loop

D7 G-loop

G-loop G

CD

reverse (T1 T2)

CelApt₇₂

CD Figure 4-6 20 °C

CelApt₇₂ T1 265 nm

245 nm G

D8,D10 (Figure 4-6 (c))

20 °C T2 reverse 250 nm 280 nm

DNA

DNA G T1

v

v

v

C A C C A C T

A

A A

G

G G

G GG G

A

A C G T C C

T G C A G G T T T T T T T T T T G

GG G

G

G G G

T

T T T

T T

G T G G T G T G C G G

A T G C C G

5’

3’

G-loop

T1 T2

Figure 4-5 CelApt₇₂ . [Reprinted with permission from Biomacromolecules, 17, 3356-3362 (2016). Copyright 2016 American Chemical Society.]

Figure 4-6 CD . (a) (b) 50 °C (c) 20 °C.

. CelApt72 ( ) T1 ( ) T2 ( ) reverse ( ).

[Reprinted with permission from Biomacromolecules, 17, 3356-3362 (2016). Copyright 2016 American Chemical Society.]

-6 -4 -2 0 2 4 6 8

210 230 250 270 290 310

Ellipticity (mdeg)

Wavelength (nm)

T1 T2 T1 (-) T2 (-)

-8 -6 -4 -2 0 2 4 6 8 10 12

210 230 250 270 290 310

Ellipticity (mdeg)

Wavelength (nm)

(b)

-4 -3 -2 -1 0 1 2 3 4 5 6

210 230 250 270 290 310

Ellipticity (mdeg)

Wavelength (nm)

CelApt Reverse CelApt (-) Reverse (-)

CelApt72

CelApt72 Reverse

Reverse (+) NaCl, MgCl₂ (-) NaCl, MgCl2

T1

T1 T2

T2 (+) NaCl, MgCl2 (-) NaCl, MgCl2

(a)

(c)

- T2 50 °C (100 mM NaCl 5 mM MgCl₂)

CelApt72 T1 265 nm 245 nm

(Figure 4-6 (a) (b)) 50 °C G

reverse G

CelApt72 G

CD Figure 4-7

MgCl₂ NaCl 20 °C 50 °C G

NaCl - (Figure 4-7 (a) (b))

NaCl MgCl2 20 °C 50 °C MgCl2 265

nm 245 nm G Mg²⁺

(Figure 4-7 (c) (d))

G K⁺ Mg²⁺ DNA

Mg²⁺ G

D15 CelApt Ca²⁺ Cranston

D16 CelApt72 Na⁺ Mg²⁺ G

Figure 4-7 CD . (a) (c) 20 °C (b) (d) 50 °C. (a b) NaCl

. (c d) 100 mM NaCl MgCl₂ .

[Reprinted with permission from Biomacromolecules, 17, 3356-3362 (2016). Copyright 2016 American Chemical Society.]

(a)

-8 -6 -4 -2 0 2 4 6 8 10 12

210 230 250 270 290 310

Ellipticity (mdeg)

Wavelength (nm)

0 mM 5 mM 20 mM 100 mM

0 mM MgCl2

20 mM MgCl2 100 mM MgCl₂ 5 mM MgCl₂

(d)

-6 -4 -2 0 2 4 6 8

210 230 250 270 290 310

Ellipticity (mdeg)

Wavelength (nm) 0 mM

5 mM 20 mM 100 mM

0 mM MgCl₂

20 mM MgCl2 100 mM MgCl2 5 mM MgCl2 -6

-4 -2 0 2 4 6 8

210 230 250 270 290 310

Ellipticity (mdeg)

Wavelength (nm) 0 mM

5 mM 20 mM 100 mM

0 mM NaCl

20 mM NaCl 100 mM NaCl 5 mM NaCl

-4 -3 -2 -1 0 1 2 3 4 5

210 230 250 270 290 310

Ellipticity (mdeg)

Wavelength (nm) 0 mM

5 mM 20 mM 100 mM

0 mM NaCl

20 mM NaCl 100 mM NaCl 5 mM NaCl (b)

(c)

-77- 4-3-1-3. CD

G 265 nm CD (Figure

4-8) Tm CelApt72 50% G CD

T_m 40.0 (0 mM MgCl₂) 50.2 (5 mM MgCl₂) 49.9 (20 mM MgCl₂) 53.3 (100 mM MgCl₂)

Mg²⁺ Cel6A_CD 50 °C G 50%

CelApt72 G 50 °C Mg²⁺

50% 5 mM Mg²⁺ G

100 mM NaCl 5 mM MgCl₂

Figure 4-8 CD .

