BAP-aptamer conjugates - 固液界面で機能する核酸－酵素コンジュゲートの設計とその高度利用

-41- 2-3-4. BAP -aptamer

BAP-aptamer ELAA (Figure 2-17 (a))

100 nM 50 nM BAP-aptamer 1.0

BAP-aptamer

-BAP Figure 2-17 (b)

lane 5 lane 1-3

(BSA) ELAA

(lane 4) BAP-aptamer

(lane 5)

Figure 2-16 MTG . (a) FITC- (b)

NBT/BCIP . (1) Z-QG-aptamer (2) Z-QG-TEO-aptamer (3) (Z-QG)m-aptamer (4) (Z-QG-TEO)m-aptamer. M: FITC-labeled φX174 DNA-HaeIII Digest.

[Reprinted from J. Biosci. Bioeng., 116, M. Takahara, H. Hayashi, M. Goto and N. Kamiya, Tailing DNA aptamers with a functional protein by two-step enzymatic reaction, 660-665, Copyright 2013, with permission from Elsevier.]

(bp)

872 310

M

(a)

1 2 3 4 5 M 6 7 8 9

Single labeling Multiple labeling

Unconjugated BAP

BAP-aptamer

(BAP)_n-aptamer BAP-aptamer 4 BAP

(0-100 nM) ELAA BAP-aptamer

Figure 2-18 BAP-aptamer

BAP-aptamer (BAP)n-aptamer 1 nM

25 nM 1-25 nM

BAP

(DL)

3 BAP-aptamer 0.347

± 0.0273 DL 0.61 nM (BAP)n-aptamer DL 0.12 nM

Figure 2-17 ELAA . (a) ELAA (b) BAP . (1) BAP-aptamer

(2) (3) (4) BSA

(5) (a) . BAP-aptamer ( ) (BAP)_m-aptamer ( )

BAP-aptamer (5) 1.0 ANOVA

(*p <0.05).

Substrate

Product Thrombin

BAP-aptamer conjugate

Streptavidin Biotin

(a) (b)

*

-43- //

Figure 2-18 BAP-aptamer (0-100 nM) .

BAP-aptamer 100 nM 1.0 . BAP-aptamer (○)

BAP-TEO-aptamer ( ) (BAP)m-aptamer (●).

Figure 2-19 BAP-TEO-aptamer (0-100 nM) .

BAP-aptamer 100 nM 1.0 . BAP-TEO-aptamer ( )

(BAP-TEO)m-aptamer (▲).

BAP-TEO-aptamer (Figure 2-19) TEO

BAP-TEO-aptamer 1 - 25 nM DL 0.36 nM BAP-aptamer

(BAP-TEO)n-aptamer 1-100 nM DL 0.65 nM (BAP-TEO)n-aptamer BAP (BAP)n-aptamer

2-4.

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

-45- 2-5.

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-47- 3-1.

3-1-1. DNA

Tang

HRP (CEA)

C1 (BAP)_n-aptamer

BAP-aptamer ^C2

DNA

DNA Kitaoka DNA-( )_n

DIG (Figure 3-1 (a))^C3 Mori DNA-(

)_n 5.7 (Figure 3-1 (b))^C4

DNA

DNA PCR

DNA DNA

DNA: 1:1 ^C5

-C6, C7 (SA)

SA DNA-

3´-Figure 3-1 MTG DNA-( )n .

(a) DNA (b) DNA

NH O

O N O OH O P O

O -O NH HN NH O

O NH2 O O

P O

O -O P O O --O

Z-QG-dUTP

O NH₂ NH2 O

NH2 O

O NH₂

PCR

w/dNTPs ^O ^NH

N O H

O NH N O H

(a) (b)

NH O

N O H

Z-QG-labeled DNA

DNA-(enzyme)_n conjugate MTG-mediated

cross-linking

NH2

K-tagged enzyme Z-QG

incorporation

3-1-2.

