Synthesis of Building Blocks and Oligonucleotides Containing {T}O4‐Alkylene‐O4{T} Interstrand Cross‐Links实验方法详情页

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Synthesis of Building Blocks and Oligonucleotides Containing {T}O4‐Alkylene‐O4{T} Interstrand Cross‐Links

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Abstract

This protocol describes the preparation of O 4 ?thymidine?alkylene?O 4 ?thymidine dimer bis?phosphoramidites and precursors for incorporation into DNA sequences to produce site?specific DNA interstrand cross?links. Linkers are introduced at the 4?position of thymidine by reacting the sodium salt of a diol with a pyrimidinyl?convertible nucleoside to produce mono?adducts, which then undergo reaction with a stoichiometric equivalent of a pyrimidinyl?convertible nucleoside under basic conditions to form O 4 ?thymidine?alkylene?O 4 ?thymidine dimers. Bis?phosphoramidites are incorporated into oligonucleotides by solid?phase synthesis, and mild conditions for deprotection and cleavage from the solid support are employed to prevent degradation of the thymidine modifications. Purification of these cross?linked oligonucleotides is performed by denaturing polyacrylamide gel electrophoresis. This approach allows for the preparation of cross?linked DNA substrates in quantities and purity sufficient for a wide range of biophysical experiments and biochemical studies as substrates to investigate DNA repair pathways. Curr. Protoc. Nucleic Acid Chem . 55:5.13.1?5.13.19. © 2013 by John Wiley & Sons, Inc.

Keywords: DNA interstrand cross?link; chemically modified oligonucleotide; oligonucleotide synthesis; solid?phase synthesis; DNA damage; DNA repair

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Introduction
Basic Protocol 1: Synthesis of O4‐Hydroxyalkyl‐Thymidine‐3′‐O‐Phosphoramidites
Basic Protocol 2: Synthesis of O4‐Thymidine‐Alkylene‐O4‐Thymidine‐3‐O‐Bis‐Phosphoramidites
Basic Protocol 3: Solid‐Phase Synthesis and Deprotection of Oligonucleotides Containing O4‐Hydroxyalkyl‐Thymidine Mono‐Adducts and O4‐Thymidine‐Alkylene‐O4‐Thymidine Cross‐Link Modifications
Basic Protocol 4: Purification of Oligonucleotides Containing O4‐Alkylene‐Thymidine Mono‐Adducts and O4‐Thymidine‐Alkylene‐O4‐Thymidine Cross‐Link Modifications
Commentary
Literature Cited
Figures

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Basic Protocol 1: Synthesis of O4‐Hydroxyalkyl‐Thymidine‐3′‐O‐Phosphoramidites Materials

3′‐O ‐(tert ‐Butyldimethylsilyl)‐5′‐O ‐(4,4′‐dimethoxytrityl)‐thymidine (1 ; Wilds et al., )
Acetonitrile (MeCN, EMD Millipore)
Dichloromethane (reagent‐grade DCM, EMD Millipore)
1,2,4‐Triazole (C 2 N 3 H 3 , Sigma‐Aldrich)
Triethylamine (TEA, Sigma‐Aldrich)
Phosphorus (V) oxychloride (POCl 3 , Sigma‐Aldrich)
3% (w/v) aqueous sodium bicarbonate (NaHCO 3 )
Sodium sulfate (Na 2 SO 4 , Sigma‐Aldrich)
1,7‐Heptanediol (Sigma‐Aldrich)
Tetrahydrofuran (THF, EMD Millipore)
Sodium metal (Sigma‐Aldrich)
Argon gas
Dioxane (Sigma‐Aldrich)
99% ethanol (reagent‐grade EtOH, EMD Millipore)
Ethyl acetate (reagent‐grade EtOAc, EMD Millipore)
Silica gel (60 Å, 230 to 400 mesh)
Hexanes (reagent‐grade Hex, EMD Millipore)
Tetrabutylammonium fluoride (1 M in THF, Sigma‐Aldrich)
Phenoxyacetyl chloride
Methanol (reagent‐grade MeOH, EMD Millipore)
Diisopropylethylamine (DIPEA, Sigma‐Aldrich)
N,N‐Diisopropylamino cyanoethyl phosphonamidic chloride (Cl‐P(OCE)Ni Pr 2 , ChemGenes)
Saturated NaCl aqueous solution