[Reprinted with permission from Biomacromolecules, 17, 3356-3362 (2016). Copyright 2016 American Chemical Society.]

Temperature (˚C)

Ellipticity (mdeg)

0 mM MgCl2

20 mM MgCl2

100 mM MgCl₂ 5 mM MgCl₂

4-3-1-4.

Brekaer SELEX CelApt

CelApt (100 mM NaCl 5 mM MgCl2) (20 mM Tris-HCl, 0.01% (w/v) SDS, pH 7.5)

β-1,4-

(α-1,4-) ^D7 β-1,4- CelApt

SDS DNA CelApt

β-1,4- (Avicel PASC) CelApt

FITC-DNA 50 °C Avicel

PASC CelApt₇₂ T1 T2 reverse CLSM

CLSM Figure 4-9 Figure 4-10 CLSM

FITC-CelApt₇₂

(Figure 4-9) CelApt₇₂ Avicel

PASC reverse

CelApt72

(Figure 4-10) CelApt₇₂ T1 T2

T2 Avicel PASC T1

Avicel PASC (Figure 4-10 (a) (c))

(1) CelApt₇₂ CelApt

(2) T1 G

(3) T1 PASC Avicel

CelApt₇₂ CelApt₇₂

Figure 4-9 CLSM CelApt₇₂ . (a) Avicel (b) PASC.

[Reprinted with permission from Biomacromolecules, 17, 3356-3362 (2016). Copyright 2016 American Chemical Society.]

(a)

Avicel + CelApt₇₂

+ salt

FITC (DNA) Bright field Merge

Avicel + CelApt₇₂

(b)

PASC + CelApt₇₂

+ salt

FITC (DNA) Bright field Merge

PASC + CelApt₇₂

-79-

Figure 4-10 CLSM T1 T2 reverse . (a b) Avicel (c

d) PASC. (a) (c) (b) (d) .

[Reprinted with permission from Biomacromolecules, 17, 3356-3362 (2016). Copyright 2016 American Chemical Society.]

(a) FITC Bright field Merge

Avicel alone

Avicel + reverse

Avicel + T1

Avicel + T2

(b) FITC Bright field Merge

Avicel + T1

Avicel + T2 Avicel alone

Avicel + reverse

PASC + T1

PASC + T2 PASC alone

PASC + reverse PASC

alone

PASC + reverse

PASC + T1

PASC + T2

(c) FITC Bright field Merge (d) FITC Bright field Merge

4-3-1-5.

CelApt₇₂ Figure 4-11 CelApt₇₂

Avicel PASC

Langmuir ^D16 CelApt₇₂

CelApt72

PASC 10 nM Avicel 6 nM CelApt72

Avicel PASC CelApt₇₂

PASC 30 nM Avicel 50 nM PASC

CelApt72

4-3-2.

4-3-2-1. TdT Z-QG-CelApt72

PAGE Figure 4-12 TdT TdT

T1 T2 CelApt₇₂ Z-QG-ddUTP

DNA Z-QG 1:1 Reverse CelApt₇₂ TdT

Z-QG-ddUTP reverse (data is not shown)

Figure 4-11 CelApt₇₂ .

[Reprinted with permission from Biomacromolecules, 17, 3356-3362 (2016). Copyright 2016 American Chemical Society.]