TdT MTG (1)

(2) TdT

DNA Z-QG

Z-QG-dUTP TdT (dNTP)

Z-QG-dUTP TdT

Z-QG-dUTP Z-QG

MTG

TdT dNTP Z-QG-dUTP

Z-QG-dUTP dNTP (Z-QG)m-(dN)l-aptamer MTG

NK14-BAP (Figure 3-2)

(ELAA)

Z-QG-dUTP:dNTP TdT DNA

Z-QG (Z-QG)m-aptamer BAP

ELAA 29-mer

15-mer (Table 3-1)

T20 TEG linker

O NH NH O

NH O

NH2

Site-specific cross-linking

by MTG Z-QG-dUTP (25%)

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

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

K-tagged BAP (K-tag: MKHKGS)

Z-QG-dU and dN tailing by TdT

dTTP (75%)

Tail-specific assembly of multiple enzymes on single-strand DNA

DNA aptamer sequence Tail-specific enzyme addition

5’ 3’-OH

NH O

O N O OH O P O

O -O

NH HN NH O

O NH2 O O

P O

O -O P O O --O

NH O

O N O OH O P O

O -O P O

O -O P O O --O

O NH2 NH₂ O

NH₂ O

Figure 3-2 3´- .

-49-

Table 3-1 .

Abbreviation DNA sequence

Biotin-TEG-TBA

(29-mer, primary aptamer)

5´-biotin-TEG-AGTCCGTGGTAGGGCAGGTTGGGGTGAC -3´

FITC-TBA

(30-mer, secondary aptamer)

5´-FITC-GGTTGGTGTGGTTGGTTTTTTTTTTTTTTT-3´

*Underlined sequences were thrombin binding aptamers (abbreviated as TBA).

3-2.

3-2-1.

(dNTP) Roche Applied Science Penicillium citrinum

P1 Crotalus adamanteus venom I Tween20 Sigma-Aldrich

(CIAP)

FITC-TBA biotin-TEG-TBA

NK14-BAP His-tag (Hi Tranp^TMDEAE FF

GE ) PD-10 (GE ) 10 mM Tris-HCl (PH 8.0)

(Molecular Imager FX Pro Bio-Rad) FITC (

488 nm 530± 15 nm ) ECF LS-55 Fluorescence

Spectrometer (PerkinElmer) 430 nm 560 nm 10 nm

3-2-2. TdT Z-QG-aptamer 3-2-2-1. (Z-QG)m-(dN)l-aptamer

TdT FITC-TBA (5 µM) Z-QG-dUTP (0.5 mM) dNTP (1.5 mM) 5 mM CoCl2 TdT (20 U/µL) TdT (200 mM potassium cacodylate 25 mM Tris-HCl 0.25 mg/mL BSA pH 6.6 50 µL)

37 °C 1 94 °C 15 TdT

Probe Quant G-50 spin column

15 % PAGE Z-QG-aptamer

96 LS-55 fluorescence spectrometer FITC-TBA

( 495 nm 520 nm 10 nm)

TdT 1 µL 5 µL 15 %

PAGE (1×TBE buffer 7 M Urea 280 V 60 min) TdT 5´-FITC

TdT (Z-QG)_m-(dT)_l-aptamer TdT 20 µL

37 °C 3 5 10 30 60 120 180 1 µL

0.5 mM EDTA 1 µL 15 % PAGE

3-2-2-2. (Z-QG)m(X%)-(dT)l-aptamer

TdT FITC-TBA (5 µM) Z-QG-dUTP (0. 5 mM) dTTP (1.5 mM) 5 mM CoCl₂ TdT (20 U/µL)

TdT (50 µL) Z-QG-dUTP X% (X%: 16.6

20 25 33.3 50 66.6 100) 37 °C 1 X% (16.6-100)

[Z-QG-dUTP]/[dTTP] = 1:5 1:4 1:3 1:2 1:1 2:1 1:0 94 °C 15

TdT Probe Quant G-50 spin column (GE )

15 % PAGE

96 LS-55 fluorescence spectrometer

FITC-TBA ( : 495 nm : 520 nm : 10

nm)

TdT 1 µL 5 µL 15 %

PAGE (1×TBE buffer 7 M Urea 280 V 60 min) TdT 5´-FITC

3-2-2-3. Z-QG-dUTP

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

—

(LC-ESI-MS) ^{C3, C8} Z-QG-dU

TdT (Z-QG)m-(dN)l-aptamer QIA Quick Nucleotide Removal Kit (Qiagen)

