Preparation of DNA and RNA Fragments Containing Guanine N2‐Thioalkyl Tethers

Xiaorong Hou1, Gang Wang1, Barbara L. Gaffney1, Roger A. Jones1

1 Rutgers, The State University of New Jersey, Piscataway, New Jersey
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.8
DOI:  10.1002/0471142700.nc0508s41
Online Posting Date:  June, 2010
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Abstract

This unit describes procedures for preparation of deoxyguanosine and guanosine derivatives in which the guanine N2 contains a thiopropyl tether, protected as a tert‐butyl disulfide. After incorporation into a DNA or RNA fragment, this tether allows site‐specific cross‐linking to a thiol of a protein or another nucleic acid. Curr. Protoc. Nucleic Acid Chem. 41:5.8.1‐5.8.23. © 2010 by John Wiley & Sons, Inc.

Keywords: oligonucleotides; disulfide cross‐links

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Diisopropyl‐1‐(tert‐Butylthio)‐1,2‐Hydrazinedicarboxylate (S.4)
  • Basic Protocol 2: Preparation of the 2′‐Deoxyguanosine N2‐Propyl‐tert‐Butyl Disulfide Phosphoramidite Derivative (S.12)
  • Basic Protocol 3: Preparation of the Guanosine N2‐Propyl‐tert‐Butyl Disulfide Phosphoramidite Derivative (S.20)
  • Basic Protocol 4: Preparation of DNA Fragments Containing N2‐Propyl‐tert‐Butyl Disulfide Guanine
  • Alternate Protocol 1: Preparation of RNA Fragments Containing N2‐Propyl‐tert‐Butyl Disulfide Guanine
  • Basic Protocol 5: Procedures for Converting the N2‐Propyl‐tert‐Butyl Disulfide to the Ethylamine Disulfide, 5‐Thio‐2‐Nitrobenzoic Acid (TNB) Disulfide, or Free Thiol
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Diisopropyl‐1‐(tert‐Butylthio)‐1,2‐Hydrazinedicarboxylate (S.4)

  Materials
  • 3‐Chloropropylammonium hydrochloride ( S.1; Aldrich)
  • Sodium thiosulfate pentahydrate (Na 2S 2O 3), reagent grade
  • Iodine
  • Methanol (MeOH), reagent grade
  • 6 N NaOH
  • Methylene chloride
  • Toluene
  • Dithiothreitol (DTT; Aldrich)
  • Concentrated HCl
  • 2‐Methyl‐2‐propanethiol (tert‐butyl thiol)
  • Diisopropyl azodicarboxylate ( S.5; Aldrich)
  • Diethyl ether, anhydrous
  • Nitrogen and argon sources
  • 25% (w/v) sodium methoxide in methanol
  • 6 N HCl
  • Dimethylformamide (DMF), anhydrous reagent, argon degassed
  • Triethylamine, anhydrous
  • Magnetic stir bars
  • Magnetic stir plate
  • Reflux condenser
  • Addition funnel
  • Rotary evaporator with diaphragm pump or water aspirator
  • 125‐mL separatory funnel
  • Falling film distillation apparatus (available from Aldrich; see Sigma‐Aldrich, )
  • Shaker
  • Silica gel column: 40 to 63 µm in 40 × 150–mm prepackaged cartridges (SciPro, cat. no. CO2001; http://www.scipro.com/)

Basic Protocol 2: Preparation of the 2′‐Deoxyguanosine N2‐Propyl‐tert‐Butyl Disulfide Phosphoramidite Derivative (S.12)

  Materials
  • 2′‐Deoxyguanosine monohydrate ( S.7; Aldrich)
  • 1,4‐Dioxane
  • Trimethylsilylimidazole (Aldrich)
  • Trimethylsilylethanol, Aldrich
  • Diethyl azodicarboxylate (DEAD; Aldrich)
  • Concentrated aqueous ammonia
  • Methylene chloride
  • Methanol (MeOH), reagent grade
  • 0.1 M triethylammonium chloride (TEAA), pH 6.8
  • Acetonitrile (CH 3CN)
  • Phosphorus pentoxide (P 2O 5)
  • Pyridine, anhydrous
  • Toluene, anhydrous
  • 70% HF⋅pyridine (Aldrich)
  • tert‐Butyl nitrite (Aldrich)
  • Dry ice/acetone bath
  • K 2CO 3
  • Ethyl acetate
  • Triethylamine
  • 4, 4′‐Dimethoxytrityl chloride (Aldrich)
  • Nitrogen source
  • 5% (w/v) sodium bicarbonate
  • Diethyl ether
  • S.4 ( protocol 1)
  • 1 M NaOH
  • Hexane
  • 2‐Cyanoethyl (N,N,N′,N′‐tetraisopropyl)phosphordiamidite (Cambridge Major, http://www.c‐mlabs.com)
  • Pyridinium trifluoroacetate (Aldrich)
  • Magnetic stir bars
  • Magnetic stir plate
  • 250‐mL separatory funnel
  • Rotary evaporator with diaphragm pump or water aspirator
  • Silica gel column: 40 to 63 µm in 40 × 150–mm prepackaged cartridges (SciPro, cat. no. CO2001; http://www.scipro.com/)
  • Reversed‐phase HPLC system with ESI‐MS
  • XTerra MS or Atlantis µC18 reversed‐phase columns