10‐, 50‐, and 100‐mL round‐bottom flasks with rubber septa
Magnetic stir plate and stir bar
1‐mL disposable syringes and oven‐dried, 1‐in., 12‐G needles
Rotary evaporator and chemically resistant dry vacuum pump
250‐mL separatory funnels
Vacuum manifold
Vacuum pump
Pasteur pipets
2 × 50– and 10 × 70–cm flash chromatography columns
TLC aluminum sheets (silica 60 F 254 )
UV lamp (254 nm)
Automatic dispensing pipet
Glass funnel and filter paper

Basic Protocol 2: Synthesis of O4‐Thymidine‐Alkylene‐O4‐Thymidine‐3‐O‐Bis‐Phosphoramidites Materials

3′‐O ‐(tert‐ Butyldimethylsilyl)‐5′‐O ‐(4,4′‐dimethoxytrityl)‐O 4 ‐(1,2,4‐triazolyl)‐thymidine (2 ; see Basic Protocol 1)
3′‐O ‐(tert‐ Butyldimethylsilyl)‐5′‐O ‐(4,4′‐dimethoxytrityl)‐O 4 ‐(hydroxyheptyl)‐thymidine (3 ; see Basic Protocol 1)
Pyridine (Sigma‐Aldrich)
1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU, Sigma‐Aldrich)
Argon gas
Ethyl acetate (EtOAc, reagent‐grade)
3% (w/v) aqueous sodium bicarbonate (NaHCO 3 )
Sodium sulfate (Na 2 SO 4 )
Silica gel (60 Å, 230 to 400 mesh)
Acetonitrile (MeCN)
Dichloromethane (DCM)
Tetrahydrofuran (THF, EMD Millipore)
Tetrabutylammonium fluoride (1 M in THF, Sigma‐Aldrich)
Methanol (MeOH, reagent‐grade)
Hexanes (Hex, reagent‐grade)
Diisopropylethylamine (Sigma‐Aldrich)
N,N‐Diisopropylamino cyanoethyl phosphonamidic chloride (Cl‐P(OCE)Ni Pr 2 , ChemGenes)

10‐ and 25‐mL round‐bottom flasks with rubber septa
Magnetic stir plate and stir bar
Automatic dispensing pipet
Rotary evaporator and chemically resistant dry vacuum pump
250‐mL separatory funnels
2 × 50– and 10 × 70–cm flash chromatography columns
TLC aluminum sheets (silica 60 F 254 )
UV lamp, 254 nm
Vacuum manifold
Vacuum pump
Glass funnel and filter paper

Basic Protocol 3: Solid‐Phase Synthesis and Deprotection of Oligonucleotides Containing O4‐Hydroxyalkyl‐Thymidine Mono‐Adducts and O4‐Thymidine‐Alkylene‐O4‐Thymidine Cross‐Link Modifications Materials

5‐O ‐(4,4′‐Dimethoxytrityl)‐2′‐deoxynucleoside‐3‐O ‐succinate long‐chain alkylamine controlled‐pore glass (LCAA‐CPG, 500 Å; Glen Research)
2′‐Deoxyribonucleoside‐3‐phosphoramidites (Glen Research)
Anhydrous acetonitrile (MeCN; EMD Millipore)
O 4 ‐Hydroxyalkyl‐thymidine mono‐phosphoramidite (6 ; see protocol 1 ) or O 4 ‐thymidine‐alkylene‐O 4 ‐thymidine bis‐phosphoramidite (9 ; see protocol 2 )
4‐Å activated molecular sieves (Sigma‐Aldrich)
Argon
1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU, Sigma‐Aldrich)
1,7‐Heptanediol (Sigma‐Aldrich)
50% aqueous acetic acid (50% AcOH(aq), EMD Millipore)
Anhydrous ethanol (EtOH)

Screw‐cap columns
Brown bottles
DNA synthesizer (Applied Biosystems)
Vacuum pump
2‐mL screw‐capped microcentrifuge tubes (Fisher Scientific)
Automatic dispensing pipet
Storage box
DNA concentrator

Basic Protocol 4: Purification of Oligonucleotides Containing O4‐Alkylene‐Thymidine Mono‐Adducts and O4‐Thymidine‐Alkylene‐O4‐Thymidine Cross‐Link Modifications Materials