50 µL MV-100 ( )

(50 µL 100 mM pH 4.5) (Z-QG)_m-(dN)_l-aptamer (2 - 20 µg) P1 (1U)

37 °C 3 3´-5´- I (0.01 U)

(100 µL 100 mM pH 9.5) 37 °C 2

CIAP (100 U) 37 °C 1 TdT

dATP dGTP dCTP dTTP CIAP

2´-deoxyadenosine (dA) 2´-deoxyguanosine (dG) 2´-deoxythymidine (dT) 2´-deoxycytidine (dC) 5´-Z-QG-deoxyuridylate (Z-QG-dU)

(Z-QG)_m-(dN)_l-aptamer 20 µL (0.4–5 µg) LC-ESI-MS (

) HPLC

(SPD-M20A ) ESI- (microTOF QIII Bruker Daltonics)

HPLC 2.0 × 150 mm COSMOSIL 5C18-AR-II ( ) Nexera X2 (

) 30 °C 10 mM (pH 6.7)/ (95/5 (v/v))

10 100% 10 mM (pH 6.7)/

30 50% 300 µL/min

ESI endplate offset (500 V) capillary (4.5 kV) nebulizer (1.6 bar) dry gas (8.0 L/min) dry temperature

(180°C) 190–400 nm 50-3000 m/z

ESI 112 (dC) 127 (dT) 152 (dG) 252 (dA) 620.23 (Z-QG-dU) Z-QG-dU

dN (1–500 µM)

-51-

Z-QG-dU (1–200 µM) LC-ESI-MS dN Z-QG-dU

(N = 8[dN] /[dG]) (N)

FITC-TBA 21-nt dT 8-nt dG ( dG FITC )

dN [dG]

3-2-3. MTG (Z-QG)m-(dN)l-aptamer NK14-BAP 3-2-3-1. MTG

MTG [BAP]/[(Z-QG)_m-(dN)_l-aptamer] (Z-QG)_m-(dN)_l-aptamer MTG (Z-QG)_m-(dN)_l-aptamer (0.5 µM) NK14-BAP (0–10 µM) MTG (0.1 U/mL) 20 mM Tri-HCl (pH 7.4) [NK14-BAP]/[ (Z-QG)m-(dN)l-aptamer r] = 0 1 1.25 2 2.5 5 10 20 (Z-QG)_m-(dN)_l-aptamer (0.25 µM) NK14-BAP (12.5 µM

25 µM) MTG (0.1 U/mL) 20 mM Tri-HCl (pH 7.4)

[NK14-BAP]/[(Z-QG)_m-(dN)_l-aptamer] = 50 100 MTG 4 °C 3 1 mM N-ethylmaleimide (NEM)

10 µL 2.0 % (1×TBE 135 V 20 ) MTG

5´-FITC

3-2-3-2. (BAP)n-(dN)l-aptamer

MTG NK14-BAP [NK14-BAP]/[ (Z-QG)m-(dA)l-aptamer] = 20 [NK14-BAP]/[ (Z-QG)_m-(dT)_l-aptamer] = 50

(Z-QG)m (X%)-(dT)l-aptamer [NK14-BAP]/[ (Z-QG)_m-(dT)_l-aptamer] = 50

MTG

MTG (Z-QG)m-(dA)l-aptamer (0.5 µM) NK14-BAP (10

µM) (Z-QG)_m-(dT)_l-aptamer (0.5 µM) NK14-BAP (25 µM) MTG (0.1 U/mL) 20 mM Tris-HCl

(pH 7.4) 4 °C 3 1 mM NEM

MTG 10 µL 2.0 % (1×TBE 135 V 20

)

5´-FITC NBT/BCIP (p-nitroblue tetrazolium

chloride/5-bromo-4-chloro-3-indolyl phosphate) NK14-BAP NBT/BCIP NTM 37 °C 40

3-2-3-3. BAP

(BAP)_n-(dN)_l-aptamer BAP (SEC)

400 µL MTG 30 kDa ( ) 50

µL BAP-aptamer (BAP)n-aptamer

5 µM NK14-BAP 1 U/mL MTG Nexera X2 series LC ( ) 40 µL 2.0 mm × 30 cm TSKgel SuperSW 3000 column ( )

30 °C 100 mM NaCl 5 mM Tris-HCl (pH 7.5) 65 µL/min

280 nm SEC Gel Filtration LMW

Calibration Kit (GE ) blue

dextran (>200 MDa 1 mg/mL) aldorase (158 kDa 0.3 mg/mL) ovalbumin (43 kDa 3.0 mg/mL) carbonic anhydrase (29 kDa 1.5 mg/mL) ribonuclease A (13.7 kDa 3.0 mg/mL)

3-2-4. BAP-aptamer 3-2-4-1.

