Synthesis and Characterization of Chimeric 2‐5A‐DNA Oligonucleotides

Mark R. Player1, Paul F. Torrence1

1 Northern Arizona University, Flagstaff, Arizona
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.4
DOI:  10.1002/0471142700.nc0404s01
Online Posting Date:  May, 2001
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Abstract

This unit provides protocols for the synthesis and characterization of 2‐5A‐antisense nucleic acids. These chimeric oligonucleotides consist of 2′,5′‐phosphodiester‐linked oligoadenylates ligated to 3′,5′‐deoxyribonucleotides and are readily prepared using phosphoramidite chemistry on CPG solid supports. The 3′,5′‐deoxyribonucleotide functions as the antisense domain to target a given mRNA sequence, while the 2′,5′‐phosphodiester‐linked oligoadenylate serves to locally activate 2‐5A‐dependent RNase L, causing the targeted sequence to be cleaved.

     
 
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Table of Contents

  • Basic Protocol 1: Automated Synthesis of Chimeric 2‐5A‐Antisense Oligonucleotides According to Solid‐Phase Phosphoramidite Chemistry
  • Support Protocol 1: Preparation of Key Intermediate for Synthesis of the (2′,5′)‐Oligoadenylate (2‐5A) Domain
  • Support Protocol 2: Preparation of a Linker Intermediate that Joins (2′‐5′)‐Oligoadenylate (2‐5A) to Antisense (3′‐5′)‐Oligodeoxyribonucleotides
  • Basic Protocol 2: Purification and Chromatographic Characterization of Chimeric 2‐5A Antisense Oligonucleotides
  • Basic Protocol 3: Composition Analysis of Chimeric 2‐5A‐Antisense Oligonucleotides Using Snake Venom Phosphodiesterase Digestion and Reversed‐Phase HPLC
  • Basic Protocol 4: Sequencing Chimeric 2‐5A‐Antisense Oligonucleotides According to a Modified Maxam‐Gilbert Procedure
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Automated Synthesis of Chimeric 2‐5A‐Antisense Oligonucleotides According to Solid‐Phase Phosphoramidite Chemistry

  Materials
  • Reagents for oligonucleotide synthesis:
  •  Acetonitrile (CH 3CN; diluent; Cruachem)
  •  Tetrazole/acetonitrile (activator/coupling solution; PE Biosystems)
  •  Acetic anhydride/lutidine/tetrahydrofuran (capping solution; PE Biosystems)
  •  N‐Methylimidazole/tetrahydrofuran (capping solution; PE Biosystems)
  •  Trichloroacetic acid/CH 2Cl 2 (detritylation solution; PE Biosystems)
  •  Iodine/H 2O/pyridine/tetrahydrofuran (oxidizer; PE Biosystems)
  •  Tetraethylthiuram disulfide (TETD)/CH 3CN (PE Biosystems; for sulfurization)
  • 0.1 M phosphoramidite solutions (see recipe):
  •  Linker CE phosphoramidite: 4‐O‐(4,4′‐dimethoxytrityl)oxybutyl‐1‐ [(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite] (see protocol 3)
  •  RNA CE phosphoramidite (2‐5A): N6‐benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐ 3′‐O‐(tert‐butyldimethylsilyl)adenosine‐2′‐(N,N‐diisopropyl‐2‐ cyanoethyl)phosphoramidite (see protocol 2)
  •  Phosphorylation reagent: 2‐[2‐(4,4′‐Dimethoxytrityl)ethylsulfonyl]ethyl‐ (2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite
  •  5′‐O‐Dimethoxytrityl‐N6‐benzoyl‐2′‐deoxyadenosine‐3′‐ (2‐cyanoethyl‐N,N‐diisopropyl)phosphoramidite (dABz)
  •  5′‐O‐Dimethoxytrityl‐N2‐isobutyryl‐2′‐deoxyguanosine‐3′‐(2‐cyanoethyl‐ N,N‐diisopropyl)phosphoramidite (dGi‐Bu)
  •  5′‐O‐Dimethoxytrityl‐N4‐benzoyl‐2′s‐deoxycytidine‐3′‐(2‐cyanoethyl‐N,N‐ diisopropyl)phosphoramidite (dCBz)
  •  5′‐O‐Dimethoxytrityl‐2′‐deoxythymidine‐3′‐(2‐cyanoethyl‐N,N‐ diisopropyl)phosphoramidite (T)
  • 3:1 (v/v) concentrated NH 4OH/ethanol
  • 1 M n‐tetrabutylammonium fluoride (TBAF)/tetrahydrofuran (THF; Aldrich)
  • 10 mM n‐tetrabutylammonium phosphate (TBAP), pH 7.5, in H 2O
  • Automated DNA synthesizer (ABI Biotechnologies model 391 or 392) with 5′ or 3′ solid supports:
  •  5′‐O‐Dimethoxytrityl‐N6‐benzoyl‐2′‐deoxyadenosine‐3′‐lcaa‐CPG (1 µmol; dABz‐lcaa‐CPG; ABI)
  •  5′‐O‐Dimethoxytrityl‐N4‐benzoyl‐2′‐deoxycytidine‐3′‐lcaa‐CPG (1 µmol; dCBz‐lcaa‐CPG; ABI)
  •  5′‐O‐Dimethoxytrityl‐N2‐isobutyryl‐2′‐deoxyguanosine‐3′‐lcaa‐CPG (1 µmol; dGi‐Bu‐lcaa‐CPG; ABI)
  •  5′‐O‐Dimethoxytritylthymidine‐3′‐lcaa‐CPG (1 µmol; T‐lcaa‐CPG; ABI)
  •  3′‐O‐Dimethoxytrityl‐N6‐benzoyl‐2′‐deoxyadenosine‐5′‐lcaa‐CPG (1 µmol; Glen Research)
  •  3′‐O‐Dimethoxytrityl‐N4‐benzoyl‐2′‐deoxycytidine‐5′‐lcaa‐CPG (1 µmol; Glen Research)
  •  3′‐O‐Dimethoxytritylthymidine‐5′‐lcaa‐CPG (1 µmol; Glen Research)
  •  3′‐O‐Dimethoxytrityl‐N2‐isobutyryl‐2′‐deoxyguanosine‐5′‐lcaa‐CPG (1 µmol; Glen Research).
  • Water bath at 55°C
  • Speedvac evaporator
NOTE: The above‐mentioned 5′ solid supports are used in the steps below. The 3′ solid supports are used in place of the 5′ supports when synthesizing oligonucleotides with terminal (3′‐3′)‐phosphodiester bonds.

