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

九州大学学術情報リポジトリ

Kyushu University Institutional Repository

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

髙原, 茉莉

http://hdl.handle.net/2324/1807002

出版情報：Kyushu University, 2016, 博士（工学）, 課程博士 バージョン：

権利関係：

博士論文   

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 

九州大学  大学院工学府  化学システム工学専攻  後藤・神谷研究室  博士 3 年  高原  茉莉 

指導教官  神谷  典穂  教授 

第一章  序論 

1-1.  核酸の性質    1 

1-1-1.  核酸の構造  2 

1-1-2.  核酸アナログ  3 

1-1-3.  機能性核酸  5 

1-1-3-1.  遺伝子発現の抑制作用  5 

1-1-3-2.  触媒作用  7 

1-1-3-3.  核酸の超分子化学  7 

1-2.  核酸アプタマー    8 

1-2-1.  試験管内進化法  9 

1-2-2.  核酸アプタマーの化学修飾  10 

1-2-3.  医薬品への応用  11 

1-2-3-1.  阻害作用・アゴニスト活性  11 

1-2-3-2.  標的指向性  11 

1-2-4.  分析分野への応用  12 

1-2-4-1.  バイオセンサー  12 

1-2-4-2.  クロマトグラフィー・キャピラリー電気泳動  13   

1-3. DNA-タンパク質コンジュゲート  14 

1-3-1.  非共有結合的 DNA-タンパク質コンジュゲート  14 

1-3-1-1.  ビオチン-アビジン相互作用  14 

1-3-1-2.  ニトリロ三酢酸  (NTA)-Ni²⁺-ヘキサヒスチジン相互作用  15 

1-3-1-3.  抗原-抗体相互作用  15 

1-3-1-4.  タンパク質-アプタマー相互作用  16 

1-3-1-5.  アポ酵素再構成  16 

1-3-1-6. DNA 結合性タンパク質の利用  17 

1-3-2.  共有結合的 DNA-タンパク質コンジュゲート  17  1-3-2-1.  野生型タンパク質とのコンジュゲーション  17  1-3-2-2. Expressed protein ligation  18 

1-3-2-3.  生体直交性化学  18 

1-3-2-4.  自己連結性タンパク質タグ  19 

1-3-2-5.  酵素反応によるライゲーション  20 

1-4.  本研究の目的及び本小論文の構成  22 

1-5.  参考文献  24 

第二章  DNA アプタマー-酵素コンジュゲートの設計 

2-1.  核酸合成及び修飾技術    27 

2-1-1.  固相合成法  27 

2-1-2.  酵素合成法  28 

2-1-3. Terminal deoxynucleotidyl transferase (TdT)  29 

2-1-3-1.  金属イオン依存性  29 

2-1-3-2.  基質選択性  30 

2-1-3-3. TdT を用いた核酸修飾技術  31 

2-1-4.  本論文における核酸修飾技術  32 

2-2.  実験操作    34 

2-2-1.  試薬及び装置  34 

2-2-2. NK14-BAP の発現及び精製  34 

2-2-3. TdT による Z-QG-aptamer の調製  34  2-2-4. MTG による Z-QG-aptamer と NK14-BAP の複合化  35 

2-2-4-1. MTG 反応における仕込み比検討  35 

2-2-4-2. BAP-aptamer コンジュゲートの調製  35 

2-2-5. BAP-aptamer の機能評価  36 

2-3.  結果及び考察    37 

2-3-1. NK14-BAP の発現及び精製  37 

2-3-2. TdT による Z-QG-aptamer の調製  38 

2-3-2-1. TdT 反応の経時変化  38 

2-3-2-2. Z-QG-aptamer の調製  39 

2-3-3. MTG による Z-QG-aptamer と NK14-BAP の複合化  39 

2-3-3-1. MTG 反応における仕込み比検討  39 

2-3-3-2. BAP-aptamer コンジュゲートの調製  40 

2-3-4. BAP-aptamer の機能評価  41 

2-4.  結論  44 

2-5.  参考文献  45 

第三章  核酸アプタマーを認識部位とする高感度分析試薬の開発 

3-1.  緒言    47 

3-1-1. DNA における酵素の集積化  47 

3-1-2.  本章の目的及び戦略  48 

3-2.  実験操作    49 

3-2-1.  試薬及び装置  49 

3-2-2. TdT による(Z-QG)_m-aptamer の調製  49  3-2-2-1. (Z-QG)m-(dN)l-aptamer の調製  49  3-2-2-2. (Z-QG)m(X%)-(dT)l-aptamer の調製  50 