MTG NK14-BAP BAP

NK14-BAP NK14-BAP

2% BSA 1-5%

37 °C 1 1 µM NK14-BAP (100 µM/well) 37 °C 1

1 µM NK14-BAP (100 µM/well) 37 °C 1

BAP ECF NTM 0.2 mM

100 µL/well NK14-BAP

(37 °C 20 )

3-2-4-2.

NK14-BAP 1%

ELAA MTG (BAP)n-(dW)l-aptamer

BAP-aptamer (BAP)_n-aptamer

(SA )

ELAA

TBS (biotin-TEG-TBA 200 nM) 100 µL/well

- (37 °C 1 )

TBS (0-100 nM) 100 µL/well

(37 °C 1 )

TBS (1% ) ((BAP)_n-(dN)_l-aptamer 50 nM)

100 µL/well (37 °C 1

)

BAP ECF NTM 0.2 mM 100 µL/well

BAP-aptamer (37 °C 30 )

LS-55 Fluorescence Spectrometer ECF ( 430 nm 560 nm)

( TBST (0.05 v/v %

Tween 20 TBS) 300 µL/well 3

-53-

- 4

KaleidaGraph^TM ^C9,C10 IUPAC

3-3.

3-3-1. TdT Z-QG-aptamer 3-3-1-1. TdT

PAGE Figure 3-3

Z-QG-dUTP (25%)/dTTP (Z-QG)m-(dT)l-aptamer 60

120

872 bp

603 bp (Z-QG)m–

aptamer ( Figure 2-13) TdT

3-3-1-2. (Z-QG)m-(dN)l-aptamer TdT

DNA 3´-OH TdT

Mg²⁺ (dATP dGTP) Co²⁺

(dTTP dCTP) ^C11 Co²⁺

dCTP dTTP

TdT TdT

C12 dN

Z-QG dNTP Z-QG-dUTP

TdT

Figure 3-4 TdT PAGE Z-QG-aptamer

(lane 2) (Z-QG)_m-aptamer (lane 3) Figure 3-4 lane 2 Z-QG lane 3-7 (TdT )

(lane 1) Z-QG

dT>dA>dC>dG>Z-QG-dUTP >Z-QG-ddUTP (dN

) dNTP TdT

dGTP dWTP (dWTP: dATP or dTTP)

(dG)_l TBA G TdT 3´-OH

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

Kessier dATP

C13 dTTP Co²⁺

310 872

5 10

72 118

0 (bp)

Time (min) 120 180

Figure 3-3 TdT

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

FITC-labeled φX174 DNA-HaeIII

dTTP dATP dCTP

(Z-QG)_m-(dW)_l-aptamer (dW: dA or dT)

(Z-QG)m-(dW)l-aptamer (Z-QG)m-aptamer Z-QG BAP

3-3-1-2. (Z-QG)m(X%)-(dT)l-aptamer

Figure 3-5 TdT PAGE Z-QG-dUTP (0.5 mM) dTTP

(lane 1-7) dTTP

Figure 3-4 PAGE TdT . (1) FITC-TBA (2) Z-QG-ddUTP (3)

Z-QG-dUTP (4) +dATP (5) +dTTP (6) +dGTP (7) dCTP. M. FITC-labeled φX174 DNA-HaeIII Digest.