Support Protocol 1: Preparation of Key Intermediate for Synthesis of the (2′,5′)‐Oligoadenylate (2‐5A) Domain

  Materials
  • Pyridine (Aldrich) dried over molecular sieves
  • Adenosine (Aldrich) dried overnight in vacuo over P 2O 5 (Aldrich)
  • 4‐Dimethylaminopyridine (DMAP; Aldrich)
  • Triethylamine (Aldrich)
  • 4,4′‐Dimethoxytrityl chloride (DMTr⋅Cl; Aldrich)
  • Chloroform (CHCl 3; Aldrich)
  • Methanol (Aldrich)
  • Trimethylsilyl chloride (Aldrich)
  • Benzoyl chloride (Aldrich)
  • Concentrated ammonium hydroxide (Aldrich)
  • Diethyl ether (Aldrich)
  • MgSO 4 (anhydrous; Aldrich)
  • Kieselgel 60 silica gel (220 to 440 mesh; Fluka)
  • Ethyl acetate (Aldrich)
  • Toluene (Aldrich)
  • Imidazole (Aldrich)
  • tert‐Butyldimethylsilyl chloride (TBDMS⋅Cl; Aldrich)
  • N,N‐Dimethylformamide (DMF; anhydrous; Aldrich)
  • Cyclohexane (Aldrich)
  • 5% (w/v) aqueous Na 2CO 3
  • Methylene chloride (CH 2Cl 2; Aldrich)
  • 1H‐Tetrazole (Aldrich)
  • P 2O 5 (Aldrich)
  • Dry nitrogen
  • 2‐Cyanoethyl‐(N,N,NN′‐tetraisopropyl)phosphoramidite(Aldrich)
  • Benzene
  • 250‐mL flask with ground‐glass stopper
  • Rotary evaporator with vacuum pump
  • 2 × 15‐cm, 5 × 15‐cm, and 5 × 20‐cm chromatography columns
  • 25‐mL two‐arm reaction flask fitted with a rubber septum
  • 5‐mL hypodermic syringe
  • Additional reagents and equipment for thin‐layer chromatography (TLC) and fast silica gel column (flash) chromatography

Support Protocol 2: Preparation of a Linker Intermediate that Joins (2′‐5′)‐Oligoadenylate (2‐5A) to Antisense (3′‐5′)‐Oligodeoxyribonucleotides