3-2-2-3. Z-QG-dUTP の取込み量評価  50 

3-2-3. MTG による(Z-QG)_m-aptamer と NK14-BAP の複合化  51 

3-2-3-1. MTG 反応における仕込み比検討  51 

3-2-3-2. (BAP)n-(dN)l-aptamer コンジュゲートの調製  51 

3-2-3-2. BAP 標識効率の評価  51 

3-2-4. BAP-aptamer の機能評価  52 

3-2-4-1.  ブロッキング条件の最適化  52 

3-2-4-2.  生成コンジュゲートによるトロンビン検出  52   

3-3.  結果及び考察    53 

3-3-1. TdT による(Z-QG)_m-aptamer の調製  53 

3-3-1-1. TdT 反応の経時変化  53 

3-3-1-2. (Z-QG)m-(dN)l-aptamer の調製  53  3-3-1-3. (Z-QG)m(X%)-(dT)l-aptamer の調製  53 

3-3-1-4. Z-QG-dUTP 取込み量評価  55 

3-3-2. MTG による(Z-QG)_m-aptamer と NK14-BAP の複合化  57 

3-3-2-1. MTG 反応における仕込み比検討  57 

3-3-2-2. (BAP)n-(dN)l-aptamer コンジュゲートの調製  58 

3-3-2-3. BAP 標識効率の評価  59 

3-3-3. BAP-aptamer の機能評価  61 

3-3-3-1.  ブロッキング条件の最適化  61 

3-3-3-2.  生成コンジュゲートによるトロンビン検出  62   

3-4.  結論    64 

3-5.  参考文献  66 

第四章  セルロース結合性アプタマーを利用した人工バイオマス          分解酵素の開発 

4-1.  緒言    67 

4-1-1.  アプタマーを利用した触媒  67 

4-1-2.  バイオマス分解酵素触媒  67 

4-1-3.  本章の目的及び戦略  68 

4-2.  実験操作    69 

4-2-1.  試薬  69 

4-2-2.  セルロース結合性アプタマーの設計  70 

4-2-2-1. Mfold による高次構造予測  70 

4-2-2-2.  円二色性  (CD)  スペクトル測定  71 

4-2-2-3. CD による融点測定  71 

4-2-2-4.  セルロース基質への結合観察  71 

4-2-2-5.  セルロース基質への吸着量評価  71 

4-2-3.  人工セルラーゼの合成  72 

4-2-3-1. Cel6A、Cel6ACDの発現及び精製  72  4-2-3-2. TdT による Z-QG-CelApt72の調製  72  4-2-3-2. MTG による Z-QG-CelApt72と Cel6ACDの複合化  72 

4-2-4.  人工セルラーゼの機能評価  73 

4-3.  結果及び考察  74 

4-3-1.  セルロース結合性アプタマーの設計  74 

4-3-1-1. Mfold による高次構造予測  74 

4-2-2-2. CD スペクトル測定  75 

4-2-2-3. CD による融点測定  77 

4-2-2-4.  セルロース基質への結合観察  78 

4-2-2-5.  セルロース基質への吸着量評価  80 

4-2-3.  人工セルラーゼの合成  80 

4-2-3-2. TdT による Z-QG-CelApt72の調製  80  4-2-3-2. MTG による Z-QG-CelApt72と Cel6ACDの複合化  81 

4-2-4.  人工セルラーゼの機能評価  82 

4-4.  結論    85 

4-5.  参考文献  87 

第五章  結論 

謝辞 

-1-

19 1869

F. Miescher ( )

1944 O. Avery

(DNA) 1953 J. Watson

F. Crick DNA

DNA

Watson-Crick DNA

DNA

(RNA) RNA DNA

DNA RNA RNA

( )^A1

(Figure 1-1)

1980 Cech

RNA

RNA RNA (ribozyme)

1990 Gold (SELEX ) ^A2 Szostak in vitro selection ^A3

(aptamer)

DNA

DNA DNA

Figure 1-1 .