Dried oligonucleotide (see protocol 3 )
0.1 M sodium acetate (NaOAc, reagent‐grade)
C‐18 SEP‐PAK cartridges (Waters)
Acetonitrile (MeCN, HPLC‐grade)
18 MΩ water
Methanol (MeOH, HPLC‐grade)
20% 7 M urea denaturing polyacrylamide solution (19:1 acrylamide/bis‐acrylamide, Bioshop Canada)
95% ethanol (EtOH, reagent‐grade)
Tetramethylethylenediamine (TEMED, reagent‐grade)
20% (w/v) aqueous ammonium persulfate (20% APS, reagent‐grade)
Running buffer: 89 mM Tris·Cl, 89 mM boric acid, 2 mM EDTA, pH 8.0 (TBE, Bioshop Canada)
Formamide (Bioshop Canada)
Xylene cyanol (Bioshop Canada)
Bromophenol blue (Fisher Scientific)

5‐, 15‐, and 50‐mL conical tubes (VWR or Greiner Bio‐One)
10‐ and 60‐mL disposable syringes (BD Biosciences)
Vortexer
UV spectrophotometer (Varian Cary 300) and quartz cuvettes
DNA concentrator
Glass plates (18 × 16–cm, Amersham Biosciences)
Standard vertical electrophoresis unit (Amersham Biosciences)
One‐well comb
1‐in., 20‐G disposable needles
200‐μL micropipettor
Electrophoresis power supply (Amersham Biosciences)
Cling plastic wrap
UV lamp, 254 nm
Scalpel

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Figure 5.13.1 Chemical structure of (A ) the O 4 ‐hydroxyalkyl‐thymidine mono‐adduct and (B ) the O 4 ‐thymidine‐alkylene O 4 ‐thymidine cross‐link.

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Figure 5.13.2 Preparation of 6 . (a) 1,2,4‐triazole, TEA, POCl3 , MeCN:DCM (1:1 v/v); (b) sodium salt of 1,7‐heptanediol, dioxane; (c) Pac‐Cl, TEA, THF; (d) TBAF (1 M in THF), THF; (e) Cl‐P(OCE)N i Pr2 , DIPEA, THF. Abbreviations: TEA, triethylamine; POCl3 , phosphorus(V) oxychloride; MeCN, acetonitrile; DCM, dichloromethane; Pac‐Cl, phenoxyacetyl chloride; THF, tetrahydrofuran; TBAF, tetrabutylammonium fluoride; Cl‐P(OCE)N i Pr2 , N,N‐diisopropylamino cyanoethyl phosphonamidic chloride; DIPEA, diisopropylethylamine; DMT, 4,4′‐dimethoxytrityl; TBDMS, tert ‐butyldimethylsilyl; Pac, phenoxyacetyl.

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Figure 5.13.3 Preparation of 9 . (a) DBU, pyridine; (b) TBAF (1 M in THF), THF; (c) Cl‐P(OCE)N i Pr2 , DIPEA, THF. Abbreviations: DBU, 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene; TBAF, tetrabutylammonium fluoride; THF, tetrahydrofuran; Cl‐P(OCE)N i Pr2 , N,N‐diisopropylamino cyanoethyl phosphonamidic chloride; DIPEA, diisopropylethylamine; DMT, 4,4′‐dimethoxytrityl; TBDMS, tert ‐butyldimethylsilyl.

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Figure 5.13.4 Mono‐phosphoramidite approach for assembly of oligonucleotides containing a O 4 ‐hydroxyalkyl‐thymidine by solid‐phase synthesis. (a) Oligonucleotide synthesis with 2′‐deoxyribonucleoside‐3′‐ O ‐phosphoramidites; (b) coupling with phosphoramidite 6 (labeled as T* ); (c) chain extension with 2′‐deoxyribonucleoside 3′‐phosphoramidites; (d) cleavage from the solid‐support and deprotection with 10% DBU in 1,7‐heptanediol for oligonucleotides containing 6 .

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Figure 5.13.5 Bis‐phosphoramidite approach for assembly of oligonucleotides containing an O 4 ‐ thymidine‐alkylene‐ O 4 ‐thymidine cross‐link by solid‐phase synthesis in a (A ) directly opposing fashion and in a (B ) staggered fashion. (a) Oligonucleotide synthesis with 2′‐deoxyribonucleoside 3′‐phosphoramidites; (b) coupling with bis‐phosphoramidite 9 (labeled as T‐T ); (c) chain extension with 2′‐deoxyribonucleoside 3′‐phosphoramidites; (d) cleavage from the solid support and deprotection with 10% DBU in anhydrous ethanol.

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