-2 0 2 4 6 8 10 12

0 20 40 60 80

Adsorbed CelApt72 (nM)

CelApt₇₂ (nM)

Avicel salt(+) PASC salt (+) Avicel salt(-) PASC salt(-)

Avicel PASC PASC

(+) NaCl, MgCl₂ Avicel (-) NaCl,

MgCl₂

-81- 4-3-3-2. MTG Z-QG-CelApt72 Cel6ACD

SDS-PAGE Figure 4-13 MTG Z-QG-T1 Z-QG-T2

Z-QG-T1 Z-QG-T2 MTG Cel6ACD

(Figure 4-13 (a)) Z-QG-CelApt72 MTG

Z-QG-CelApt₇₂ T1 T2 MTG

Z-QG DNA T1 T2 42-mer

Z-QG-CelApt72 72-mer DNA MTG Q

500 (bp)

200

1 2 1 M

100

40

T1 T2

- + - + TdT

2 CelApt

- + 1 2 M

Figure 4-12 PAGE TdT . M. 20 bp DNA ladder (Takara Bio).

[Reprinted with permission from Biomacromolecules, 17, 3356-3362 (2016). Copyright 2016 American Chemical Society.]

Figure 4-13 SDS-PAGE MTG . (a) GelGreen (b) . (1) Z-QG-T1 (2) Z-QG-T2 (3) Z-QG-CelApt₇₂ M. 20 bp DNA ladder (Takara Bio).

[Reprinted with permission from Biomacromolecules, 17, 3356-3362 (2016). Copyright 2016 American Chemical Society.]

100 (kDa)

65 41 155

23

M 1 2 3 1 w/o MT

G

w/ MT G

100 (kDa)

65 41 155

23

(a) (b)

2 3 4 M M 1 2 3 1

w/o MT G

w/ MT G

2 3 4 M

CelApt-Cel6A_CD T1-Cel6A_CD or T2-Cel6A_CD Cel6A_CD and MTG

MTG DNA

Z-QG DNA K-tag MTG

30-mer Z-QG-TBA NK14-BAP ^D12 DNA-Cel6A_CD

DNA Cel6A_CD

(Figure 4-13 (b)) Cel6ACD MTG 37 kDa MTG Cel6ACD (35 kDa)

SEC Cel6ACD MTG

SEC Figure 4-14 SEC (1) Cel6A_CD (11 min) (3) MTG (11.5 min)

Cel6A_CD MTG Cel6A_CD (1) MTG (2)

CelApt72-Cel6ACD conjugate (8.8 min) Cel6ACD MTG CelApt72

(2) MTG Cel6A_CD

Reverse

Cel6A_CD (data is not shown)

4-3-3.

Cel6ACD (Cel6A) Cel6A CBM

CBM

Cel6A_CD CelApt₇₂-Cel6A_CD reverse-Cel6A_CD 48

Figure 4-15 (100 mM NaCl

5 mM MgCl2) CelApt72-Cel6ACD 48 Avicel 0.66 mg/mL (Figure 4-15 (a)) PASC

0.31 mg/mL (Figure 4-15 (b)) Cel6A

CelApt₇₂ reverse reverse-Cel6A_CD 48 Cel6A_CD CelApt72

CelApt72-Cel6ACD Cel6A

CelApt₇₂-Cel6A_CD 24 Figure 4-14 SEC . (1) Cel6A_CD (2) CelApt₇₂-Cel6A_CD conjugate (3) MTG.

[Reprinted with permission from Biomacromolecules, 17, 3356-3362 (2016). Copyright 2016 American Chemical Society.]

Time (min) 12 11

8 9 10

5

14 10

15 20

Absorbance at 280 nm (mAU) 7

2

0

13

1 3

-83-

Cel6A Cel6A CelApt₇₂

Figure 4-15 (c) (d)

48 Cel6A_CD

1. 0 Cel6A_CD (1) Cel6A (4) Avicel PASC

Cel6A_CD

Cel6A reverse-Cel6ACD (2)

CelApt₇₂-Cel6A_CD (3) Avicel PASC

Cel6A Cel6A_CD

DNA CBM

(T1 T2)

Figure 4-15 . (a) Avicel (b) PASC. 48

. (c) Avicel (d) PASC. ( ) ( ). (1) Cel6A_CD (2)

reverse-Cel6A_CD conjugate (3) CelApt₇₂-Cel6A_CD conjugate (4) Cel6A.

[Reprinted with permission from Biomacromolecules, 17, 3356-3362 (2016). Copyright 2016 American Chemical Society.]