Amino acid

Polypeptide

Protein Antibody

IgG

Catalysis Alkaline phosphatase (AP) Green

Fluorescent Protein (GFP)

PDB ID:

1EMA PDB ID:

1IGT PDB ID:

1ALK

1-1.

1-1-1.

DNA RNA

(Figure 1-2)

DNA β-D-

RNA β-D-

1 β-N-

5´ 1 ~ 3

3´-5´

5’- 3´-

2

(Figure 1-3 (a)) DNA 2

DNA 2

(Adenine; A) (Thymine; T)

(Guanine; G) (Cytosine; C)

RNA T (Uracil; U) (Figure 1-3 (b)) A-T G-C

Watson-Crick A-T 2 G-C 3

Sugar O HO R O

P O P O P

O O O O

O O O

Base Phosphate group

Glycosidic bond R = H: Deoxyribose R = OH: Ribose Nucleoside Nucleotide

Figure 1-2 .

Figure 1-3 . (a) DNA (b) RNA .

(a) DNA polynucleotide strand (b) RNA polynucleotide strand

In RNA, uracil (U) replaces thymine (T)

5´-to 3´ d

irecti on A phosphodiester linkage (5´-phosphate group and

3´-OH group)

5´-to 3´ d

irecti on

5´-to 3´ d

irecti on

N C N C C HC

O H₃C

H O

T

T-A pairs have two hydrogen bonds

G-C pairs have three hydrogen bonds

O

H O

H H

H CH₂

H

P O

O O

O

H O

H H

H H2C

H

P O

O O

O

H O

H H

H H2C

H

P O

O O

O

H O

H H

H CH₂

H

P O O P

O O

O

H O

H H

H

CH2 H P OH

O O

O

H O

H H

H

CH₂ H P O

O O

O

H O

H H

H

CH₂ H P O

O O

O

H O

H H

H

H₂C H P O

O O

O

OH O

H H

H CH₂

H

P O

O O

O

OH O

H H

H H₂C

H

P O

O O

O

OH O

H H

H H2C

H

P O

O O

O

OH O

H H

H CH2

H

P O O P

O O

O H

CH₃ O

CH C C N

C N

T

O H

CH C C

N C N NH H

C

O H

HC C C N C N

HN H

C

C U

HN C

CH CH N O

O

C N C

N CH

N C N O

H

NH H

G

N C C HC N

N C N O

H

HN H

G

N C C HC N

N C N C

HN

H H

A

H

H NH

C N

C N N CH C

C N

A

N C C HC N

N C N O

H

HN H

G

N C C HC N

N C N C HN

H H

A

O H

HC C C

N N C

HN H

C

-3- A-T G-C

3 2

DNA

A4,5

J. Watson F. Crick B DNA

2.37 nm 3.4 Å 3.4 nm 36°

10.5 β-N-

3´ 4´ DNA

2’ RNA DNA-RNA RNA-RNA A

B C2´-endo A

C3´-endo

A RNA-RNA DNA-RNA

DNA-DNA Z DNA Hoogsteen

1-1-2.

(10 ) ^A6

(glycerol-derived nucleic acid; GNA) ^A7 (Figure 1-4 (a))

O P

O

O O

O

P O

O O

O O

P O

O O

O O

O

B

B

B

(a) (b) (c) (d)

(N) NH₂

N O

NH

N O O OH O

(C)

B

B

O OR

O

O P

O O

O

O OR'O B

B

O OR

O P O S

O O OR'

B B

Figure 1-4 . (a) GNA (b) PNA (c) LNA (d) PS.