(bp)

72 603

1 2 3 4 5 M

Z-QG-dUTP mixed 1:3 with dNTP

118 310

6 7 872

Z-QG-dUTP (X%) mixed with dTTP (bp)

72 872

1 2 3 4 5 M

118 310

6 7 8 M

Figure 3-5 PAGE TdT . (Z-QG)m-(dT)l(X%)-aptamer X = (2) 100 (3) 66.6 (4) 50 (5) 33.3 (6) 25 (7) 20 (8) 16.6. X% = 100-16.6. M. FITC-labeled φX174 DNA-HaeIII Digest. [From Biotechnol. J., 11, 814-823 (2016). Copyright © 2016 by John Wiley Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc.]

-55-

Z-QG lane 7 lane 8

TdT X = 20% X = 20% 16.6%

Z-QG-dUTP

3-3-1-3. Z-QG PAGE

LC-ESI-MS Z-QG-dU dW

LC-ESI-MS Figure 3-6

Figure 3-7 Figure 3-6 (a) dC 2.3 dG 4.9 dT 6.4 dA 12.3

Z-QG-dU 19.6 HPLC

MS (Figure 3-6 (c))

(Figure 3-7) 1-500 µM R²> 0.99

(Z-QG)m-(dW)l-aptamer

(Z-QG)_m-aptamer (Figure 3-6 (b)) Z-QG

Figure 3-6 HPLC . (a) 260 nm ( ) 300 nm (b)

MS (c) MS .

Template aptamer Z-QG-dU (100%)

Z-QG-dU (25%)/dT (75%) Z-QG-dU (25%)/dA(75%)

Z-QG-dU (25%)/dC (75%) Z-QG-dU (25%)/dG (75%)

Standard mixture

dC dG dT dA Z-QG-dU

0 4.9 6.4 12.3 19.6

t (min)

0 4.9 6.4 12.3 19.6

t (min)

dC dG dT dA Z-QG-dU

(a)

Template aptamer Z-QG-dU (100%)

Z-QG-dU (25%)/dT (75%) Z-QG-dU (25%)/dA (75%)

Z-QG-dU (25%)/dC (75%) Z-QG-dU (25%)/dG (75%)

Standard mixture

0 4.9 12.3 19.6

t (min) 6.4

dC dG dA

Z-QG-dU

5 µM dN and 10 µM Z-QG-dU

0 4.9 12.3 19.6

t (min) 6.4

dC dG

Z-QG-dU

10 µM dN and 20 µM Z-QG-dU 50 µM dN and 40 µM Z-QG-dU

100 µM dN and 60 µM Z-QG-dU 200 µM dN and 80 µM Z-QG-dU

500 µM dN and 100 µM Z-QG-dU 1 mM dN and 120 µM Z-QG-dU

(b) (c)

Figure 3-8 Z-QG dW (Z-QG)_m-(dW)_l-aptamer

100 Z-QG-dUTP 100 Z-QG-dUTP TdT

Z-QG Z-QG dT>dA>Z-QG-dU

PAGE (Z-QG)m-(dW)l-aptamer

(Z-QG)_m-aptamer Z-QG Z-QG

dW TdT (Z-QG)m-(dW)l-aptamer

m:l = 1:3 Z-QG-dU dW TdT

Figure 3-7 . (a) dC (b) dG (c) dT (d) dA (e) Z-QG-dU.

y = 1102.8x + 48604 R² = 0.99138

0 100000 200000 300000 400000 500000 600000 700000

0 100 200 300 400 500

Peak area (-)

dC concentration (µM)

y = 2476.8x + 42856 R² = 0.99057

0 200000 400000 600000 800000 1000000 1200000 1400000

0 100 200 300 400 500

Peak area (-)

dG concentration (µM)

y = 2330.8x + 15677 R² = 0.99889

0 200000 400000 600000 800000 1000000 1200000 1400000

0 100 200 300 400 500 600

Peak area (-)

dT concentration (µM)

y = 6800.8x + 325786 R² = 0.99198

0 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000

0 100 200 300 400 500

Peak area (-)

dA concentration (µM)

y = 5476.2x + 30140 R² = 0.99334

0 200000 400000 600000 800000 1000000 1200000

0 50 100 150 200

Peak area (-)

Z-QG-dU concentration (µM)

(a) (b)

(c) (d)

(e)

-57-

3-3-2. MTG (Z-QG)m-(dN)l-aptamer NK14-BAP 3-3-3-1. MTG [BAP]/[DNA aptamer]