(a)

0 0.2 0.4 0.6 0.8

0 12 24 36 48

Reducing sugars (mg/mL)

Reaction time (h)

Free CD alone CelApt-CD Reverse-CD CD-CBM CelApt-CD Reverse-CD

Free Cel6A_CD alone CelApt₇₂-Cel6A_CD Reverse-Cel6A_CD Cel6A

CelApt₇₂-Cel6A_CD Reverse-Cel6A_CD

(+) NaCl, MgCl₂

(-) NaCl, MgCl₂

(b)

0 0.1 0.2 0.3 0.4

0 12 24 36 48

Reducing sugars (mg/mL)

Reaction time (h)

Free CD alone CelApt-CD Reverse-CD CD-CBM CelApt-CD Reverse-CD

0 1 2 3

1 2 3 4

Relative activity (-)

0 1 2 3 4 5

1 2 3 4

Relative activity (-)

(c) (d)

Free Cel6A_CD alone CelApt₇₂-Cel6A_CD Reverse-Cel6A_CD Cel6A

CelApt₇₂-Cel6A_CD Reverse-Cel6A_CD

(+) NaCl, MgCl₂

(-) NaCl, MgCl₂

Cel6A_CD

alone

Reverse-Cel6A_CD CelApt₇₂

-Cel6A_CD Cel6A Cel6A_CD

alone

Reverse-Cel6A_CD CelApt₇₂

-Cel6A_CD Cel6A

T1-Cel6A_CD T2-Cel6A_CD

(Figure 4-16 (a) (b)) Avicel PASC

T1-Cel6A_CD T2-Cel6A_CD Cel6A_CD CelApt₇₂-Cel6A_CD Cel6A

CelApt₇₂ G

- CelApt72-Cel6ACD

Cel6A

Mfold CD

Cranston Ca²⁺ DNA

D16 DNA

CBM (

)

CelApt₇₂-Cel6A_CD Cel6A

Cel6ACD Avicel 3.0 PASC 4.5

CelApt₇₂ CBM PASC

CelApt72

CelApt β-1,4-

α-1,4-β-1,4- ^D7 CelApt

Figure 4-16 48 . (A) Avicel (B) PASC (C) CMC.

( ) ( ). (A B) (1) Cel6A_CD (2) T1-Cel6A_CD conjugate (3) T2-Cel6A_CD conjugate (4) Cel6A. (C) (1) Cel6A_CD (2) T1-Cel6A_CD conjugate (3) T2-Cel6A_CD

conjugate (4) reverse-Cel6A_CD conjugate (5) CelApt₇₂-Cel6A_CD conjugate (6) Cel6A.

[Reprinted with permission from Biomacromolecules, 17, 3356-3362 (2016). Copyright 2016 American Chemical Society.]

(a)

0 0.5 1 1.5 2 2.5 3 3.5

1 2 3 4

Relative activity (-)

0 1 2 3 4 5

1 2 3 4

Relative activity (-)

0 0.5 1 1.5 2

1 2 3 4 5 6

Relative activity (-)

(b)

(c)

-85-

β-1,4-CelApt₇₂ Avicel PASC

PASC Avicel CelApt

CelApt72 Figure 4-15 (a)

24

CelApt₇₂ CBM

CelApt₇₂ G

-induced-fit CBM

DNA CBM

CelApt₇₂ CBM ( ^D17

D18) CBM

CelApt₇₂ CelApt₇₂-Cel6A_CD

CBM

CMC Cel6A Cel6A_CD

(Figure 4-17)

DNA

D19

4-4.

(CBM)

CBM DNA (CelApt)

CelApt- DNA

CelApt-Cel6A_CD Cel6A_CD 50 °C

50 °C DNA

(Mfold) Breaker CelApt G-C T10

CelApt₇₂ CD (100 mM NaCl 5 mM

MgCl₂) CelApt 50 °C 50% G Na⁺

- Mg²⁺ G CelApt72

Avicel

PASC CelApt₇₂

CelApt₇₂ Cel6A_CD CelApt₇₂-Cel6A_CD

SEC Cel6ACD CelApt72

CelApt₇₂-Cel6A_CD

CelApt72-Cel6ACD Cel6ACD 4.5 DNA

-87- 4-5.