GNA

3-

(peptide nucleic acid; PNA)^A8 (Figure 1-4 (b)) (locked nucleic acid; LNA)^A9 (Figure 1-4 (c))

(phosphorothioate; PS)^A10 (Figure 1-4 (d))

(PNA)

Nielsen PNA N-(2-aminoethyl) glycine

PNA DNA RNA PNA

PNA-DNA PNA-RNA

DNA-DNA DNA-RNA ^A11 PNA

A11 PNA in vitro in vivo

fluorescent in situ hybridization (FISH)

(LNA)

LNA 2´ 4´

C3´-endo

C3´ C5´ RNase H

A12

LNA PNA

A13 PNA LNA-DNA

LNA-RNA T_m ^A14

KOD polymerase (PCR)

A15 RNA

SELEX PCR

LNA

(HIV)-1 RNA ¹⁶

(PS)

PS F. Eckstein ^A10 PS

( S Na⁺

) PS

A17

PS ^A18

W. J. Stec PS

-5-

A19

(bifunctional linker) -DNA

A20

1-1-3.

RNA

1-1-3-1.

DNA mRNA tRNA

mRNA

mRNA

RNaseH mRNA

(Figure 1-5)^A21

mRNA ( )

1998 (fomivirsen)

A22

RNA (RNAi)

A. Fire C. Mello RNA (RNA interference; RNAi)

RNA ^A23

RNAi 3´- RNA (dsRNA) RNase III Dicer

RNA (short interfering RNA; siRNA)

Figure 1-5 .

DNA

Antisense mRNA oligonucleotide

Transcription

Translation

Ribosome

Amino acid Rnase H

Antisense oligonucleotide-mediated translational inhibition or cleavage

through RNase H activation

siRNA

(Ago) RNA-inducing silencing complex (RISC)

RISC

mRNA RNase

mRNA (Figure

1-6) RNAi mRNA

mRNA

RISC mRNA siRNA

siRNA 2013

(TTR) (ATTR) (ALN-TTR02)

A24 RNAi TTR TTR 94%

DNA

(TF) TF

in vitro in vitro (Figure 1-7) TF

(NF-κB) DNA

A25 NF-κB

NF-κB NF-κB

in vivo NF-κB

Figure 1-6 RNA . Long dsRNA Dicer

siRNA

Passenger strand Guide strand

Argonaute RISC

Target mRNA

Cleaved passenger strand Guide strand

Active RISC

PDB ID:

3HXM

Argonaute

Figure 1-7 .

Decoy ODN

“Decoy” cis element

Transcription factor (TF)

TF-promotor binding Promoter

Gene transcription NF-κB

Decoy ODN

PDB ID:

2V2T

-7- (TF)

TF

TF

1-1-3-2.

RNA 1980 Cech

RNA

A26 RNA ribozyme

DNA ^A27

DNA (deoxyribozime DNAzyme)

RNA

DNA RNA O. Willner

hemin/G-quadruplex DNAzyme (Figure 1-8)^A28

1-1-3-3.

DNA

A29 DNA π-π

DNA DNA

DNA

DNA DNA

peptide-DNA

assembly ^A30 β- DNA

DNA β-

Figure 1-8 Hemin/G-quadruplex

[Reprinted with permission from Anal. Chem., 82, 4396-4402 (2010). Copyright 2010 American Chemical Society.]

(Figure 1-9)

1-2.

(Figure1-10) 1990 Tuerk Gold (SELEX ) ^A2 Ellington Szostak

in vitro selection ^A3

fit ‘aptus’

part region ‘meros’

aptamer ( )

van der waals

induced-fit -

( ) ^A31,32

theophylline caffeine

theobromine

A33

A34

SELEX

Figure 1-9 peptide-DNA .

[From Angew. Chem. Int. Ed., 55, 12003-12007 (2016). Copyright © 2016 by John Wiley Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc.]

Figure 1-10

.

(ATP) DNA

ATP.

DNA aptamer 

Target molecule 

PDB ID : 1AW4 

-9-

1-2-1. (SELEX )

SELEX SELEX

DNA RNA

SELEX

(Figure 1-11)^A35

SELEX 100

10¹⁴ 10¹⁵

(ⅰ)

(ⅱ) (ⅲ) PCR (ⅳ)

SELEX

cell-SELEX (Figure 1-12 (a))^A36

SELEX (Figure 1-12 (b))^A37 cell-SELEX

DNA

DNA negative selection

DNA

Figure 1-11 (SELEX ).

