＜20% －20%≦Δi/%＜30% ±Δi/%≧30% ＋

Classified by number of ladder

ladder =0 －0＜ ladder ＜5 ±ladder ≧5, ＋

ECTA for clinical samples

30

The change in current

increased more than 30% in

biopsy samples from most

cancer patients, whereas the

increase was 20% or lower in

most healthy individuals.

Cancer Healthy

30％

20％

Telomerase positive

Telomerase negative

BB TT

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1

TP

F

FPF

Positive “+” ： more than19%Negative“-” ： less than 19%

Setting up of cut-off value using ROC analysis

Threshold calculation with ROC analysis from ECTA results

Calculation of sensitivity

and specificity

31

0% 20% 40% 60% 80% 100%

口腔剥離

局所剥離

組織

0% 20% 40% 60% 80% 100%

口腔剥離

局所剥離

組織

TRAP ECTA

Cancer

He

althy

0% 20% 40% 60% 80% 100%

口腔剥離

局所剥離

組織 +

+

+

±

±

±

－

－

－ ＊

+

+

+

±

±

±

－

－

－

0% 20% 40% 60% 80% 100%

口腔剥離

局所剥離

組織

+

+

±

±

±

－

－

－

+

±

±

±

－

－

－

Positive predictive

value

T：50%

L： 45%

B： 14%

Positive predictive

value

T：83%

L： 67%

B： 83%

Negative predictive

value

T：50%

L： 43%

B： 59%

Negative predictive

value

T：83%

L： 68%

B： 81%

ECTA had lower “±” judgment and estimated telomerase activity with more precision.

Calculation of “Positive predictive value using TRAP and ECTA methods

32

T

L

B

T

L

B

T

L

B

T

L

B

Sensitivity SpecificityPositive

predictivevalue

Negativepredictive

value

T 93% 86% 97% 83%

L 83% 82% 83% 82%

B 93% 86% 97% 83%

Calculation of “Positive predictive value” using ECTA methods

ECTA is not only method carrying high sensitivity and

specificity for tissue, but also for exfoliated buccal cells

(B) and local exfoliated cells (L).33

Box plot of ECTA result for cancer patient, healthy volunteer, and Leukoplakia, oral lichen planus as benign adenoma

34

0

20

40

60

80

100

120

140

Di/％

Current increase percentage for benign

closed to that for cancer patient rather than

that for healthy volunteer.

Cancer

Benig

n

Healthy

Cancer

Benig

n

Healthy

Cancer

Benig

n

Healthy

Exfoliated buccal cells (B) Local exfoliated cells (L) Cancer tissue (T)

ConclusionECTA based on FND is more sensitive than

TRAP in terms of detection of cancerous samples taken from tongue cancer patients.

Telomerase activity was detected with exfoliated buccal cells (B) or saliva by ECTA based on FND .

ECTA based on FND will enable early diagnosis of tongue cancer in clinical dentistry and group medical examination.

35

http://takenaka.che.kyutech.ac.jp

K. Mori et al., Clin. Chem., 59, 289 (2013).

M. Hayakawa et al., Electroanalysis, 27, 1159 (2015).

Today’s Topics

36

1. Cancer Diagnosis using

Naphthalene diimde

2. Cancer therapy using

Naphthalene diimide

Concept of new type of anti-cancer drug

Telomeric DNA caused G-quadruplex structure to

inhibit the action of telomerase.

Ligand binding to double-stranded DNA causes a

side effect.

Ligands that strongly bind and stabilize G-quadruplex structure are expected to be

highly specific anticancer agents.

Apoptosis

Fused ring ligand to increase overlap with G-quartet

Porphyrin, perylene diimide, and telomestatin

Increased aromatic ring which can

accommodate with propeller twist of G-quartet

PDC 360A, Phen-DC3, BMVC

Threading intercalator carrying

four substituents

Naphthalene diimide

Telomerase tetraplex DNA binder reported previously

Development of new telomerase inhibitor-Cyclic intercataor-

Double stranded DNATetraplex DNA

cNDI1NDI-DM

Threading

intercalator

capable to

double stranded

DNA

Control

compound

dsDNA

Cyclic naphthalene diimide Tetraplex DNA specific ligand

Design and synthesis of cNDI3, 4 as specific teraplex DNA binder

Change to cyclohexane from benzene unit to enhance affinity for tetraplex

DNA and reduce affinity for double stranded DNA.