  Materials
  • 1,4‐Butanediol (Aldrich)
  • Pyridine (anhydrous; Aldrich)
  • Dry argon or nitrogen (Aldrich)
  • 4,4′‐Dimethoxytrityl chloride (DMTr⋅Cl; Aldrich)
  • Chloroform (Aldrich)
  • Methanol (Aldrich)
  • Ethyl acetate (Aldrich)
  • MgSO 4 (anhydrous; Aldrich)
  • Kieselgel 60 silica gel (Fluka)
  • Methylene chloride (CH 2Cl 2; Aldrich)
  • Ethyldiisopropylamine (Aldrich)
  • 2‐Cyanoethyl‐N,N‐diisopropylphosphoramidic chloride (Aldrich)
  • Benzene (Aldrich)
  • Petroleum ether (Aldrich)
  • Triethylamine (Aldrich)
  • 250‐ml stoppered flask
  • Rotary evaporator attached to a vacuum pump
  • Hand‐held UV lamp
  • Additional reagents and equipment for thin‐layer chromatography (TLC) and fast silica gel column (flash) chromatography

Basic Protocol 2: Purification and Chromatographic Characterization of Chimeric 2‐5A Antisense Oligonucleotides

  Materials
  • Crude 2‐5A‐antisense chimera (see protocol 1)
  • Solvent A: 10 mM n‐tetrabutylammonium phosphate (TBAP), pH 7.5, in H 2O (for ion‐pair chromatography)
  • Solvent B: 10 mM TBAP, pH 7.5, in 8:2 (v/v) CH 3CN/H 2O (for ion‐pair chromatography)
  • Methanol
  • Solvent C: 20 mM potassium phosphate, pH 7.0, in 8:2 (v/v) CH 3CN/H 2O (for anion‐exchange chromatography)
  • Solvent D: 20 mM potassium phosphate, pH 7.0, in aqueous 1 M KCl (for anion‐exchange chromatography)
  • Dowex 50W (Na+ form; Bio‐Rad)
  • Tris/methanol running buffer: 75 mM Tris phosphate, pH 7.6, in 9:1 (v/v) H 2O/methanol
  • Solvent E: 25 mM Tris⋅Cl, pH 7.0 ( appendix 2A) in 1:200 (v/v) CH 3CN/H 2O
  • Solvent F: 25 mM Tris⋅Cl, 1 M ammonium chloride, pH 7.0, in 1:200 (v/v) CH 3CN/H 2O
  • High‐performance liquid chromatograph (HPLC) with 270‐nm UV detection
  • 300 × 7‐mm polystyrene reversed‐phase column (PRP‐1 semi‐prep column; 10 µm, 100 Å; Hamilton; for ion‐pair chromatography)
  • Speedvac evaporator
  • SepPak C18 cartridge
  • 125 × 4–mm Nucleogen DEAE 60‐7 column (7 µm, 60 Å; Macherey‐Nagel; for anion‐exchange chromatography)
  • PolyPrep column
  • Millex‐GV 0.22‐µm filter unit (Millipore)
  • SpectraPor dialysis chamber (V t = 5 mL) with a 3500 MWCO membrane (Spectrum)
  • Capillary electrophoresis instrument (ABI model 270A‐HT) with MICRO‐GEL 100 gel‐filled capillaries (50‐µm i.d., 27‐cm effective length) and UV detection at 260 nm
  • 250 × 4‐mm Dionex PA‐100 column (Dionex)
  • Additional reagents and equipment for determining OD 260 (unit 10.3)

Basic Protocol 3: Composition Analysis of Chimeric 2‐5A‐Antisense Oligonucleotides Using Snake Venom Phosphodiesterase Digestion and Reversed‐Phase HPLC

  Materials
  • Purified 2‐5A‐antisense chimera (see protocol 4)
  • Snake venom phosphodiesterase (SVPD, Crotallus adamanteus; Amersham Pharmacia Biotech)
  • 1 M Tris⋅Cl, pH 8.0 ( appendix 2A)
  • 1 M MgCl 2
  • Solvent A: aqueous 100 mM ammonium phosphate, pH 5.5
  • Solvent B: 1:1 (v/v) methanol/H 2O
  • Microcon‐10 concentrator (Amicon)
  • High‐performance liquid chromatograph (HPLC) with 250 × 4.6–mm Ultrasphere C18 ODS column (5 µm, 80 Å; Thomson Instrument)

Basic Protocol 4: Sequencing Chimeric 2‐5A‐Antisense Oligonucleotides According to a Modified Maxam‐Gilbert Procedure