[Reprinted from Appl. Microbiol. Biotechnol., Aptamers-basic research, drug development, and clinical applications, 69, 2005,

367-374, D. Proske, M. Blank, R. Buhmann and A. Resch, © Springer-Verlag 2005 with permission of Springer.]

1-2-2.

A38 2´-deoxy

SELEX SELEX PCR

5’ 3’

SELEX PCR

PCR 1994 Latham 5- - (dU) (Figure

1-13 (a)) Bock Tasset thrombin ^A39

Vaught C5 dU (Figure 1-13 (b))

SELEX TNFRSF9 (tumor necrosis factor superfamily member 9)

A40

SELEX 30%

C5

80%

5-typtaminocarbonyl-dU

C5 dU

SOMAmer (Slow Off-rate modified aptamer)

Figure 1-12 (a) Cell-SELEX (b) SELEX. [(a) Reprinted by permission from Macmillan Publishers Ltd: Nat. protoc., 5, 1169-1185 (2010) copyright 2010, (b) Reprinted with permission

from Anal. Chem., 84, 5542-5547 (2012). Copyright 2012 American Chemical Society.]

(a) (b)

Figure 1-13 SELEX .

NH O

O O N

OH HO

R (a)

R =

(b)

NH O

NH O N NH

NH O

NH O

N

NH O

NH O HN

R =

-11- π-

1-2-3.

siRNA

1-2-3-1.

FDA VEGF (

) VEGF165RNA

(Macugen®) (Figure1-14)

A41 Macugen® (AMD)

VEGF VEGF165

2´-OMe ( ) 2´-F

( )

Macugen® 6 nmol

Sullenger T

A42 T

4-1BB Sando

c-Met (SL1) c-Met

A43 SL1 (HGF)

A44

1-2-3-2.

W. Tan

(Dox) DNA-Dox-Au (Figure 1-15 (a))

A45

DNA Dox

Dox

(Figure 1-15 (b))^A46 Figure 1-14 Macugen®

. G

G C

U C C G U G A

G C U U A A G U

C U A A

A A

U C A

3´-3´-dT-5´

40-kDa PEG-5´

1-2-4.

Drolet (enzyme-linked

aptamer assay; ELAA)^A47 enzyme-linked immunosorbent assay (ELISA) 20%

1-2-4-1.

(a)

(aptamer beacon) (b) (C)

(Figure 1-16)

A48

Figure 1-15 (a) (b)

. [(a) Reprinted within permitted use from ACS AuthorChoice, Nano Lett., 15, 457-463 (2015). Copyright 2015 American Chemical Society. (b) From Angew. Chem. Int. Ed., 52, 1472-1476 (2013). Copyright © 2013 by John

Wiley Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc.]

(b) (a)

Figure 1-16 .

(a) (aptamer beacon) (b)

(c) .

(a)

(b)

F

Q

F Q

Target

Target

AuNP

Target ^Electron _transfer

R R

Electrode

(c)

-13-

A49

A33

Sando (fApt)

A50 toc-fApt

(Figure 1-17)

1-2-4-2.

(HPLC)

Michaud

1.1 µM^-1 D ( )

L ^A51

-

Pavski HIV-1

(HIV-RT)

HIV-RT 50 nM ^A52

Figure 1-17 .

[Reprinted with permission from J. Am. Chem. Soc., 134, 9561-9564 (2012). Copyright 2012 American Chemical Society.]

1-3. DNA-

DNA Watson-Crick 10 nm

(DNA origami)^A53

DNA DNA

DNA origami (150 mM NaCl

pH 7.4 30 min )

DNA (DNA-directed immobilization; DDI) (Figure 1-18) 1990 DNA-

DNA

1-3-1. DNA-

1-3-1-1. -

- (Figure 1-19) DNA

(SA) K_d 10 fM

A54

DNA SA

-

DNA SA ^A55 Niemyer

(Cys) DNA

DNA

DNA DNA- -

DNA

DNA- SA

Figure 1-18 DNA (DNA-directed immobilization; DDI) .