Introduction of bulky substituents to disturb intercalation for double stranded

DNA.

cNDI3cNDI4

Interaction analysis of cNDI3, cNDI4 with A-core and dsDNA：UV/Vis spectra measurement under K+

0

0.05

0.1

0.15

0.2

300 350 400 450

Wavelength / nm

Ab

s.

0

0.5

1

1.5

2

2.5

3

0 0.5 1 1.5 2

r/C

r

0

0.05

0.1

0.15

0.2

300 350 400 450

Ab

s.

Wavelength / nm

A-core DNA vs. cNDI3

dsDNA12 vs. cNDI3

A-core DNA vs. cNDI3Scatchard plot

Large

hypochromic shift

was only observed

in cNDI4 upon

addition of A-core!

Binding constant K/ M-1 Selectivity for tetraplex DNA

A-core CT-DNA

NDI-DM 1.4 x 106 3.0 x 105 4.6

cNDI1 3.7 x 106 8.0 x 105 4.6

cNDI2 4.3 x 106 1.4 x 105 31

cNDI3 1.5 x 106 1.3 x 104* 115

cNDI4 6.4 x 106 4.4 x 103* 1600

No hypochromic

shift after addition

of double stranded

DNA

Achievement of

1600-times

selectivity for

tetraplex DNA！

5.0 μM cNDI3

50 mM Tris-HCl (pH 7.4)

100 mM KCl

42

K=8.6x106 M-1

n=1.9

cNDI4 + a-core cNDI4 + HP27

50 mM potassium or sodium

phosphate buffer (pH7.4).

a-core: 5’-AGGG(TTAGGG)3-3’

HP27: 5’- GCG ATT CTC GGC TTT

GCC GAG AAT CGC-3’

Isothermal Titration Calorimetry (ITC) measurements of cNDI4 with tetraplex DNA

and double stranded DNA

TRAP activity was determined with 500 ng of an extract of a TRAPeze positive control cell line. IC50s were determined as

follows: ligand concentration under half telomerase activity with no ligand.

Telomerase inhibition by cNDIs in a TRAP assaycNDI3 NDI-DMcNDI4

IC50=0.5 μM IC50= 3.0μM IC50=3.0 μM

Computer modeling of the complex of cNDI4 or cNDI3 with A-core.

cNDI4 cNDI3

46

IC50/mM

Doxorubicin 1.48

cNDI-3 0.17

cnDI-4 0.28

WST-1 Cell Proliferation Assay (EMT6)

ConclusionCyclic naphthalene diimide, cNDI4 was newly

synthesized aiming at effective anticancer drug.

cNDI4 have the binding affinity of 106 M-1 for tetraplex DNA.

cNDI4 shows 1600-times higher affinity for tetraplex DNA than for double stranded DNA.

cNDI4 inhibits telomerase activity with stabilized the tetraplex DNA of telomere DNA.

47

http://takenaka.che.kyutech.ac.jp

Yugo Esaki et al., Chem. Commun., 50: 5967 (2014).

Md. Monirul Islam et al., Bioorg. & Med.Chem., 23, 4769 (2015).

Md. Monirul Islam et al., Molecules, 20(6), 10963 (2015).

48

Telome-Chyan

Thank for your kind attention !

49

DNA10-5K/M-1 (n)

cNDI1 cNDI2 NDI-DM

a-core (K+) 15 (1.6) 86 (1.9) 16 (1.5)

a-core (Na+) 1.1* 2.2* 1.1*

dsOligo (K+) 0.30* 0.33* 6.0 (2.8)

dsOligo (Na+) - - 6.0 (3.0)

50-

times

2.7-

times

-Binding parameter and effect for Tm-

DNA Tm/˚CΔTm/˚C

1 2 3

a-core (K+) 68 2.0 3.0 6.0

a-core (Na+) 56 1.0 1.3 1.0

dsOligo (K+) 58 0 0 5.0

dsOligo (Na+) 61 0 0 5.0

cNDI1 has 260-times higher affinity for tetraplex DNA than for double stranded one.

Structural stabilization effect of cNDI1, cNDI2 is not observed for double stranded DNA.

Binding affinity of cNDI1 & 2 with a-core or dsDNA12