  Materials
  • Purified 2‐5A‐antisense chimera (see protocol 4)
  • 5× TNT buffer (see recipe)
  • [α‐32P]ddATP (3000 Ci/mmol, 10 mCi/mL; Amersham)
  • 15 U/µL terminal deoxyribonucleotide transferase (TNT) from calf thymus (Life Technologies)
  • 0.5 M EDTA, pH 8 ( appendix 2A)
  • 1 mg/mL calf thymus DNA (Clontech)
  • 0.5 M sodium phosphate buffer, pH 6.8 (see recipe)
  • Scintillation fluid
  • Diethyl pyrocarbonate (DEPC; Sigma)
  • DEPC buffer (see recipe)
  • Ethanol
  • 0.3 and 3 M sodium acetate (Quality Biological), pH 6, in water
  • 4 mg/mL yeast tRNA (Clontech)
  • Dimethyl sulfate (DMS; Aldrich)
  • DMS buffer (see recipe)
  • DMS stop buffer (see recipe)
  • A>C buffer (see recipe)
  • 1 N acetic acid
  • Hydrazine (HZ; Aldrich)
  • HZ stop buffer (see recipe)
  • 5 M NaCl (Quality Biological) in water
  • 1 M piperidine (Aldrich) in water, freshly prepared
  • Denaturing gel‐loading buffer (see recipe)
  • 10× TBE electrophoresis buffer ( appendix 2A)
  • Lyophilizer
  • ChromaSpin‐10 gel column (Clontech)
  • Whatman DE81 filters
  • Vacuum manifold
  • Siliconized tubes
  • Heating blocks at 25°, 60°, and 90°C
  • Additional reagents and equipment for polyacrylamide gel electrophoresis (PAGE; appendix 3B)
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Literature Cited

Literature Cited
   Beaucage, S.L. and Caruthers, M.H. 1981. Deoxyribonucleotide phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22:1859‐1862.
   Cirino, N.M., Li, G., Xiao, W., Torrence, P.F., and Silverman, R.H. 1997. Targeting RNA for decay in respiratory syncytial virus infected cells with 2′→5′ oligoadenylate antisense. Proc. Natl. Acad. Sci. U.S.A. 94:1937‐1942.
   Johnston, M.I. and Torrence, P.F. 1984. The role of interferon‐induced proteins, double‐stranded RNA and 2′,5′‐oligoadenylate in the interferon‐mediated inhibition of viral translation. In Interferon: Mechanism of Production and Action (R.M. Freidman, ed.) pp. 189‐298. Elsevier/ North‐Holland, Amsterdam.
   Lesiak, K., Khamnei, S., and Torrence, P.F. 1993. 2′,5′‐Oligoadenylate:antisense chimeras—Synthesis and properties. Bioconjugate Chem. 4:467‐472.
   Li, G., Xiao, W., and Torrence, P.F. 1997. Synthesis and properties of second generation 2‐5A‐anti‐sense chimeras with enhanced resistance to exonucleases. J. Med. Chem. 40:2959‐2966.
   Maitra, R.K., Li, G., Xiao, W., Dong, B., Torrence, P., and Silverman, R.H. 1995. Catalytic cleavage of an RNA target by 2‐5A antisense and RNase L. J. Biol. Chem. 270:15071‐15075.
   Maran, A., Maitra, R.K., Kumar, A., Dong, B., Xiao, W., Li, G., Williams, B.R.G., Torrence, P.F., and Silverman, R.H. 1994. Blockage of NF‐kB signaling by selective ablation of an mRNA target by 2‐5A antisense chimeras. Science 265:789‐792.
   Player, M. and Torrence, P.F. 1998. The 2‐5A system: Modulation of viral and cellular processes through acceleration of RNA degradation. Pharmacol. Therapeut. 78:55‐113.
   Torrence, P.F., Maitra, R.K., Lesiak, K., Khamnei, S., Zhou, A., and Silverman, R.H. 1993. Targeting RNA for degradation with a (2′‐5′)oligoadenylate‐antisense chimera. Proc. Natl. Acad. Sci. U.S.A. 90:1300‐1304.
   Xiao, W., Player, M.R., Li, G., Zhang, K., Lesiak, K., and Torrence, P.F. 1996. Synthesis and characterization of composite nucleic acids containing 2′,5′‐oligoriboadenylate linked to antisense DNA. Antisense Nucleic Acid Drug Devel. 6:247‐258.
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