-15- DNA

- SA

Rhizobium etli SA ( )

SA^A56

1-3-1-2. (NTA) -Ni²⁺-

(His₆) N C

Ni²⁺ Co³⁺

(NTA) His₆ NTA DNA Ni²⁺

His₆ NTA-DNA DNA-NTA-Ni²⁺-His⁶-

(Figure 1-20)^A57 EDTA NTA

DNA NTA mono-NTA di-NTA tri-NTA DNA

His₆-GFP ( ) NTA K_d 120 nM 6 nM

Turberfield ^A58

1-3-1-3. -

DNA digoxigenin (DIG) FLAG-tag (DYKDDDDK)

G (IgG)

DNA IgG -

Figure 1-19 - DNA- .

Streptavidin (SA) Biotin-DNA

+

S NH HN HN

O

O

≡ Biotin moiety

DNA-biotin-SA- protein conjugate

His₆-fused protein NTA-DNA

+

≡ NTA moiety

DNA-NTA-Ni²⁺-His₆- protein conjugate

N N

H O

O O

S

O N OH

OH

OH O O

His6

Ni²⁺

His₆ Ni²⁺

Figure 1-20 NTA-Ni²⁺-His₆ DNA- .

(Figure 1-21) Mao - DNA

DNA ^A59

FLAG-tag DNA FLAG-tag FLAG-tag

1-3-1-4. -

DNA DNA

(Figure 1-22)

DNA DNA

A60

1-3-1-5.

( )

DNA- (Figure 1-23)

(1) (2) DNA

(3) DNA DNA- -

Fruk ( IX) DNA

DNA- DNA- HRP

A61 DNA-

DNA

Figure 1-21 - DNA- - .

Antibody Hapten-DNA

+

DNA-hapten-antibody conjugate

Figure 1-22 DNA DNA- .

+

DNA aptamer

Target

protein DNA aptamer/protein complex

-17- 1-3-1-6. DNA

DNA zinc finger protein (ZFP)

nM DNA ( 10 bp)

(Figure 1-24) ZFP DNA

ZFP (zif268 AZP4)

DNA Morii ZFP

DNA ZFP ^A62

1-3-2. DNA-

1-3-2-1.

DNA

DNA DNA DNA

(Lys) (Cys)

N- (NHS)

DNA Corey Schultz staphylococcus nuclease (SN) Cys

DNA DNA-SN

A63

sulfo-succinimidyl-4-(N-maleimidomethyl)- cyclohexane-1-carboxylate (sSMCC)

A64 Lys

sSMCC DNA (Figure 1-25)

Lys Cys

DNA

Figure 1-23 DNA- - .

Holoenzyme

Cofactor-DNA

Apoenzyme DNA-cofactor-

apoenzyme

Figure 1-24 DNA (ZFP) DNA .

DNA-ZFP-protein conjugate

PDB ID: 5TDR

ZFP fusion protein

+

Double strand DNA

DNA

DNA Ni²⁺-NTA-DNA

His NTA-Ni²⁺-His DNA

His ^A65

1-3-2-2. Expressed protein ligation

C N

Cys expressed protein ligation (EPL) ^A66 Nagamune Cys DNA

DNA- (Figure 1-26)^A67

EPL C (CBD)

(1) - -CBD (2) 2-

(MESNA) C

(3) C

DNA Cys EPL

C

1-3-2-3.

( ) DNA-

Cu (I)- - (CuAAC)

NH2

N O

N

O O O

O O SO O O

HN O O

N O

O

HN O O

N O

O S

SH

Figure 1-25 (sSMCC) DNA- .

Figure 1-26 EPL DNA- .

HS SO₃^- NH

O

Intein

S SO₃^- O

NH HN

O HN

O ⁶ O

O SH

H₂N HN

O HN HS

O ⁶ O

1. Affinity purification 2. MESNA

-19- DNA Staudinger ligation

(Figure 1-27) Humelik

DNA

DNA— ^A68 Duckworth protein farnesyl transferase

(PETase) PETase

A69 PFTase

C 4 CXXX

DNA CuAAC DNA

(1) Cu (I)

(2) Cu (I) (3) Cu (I)

Staudinger ligation

dibenzocyclooctyne (DBCO) ^A70 Staudinger ligation

DNA DNA ^A71,72

1-3-2-4.