MTG [NK14-BAP]/[ (Z-QG)m-(dW)l-aptamer]

Figure 3-9 (Z-QG)_m-(dW)_l-aptamer BAP

(Z-QG)m-(dA)l-aptamer [NK14-BAP]/[(Z-QG)m-(dW)l-aptamer] = 20 (lane8) (Figure 3-9 (a)) (Z-QG)_m-(dT)_l-aptamer [NK14-BAP]/[aptamer] = 50 (lane 4)

(Figure 3-9 (b)) [NK14-BAP]/[ (Z-QG)_m-(dA)_l-aptamer] = 20 [NK14-BAP]/[ (Z-QG)_m-(dT)_l-aptamer] = 50 MTG

295

239 71

101 62

0 100 200 300 400

dATP dTTP Z-QG TBA

The relative number of each base/chain dG dT dA Z-QG-dU Template TBA

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

Figure 3-8 LC-ESI-MS Z-QG-dU dN

. dG .

Figure 3-9 MTG . (a) [NK14-BAP]/[Z-QG-aptamer] =

(1) 0 (2) 1 (3) 1.25 (4) 2 (5) 2.5 (6) 5 (7) 10 (8) 20. (b) [NK14-BAP]/[Z-QG-aptamer] = (1) 0 (2) 10 (3) 20 (4) 50 (5) 1000. M: FITC-labeled φX174 DNA-HaeIII Digest.

(bp)

310 603

1 2 3 4 5 6

M 7 8 M 1 2 3 4 5 6 7 8

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

(a) (b)

310 603 (bp)

M 2 3 4 5

1 M 1 2 3 4 5

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

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

3-3-3-2. (BAP)n-(dN)l-aptamer

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

BAP-aptamer Figure 3-10

TdT

lane 1 lane 2-6 lane 2-6 lane 1 BAP

BAP

NBT/BCIP (Figure 3-10 (b)) BAP

3-3-3-3. (BAP)n(X%)-(dT)l-aptamer

(BAP)_n (X%)-(dT)_l-aptamer Figure 3-11

MTG TdT

Figure 3-10 MTG . (A) FITC- (B)

NBT/BCIP . (1) Z-QG-aptamer (2) (Z-QG)_m-aptamer (3) (Z-QG)_m-(dT)_l-aptamer (4) (Z-QG)_m-(dA)_l-aptamer. M: FITC-labeled φX174 DNA-HaeIII Digest.

w/o MT G

with MTG

w/o MT G

with MTG

BAP-aptamer conjugates

Unconjugated BAP BAP-aptamer

conjugates 1 2 3 4 M 1 2 3 4

M 1 2 3 4 M 1 2 3 4

(a) (b)

Loading dye band (bp)

872

310 603

Figure 3-11 MTG . (a) FITC . (b)

NBT/BCIP . (Z-QG)_m-(dT)_l(X%)-aptamer X = (1) 16.6 (2) 20 (3) 25 (4) 33.3 (5) 50 (6) 66.6 (7) 100. X% = 100-16.6. M. FITC-labeled φX174 DNA-HaeIII Digest.

Unconjugated BAP BAP-aptamer

conjugates 1 2 3 4 5 6 7 1 2 3 4 5 6 7

w/o MT G

with MTG (bp)

310 603

1 2 3 4 5 M

w/o MT G

6 7 M 1 2 3 4 5 6 7 with MTG

872

Loading dye band

(a) (b)

-59-

MTG X = 20 25 (lane 2

3) Z-QG-dUTP (X%

) TdT

X% Z-QG-dUTP X = 20 25

Z-QG-dU MTG

3-3-3-4. BAP

Figure 3-12 K_av

K_av = (K_E-V₀)/(V_c-V₀)

V_E: V₀ (Blue Dextran V_E) V_c: (0.942 mL)

K_av Table 3-2 Figure 3-12 (b) (R²>

0.99)

Figure 3-12 (a) (b) .