D1. D. Sen and L. C. Poon, Crit. Rev. Biochem. Mol. Biol., 46, 478-492 (2011).

D2. E. Golub, H. B. Albada, W. -C. Liao, Y. Biniuri and I. Willner, J. Am. Chem. Soc., 138, 164-172 (2016).

D3. M. Dashtban, H. Schraft and W. Qin, Int. J. Biol. Sci., 5, 578-595 (2009).

D4. C. M. Payne, B. C. Knott, H. B. Mayes, H. Hansson, M. E. Himmel, M. Sandgren, J. Stah̊lberg and G. T.

Beckham, Chem. Rev., 115, 1308-1448 (2015).

D5. L. Tavagnacco, P. E. Mason, U. Schnupf, F. Pitici, L. Zhong, M. E. Himmel, M. Crowley, A. Cesarò and J.

W. Brady, Carbohydr. Res., 346, 839-846 (2011).

D6. H. J.Gilbert, J. P. Knox and A. B. Boraston, Curr. Opin. Struct. Biol., 23, 669-677 (2013).

D7. B. J. Boese and R. R. Breaker, Nucleic Acids Res., 35, 6378-6388 (2007).

D8. Y. Nonaka, K. Sode and K. Ikebukuro, Molecules, 15, 215-225 (2010).

D9. H. Fujita, Y. Imaizumi, Y. Kasahara, S. Kitadume, H. Ozaki, M. Kuwahara and N. Sugimoto, Pharmaceuticals, 6, 1082-1093 (2013).

D10. R. Ueki and S. Sando, Chem. Commun., 50, 13131-13134 (2014).

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DNA

DNA-DNA DNA

-DNA

-TdT MTG BAP 3´- DNA

-3´- Z-QG-dUTP/ddUTP TdT

Z-QG-aptamer ddUTP

Z-QG-dUTP Q

Z-QG-dUTP Q NK14-BAP MTG

BAP-aptamer

MTG [NK14-BAP]/[Z-QG-DNA aptamer] = 2.5

[NK14-BAP]/[(Z-QG)_m-DNA aptamer] = 5

BAP-aptamer ELAA

BAP-aptamer 0.61 nM (BAP)n-aptamer 0.12 nM

TdT MTG (1)

(2) TdT

TdT

-Z-QG MTG

3´- DNA -( )_n

TdT

DNA -( )_n

TdT 3´- Z-QG-dUTP (dNTP)

TdT (Z-QG)_m-(dN)_l-aptamer

dATP dTTP

-89-

(Z-QG)_m-(dW)_l-aptamer Z-QG

MTG

(dW) (BAP)_m-(dW)_l-aptamer

(Z-QG)_m-(dT)_l-aptamer MTG NK14-BAP

BAP (BAP)m-(dT)l-aptamer

EC₅₀ BAP-aptamer (BAP)_n-(dT)_l-aptamer

dNTP TdT (1) dTTP Z-QG-dUTP (25%)

Z-QG (Z-QG)_m-(dT)_l-aptamer

(2) (Z-QG)m-(dT)l-aptamer MTG BAP (3)

(BAP)_n-(dT)_l-aptamer

(CBM)

CBM DNA (CelApt)

CelApt- DNA

CelApt-Cel6ACD Cel6ACD 50 °C

50 °C DNA

(Mfold) Breaker CelApt G-C T10

CelApt₇₂ CD (100 mM NaCl 5 mM

MgCl₂) CelApt 50 °C 50% G Na⁺

- Mg²⁺ G CelApt₇₂

Avicel

PASC CelApt₇₂

CelApt₇₂ Cel6A_CD CelApt₇₂-Cel6A_CD

SEC Cel6ACD CelApt72

CelApt₇₂-Cel6A_CD

CelApt₇₂-Cel6A_CD Cel6A_CD 4.5

DNA

TdT MTG

TdT

DNA-DNA Z-QG

DNA MTG

2013 - (antibody-drug conjugate; ADC)

ADC

2013

ADC T-DM1 FDA 2016 50

ADC ADC

(Fc) -Fc

Fc

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