DNA (Figure 1-28)

human O⁶-alkylguanine-DNA-alkyltrans-ferase (hAGT) SNAP-tag (20 kDa)^A73 Halo-alkane dehalogenase HALO-tag (34 kDa)^A74 SNAP-tag

Figure 1-27 DNA- . (a)

(I)- - (CuAAC) (b) (c) Staudinger ligation.

HN

OCH₃ P Ph Ph

O

O NH

N O O

HN

NH P Ph Ph

O

O NN N

NH

N O O

N N N N₃

N₃

N₃

+

Cu ( )

+

+

(a)

(b)

(c)

Alkyne-DNA

DBCO-DNA

Phosphine-DNA

Azide-protein

O⁶-benzylguanine-DNA SNAP-tag Cys

HALO-tag 5-chlorohexane-DNA Cl Asp

O⁶-benzylguanine-DNA 5-chlorohexane-DNA DNA NHS DNA

DNA

DNA DNA DNA-

HALO-tag in vivo ^A75 SNAP-tag

HALO-tag DNA

1-3-2-5.

DNA

PETase DNA-

DNA

Sortase A (MTG)

Figure 1-28 DNA- . (a) SNAP-tag

(b) HALO-tag.

(a)

(b)

NH₂

NH₂

NO N

H O

O

O O

O N

N N

H2N N

NO O

O

O H

N O

O

Cl 2

NH O

O

O N

N N N H2N

HN

O^- O

PDB ID : 5LKA  PDB ID : 3I00 

-S ^NH

O O

S

HN

N

N N

H2N N

OH

NH O

HN O

O

Cl

2

NH O

HN O

O 2

Cl^-

-21- Sortase A (SrtA)

Sortase A (SrtA)

SrtA C LPETG T G (LPET↓G) N GGG

LPETG G LPTEGGGG (Figure

1-29)^A76

Wong DNA

SrtA LPETG GGG-DNA

A77 DNA PNA

Microbial transglutaminase (MTG) (MTG)

Streptomyces mobaraensis MTG

Gln (Q) γ- Lys (K) 1

A78,79

K

(Figure 1-30) MTG NH₃

-

Figure 1-29 SrtA DNA- .

PDB ID : 2RUI 

N N

N

T E P L G

H₆

NH₂

G G G

SrtA

N N

N

T E P L G G G

Figure 1-30 MTG .

MTG

NH₂ O

Gln _H2N Lys

NH O

Gln Lys

MTG

NH₂ O

Gln _H2N Lys

NH O

Gln Lys

MTG

NH₂ O

Gln _H2N Lys

NH O

Gln Lys

NH₃ +

MTG

NH₂ O

Gln _H2N Lys

NH O

Gln Lys

MTG

NH₂ O

Gln _H2N Lys

NH O

Gln Lys

MTG

NH₂ O

Gln _H2N Lys

NH O

Gln Lys

NH₃ +

MTG

NH₂ O

Gln _H2N Lys

NH O

Gln Lys

MTG

NH₂ O

Gln _H2N Lys

NH O

Gln Lys

MTG

NH₂ O

Gln _H2N Lys

NH O

Gln Lys

OH + NH₃

H₂O R

water Primary amine

Lys (K) Gln (Q)

Gln (Q)

Gln (Q)

R

MTG (90 kDa) (1) (2)

(38 kDa) (3) Ca²⁺ (4)

MTG DNA-

Kamiya K MTG K (MKHK)

DNA Q MTG Q

N-carbobenzyloxy glutaminyl glycine (Z-QG) Z-QG-DNA MTG (Figure 1-31)^A80

MTG DNA- (1)

DNA MTG

DNA (2) 4 °C

DNase RNase

(3) Ca²⁺ MTG

DNA

1-4.