Ribonuclease 13.7 kDa Carbonic

Anhydrase 29 kDa Ovalbumin

43 kDa Aldolase

158 kDa Blue Dextran

>200 MDa

Time (min)

4 5 6 7 8 9 10 11 12 13 14

Absorbance at 280 nm (mAU)

(a)

(b)

0 50 100 150 200 250

y = -0.218ln(x) + 1.2376 R² = 0.99718

0 0.2 0.4 0.6 0.8

5 50 500

Kav (-)

Molecular mass (kDa)

Figure 3-13 SEC BAP (4) (9.8 )

(1-3) 6-9 BAP DNA

(BAP)_n-(dW)_l-aptamer (2, 3)

dW MTG

(BAP)_n-(dW)_l-aptamer Z-QG NK14-BAP

BAP

(BAP)n-(dW)l-aptamers 90.8 251.8 kDa BAP

41.4 kDa (n) n = 2.2-6.1 (Table 3-3)

BAP (BAP)_n-(dT)_l-aptamer > (BAP)_n–aptamer >

(BAP)n-(dA)l-aptamer dATP BAP

Standard Elution time (min)

Molecular mass

(kDa) V_e (mL) K_av

Ribonuclease A 11.8 13 0.767 0.667

Carbonic Anhydrase 10.6 29 0.689 0.519

Ovalbumin 9.8 43 0.637 0.420

Aldorase 7.6 158 0.494 0.148

Blue Dextran 6.4 >200000 0.416

Time (min) 9 8

6 7 10

1. (BAP)_n-aptamer 2. (BAP)_n-(dT)_l-aptamer 3. (BAP)_n-(dA)_l-aptamer 4. BAP alone

11 20

30 40 50

Absorbance at 280 nm (mAU)

2,1,3

Figure 3-13 SEC . (1) (BAP)_n-aptamer (2) (BAP)_n-(dT)_l-aptamer (3) (BAP)_n-(dA)_l- aptamer (4) NK14-BAP .

Table 3-2 K_av.

-61-

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

C14 MTG

Z-QG-dU MTG

BAP (BAP)n-(dT)l-aptamer ELAA

3-3-3. BAP-aptamer 3-3-3-1.

ELISA 2% BSA 5% BSA

MTG BAP

BAP Figure 3-14

BSA NK14-BAP

1% NK14-BAP

20% 1% 80%

BAP-Sample Elution time

(min)

Molecular mass

(kDa) Ve (mL) Kav n

1 8.3 96.1 0.540 0.235 2.34

3 8.4 90.8 0.546 0.247 2.21

2 (peak) 8.2 101 0.533 0.222 2.48

2 (shoulder) 6.6 252 0.429 0.0247 6.13

4 9.8 41.1 0.637 0.420 1

Table 3-3 Kav SEC .

3-3-4-2.

ELAA Figure 3-15 (a) Figure 3-15 (b)

100 nM 50 nM BAP-aptamer 1.0

Figure 3-14 .

Fluorescence intensity [a.u]

1% 3%

2% BSA 5%

w/o Blocki ng agents Skim milk

* *

n  Pretreatment

p Simultaneous incubation

Figure 3-15 (a) ELAA (b) .

× BAP-aptamer

○ (BAP)_n-aptamer

● (BAP)_n-(dT)_l-aptamer

0 1 2 3 4 5 6 7

0.01 0.1 1 10 100

Relative BAP activity

Thrombin concentration (nM)

Relative BAP activity (-)

(a) (b)

Substrate

Product

Streptavidin

Secondary aptamer (BAP)_n-(dN)_l-aptamer

conjugate Thrombin

Primary aptamer biotinylated 29-mer TBA

-63-

(BAP)_n-(dT)_l-aptamer BAP-aptamer 6 (BAP)_n-aptamer

BAP-aptamer 4 EC₅₀ Table 3-4 BAP

(BAP)n-(dT)l-aptamer (BAP)n-aptamer BAP-aptamer

(BAP)_n-(dT)_l-aptamer 0.30 nM BAP-aptamer 5.3 nM EC₅₀

(BAP)n-(dT)l-aptamer BAP-aptamer EC50 (BAP)n-aptamer

BAP-aptamer EC50 EC50 (BAP)n-aptamer

3´-BAP dTTP

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

BAP-aptamer

Table 3-4 ELAA EC₅₀ .