DNA DNA

DNA

(TdT) (MTG)

MTG DNA

Figure 1-31 MTG 5´-Z-QG-DNA K-tag .

PDB ID : 1IU4 

MTG

NH₂ M K H K G S

NH HN

NH O O

O H₂N O

O

NH HN

NH O O

O O HN O M K H K G S

+

5´-Z-QG-DNA K-tagged protein (K-tag: MKHKGS)

-23- DNA

DNA

(BAP) BAP N

MTG Lys BAP (NK14-BAP)

MTG (Z-QG) TdT

Z-QG DNA

NK14-BAP MTG

BAP BAP

DNA TdT

TdT

Z-QG (dNTP) MTG

dNTP Z-QG

MTG Z-QG

NK14-BAP

DNA

DNA (1:1)

1-5.

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-27-

DNA -

2-1.

(1) (2) DNA RNA

(3)

2-1-1.

( )

200 DNA

DNA 3´- DNA 5´

DNA (3´→5´)

DNA (Figure 2-1)^B1

3´- CPG (Controlled Pore Glass)

5´- p- (DMT)

CH₂Cl₂ DMT

5´- OH

5´-OH 5´

( )

5´

5´-OH

( )

2,6- (THF)

( ) HPLC PAGE

DMT

3

B2

(1) DNA (2)

2-1-2.

DNA

DNA polymerase chain reaction (PCR) RNA

RNA in vitro transcription

PCR

DNA

Taq

1000 1 0.01% ^B3

PCR ^B4-B6 PCR in vitro

transcription

DNA

PCR

B7, B8

3´→5´ 3´→5´ 5´→3´

DNA T4 DNA (ATP)

Figure 2-1 . B: .

O

H O

H H

H CH₂

H P O

O O

O

H O

H H

H H₂C

H O

O NC

Solid phase B B

O O

H O

H H

H H₂C

H O

OH O

H O

H H

H H₂C

H O

N NH

N N OCH₃

H₃CO

Solid phase Solid phase

B B

O

H O

H H

H CH₂

H P O

O

NC

Ni(iPr)₂

B

O

H O

H H

H CH₂

H P O O

O

H O

H H

H H₂C

H O

O

NC B

B

Solid phase CCl₂COOH

CH₂Cl₂

l₂, H₂O, THF

-29- 2-1-3. Terminal deoxynucleotidyl transferase (TdT)

DNA

(TdT) (Figure 2-2) 3´-OH

(Figure 2-3)

B9-B11 TdT DNA

3´- DNA

DNA TdT DNA

B10 TdT DNA3´- 6

(dA) 5 (dT)

DNA 2

DNA TdT ^B12

TdT 1971 58-60 kDa ^B13

DNA

DNA V (D) J TdT DNA

B14 V (D) J

TdT TdT

2-1-3-1.

B15 TdT TdT

Coleman (dA)_n

dATP TdT Mg²⁺ Zn²⁺ Co²⁺

Figure 2-2 TdT (PDB ID: 1KEJ).

Figure 2-3 (a) DNA DNA (b) DNA TdT.

C

C A C T G

G A

C T G A

C T G A

A

G C T G A

T C T G

(a) 5´

3´

5´

Primer Template

Template

3´

3´

-OH (b)

5´

P P P

P P P

Mn²⁺ dATP ^B15 Mg²⁺ dGTP

dATP Co²⁺ dCTP dTTP

B16 Mg²⁺ µM Zn²⁺ dATP

TdT

2-1-3-2.

TdT DNA 3´-

in vitro TdT DNA

B17 TdT

B18 TdT

DNA (Figure 2-4) TdT

TdT

TdT 3´-OH

(ddNTP) TdT

ddNTP TdT

cordycepin (Figure 2-6) TdT

B19,B20

Figure 2-5 TdT .

O HO O P O P O P

O O O O

O O O

N

NH₂

N

N⁺ O

O^-

N N

F

IndTP 5-FITP 5-AITP 5-NITP

N

N N N

5-CHITP 5-CEITP 5-PhITP 5-NapITP

N N N

N

NH₂

O OH HO

Cordycepin 3´-deoxyadenosine Figure 2-6 Cordycepin

.