Conjugates EC₅₀ (nM) Detection limit (nM)

BAP-aptamer 7.4 5.3

(BAP)_n-aptamer 21 2.5

(BAP)_n-(dT)_l-aptamer 6.5 0.30

(BAP)_n-(dT)_l(X%)-aptamer X = 16.6 33.3 66.6% ELAA

(Figure 3-16) X = 66.6% (BAP)_n-aptamer X = 16.6

33.3% X = 25% (Table 3-5) Z-QG-dUTP (25%)/dTTP

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

(BAP)_n-(dT)_l-aptamer

Table 3-5 (BAP)n(X%)-(dT)l-aptamer ELAA EC50 .

X EC₅₀ (nM) Detection limit (nM)

16.6 8.2 0.57

33.3 4.0 0.59

66.6 8.0 5.2

3-4.

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

(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)

Thrombin concentration (nM)

Relative BAP activity (-)

16.6%

25%

33.3%

66.6%

100%

//�

Figure 3-16 (BAP)_n-(dT)_l(X%)-aptamer .

-65- (BAP)_n-(dT)_l-aptamer

3-5.

C1. J. Zhou, J. Tang, G. Chen and D. Tang, Biosens. Bioelectron., 54, 323-328 (2014).

C2. M. Takahara, K. Hayashi, M. Goto and N. Kamiya, J. Biosci. Bioeng., 116, 660-665 (2013).

C3. M. Kitaoka, Y. Tsuruda, Y. Tanaka, M. Goto, M. Mitsumori, K. Hayashi, Y. Hiraishi, M. Katsuyuki, S. Noji and N. Kamiya, Chem. Eur. J., 17, 5387-5392 (2011).

C4. Y. Mori, S. Ozasa, M. Kitaoka, T. Tanaka, S. Noda, H. Ichinose and N. Kamiya, Chem. Commun., 49, 6971-6973 (2013).

C5. C. M. Niemeyer and W. Bürger, Angew. Chem. Int. Ed., 396, 2265-2268 (1998).

C6. S. Rudiuk, A. Venancio-Marques, D. Baigl, Angew. Chem. Int. Ed., 51, 12694-12698 (2012).

C7. S. S. av́an Berkel, P. Floris, J. C. av́anHest and F. L. av́anDelft, Chem. Commun., 46, 97-99 (2010).

C8. A. Chango, A. M. A. Nour, C. Niquet and F. J. Tessier, Med. Princ. Pract., 18, 81-84 (2009).

C9. S. Centi, S. Tombelli, M. Minunni and M. Mascini, Anal. Chem., 79, 1466-1473 (2007).

C10. J. Shimada, T. Maruyama, M. Kitaoka, N. Kamiya and M. Goto, Anal. Biochem., 421, 541-546 (2012).

C11. E. A. Motea and A.J. Berdis, Biochim. Biophys. Acta, 1804, 1151-1166 (2010).

C12. F. J. Bollum, J. Biol. Chem., 235, 2399-2403 (1960).

C13. G. G. Schmitz, T. Walter, R. Seibl and C. Kessler, Anal. Biochem., 192, 222-231 (1991).

C14. E. Baldrich, A. Restrepo and C. K. O´Sullivan, Anal. Chem., 76, 7053-7063 (2004)

-67- 4-1.

4-1-1.

(DNAzyme) DNAzyme

(Diels-Alder Michael

) HRP

DNAzyme hemin/G-quadruplex (hGQ) ^D1 HRP hGQ

H₂O₂

DNAzyme

I. Willner DNAzyme

nucleoapzyme

DNAzyme (Figure 4-1)^D2

nucleoapzyme

(DBA) hGQ

hGQ 20

4-1-2.

(1)

(2) (3)

( ) (4)

β-1,4-(2) Figure 4-1 Nucleoaptzyme.

[Reprinted with permission from J. Am. Chem.

(Figure 4-2) ^{D3, D4} ( )

30%

(CBM)

CBM

4-1-3.

DNA

-100 mM NaCl 5 mM MgCl₂ (20 mM

Tris-HCl, 0.01% (w/v) SDS, pH 7.5) DNA ^D7

DNA (Figure 4-3) DNA

Na⁺ K⁺ (G)

D8-D10 CBM

DNA DNA

Substitution

Cellulose-binding

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