Basic Protocol 3: Preparation of the Guanosine N2‐Propyl‐tert‐Butyl Disulfide Phosphoramidite Derivative (S.20)

  Materials
  • Guanosine monohydrate ( S.13; Aldrich)
  • 1,4‐Dioxane
  • Trimethylsilylimidazole (Aldrich)
  • Nitrogen (or argon) source
  • Triphenylphosphine (Aldrich)
  • Trimethylsilylethanol (Aldrich)
  • Diethyl azodicarboxylate (DEAD; Aldrich)
  • Concentrated aqueous ammonia
  • Methylene chloride
  • Ethyl acetate
  • Pyridine, anhydrous
  • Toluene, anhydrous
  • 70% HF⋅pyridine (Aldrich)
  • Dry ice/acetone bath
  • tert‐Butyl nitrite (Aldrich)
  • K 2CO 3
  • Methanol
  • Triethylamine
  • P 2O 5
  • S.4 ( protocol 1)
  • 1 M NaOH
  • Dimethylformamide (DMF), anhydrous reagent, argon degassed
  • Di‐tert‐butylsilyl ditriflate (Aldrich)
  • Imidazole
  • tert‐Butyldimethylchlorosilane (Aldrich)
  • NaHCO 3
  • 4,4′‐Dimethoxytrityl chloride (Aldrich)
  • Acetone, HPLC grade
  • 2‐Cyanoethyl (N,N,N′,N′‐tetraisopropyl)phosphordiamidite (Cambridge Major, http://www.c‐mlabs.com)
  • Pyridinium trifluoroacetate (Aldrich)
  • Magnetic stir bars
  • Magnetic stir plate
  • 250‐mL separatory funnel
  • Rotary evaporator with diaphragm pump or water aspirator
  • Filter for vacuum filtration
  • Silica gel column: 40 to 63 µm in 40 × 150–mm prepackaged cartridges (SciPro, cat. no. CO2001; http://www.scipro.com/)
  • Vacuum desiccator

Basic Protocol 4: Preparation of DNA Fragments Containing N2‐Propyl‐tert‐Butyl Disulfide Guanine

  Materials
  • Phosphoramidite S.12 ( protocol 2)
  • Reagents specified for the synthesizer (also see appendix 3D)
  • Concentrated aqueous ammonia
  • 10% aqueous ammonia
  • Triethylamine (TEA) dried over CaH 2
  • Acetonitrile
  • 0.1 M triethylammonium acetate (TEAA)
  • 0.6 M acetic acid, HPLC grade
  • NH 4HCO 3, solid
  • 0.1 M NH 4HCO 3, pH 6.8 to 7.0
  • AG 50W‐X2 ion‐exchange resin (BioRad)
  • DNA/RNA synthesizer ( appendix 3D)
  • 50‐mL and 15‐mL conical centrifuge tubes
  • Sintered‐glass funnel, medium porosity
  • Reversed‐phase HPLC system (see unit 10.1) with ESI‐MS
  • UV/Vis spectrophotometer
  • 3‐mL syringes
  • Syringe filters, 4.5 mm, 0.45‐µm pore size
  • Semi‐preparative reversed‐phase HPLC system (see unit 10.1)
  • Anion‐exchange HPLC system
  • Glass column
  • Silica gel column: 40 to 63 µm in 40 × 150–mm prepackaged cartridges (SciPro, cat. no. CO2001; http://www.scipro.com/)
  • Additional reagents and equipment for oligonucleotide synthesis ( appendix 3D) and HPLC purification of oligonucleotides (discussed in unit 10.1)

Alternate Protocol 1: Preparation of RNA Fragments Containing N2‐Propyl‐tert‐Butyl Disulfide Guanine

  • Phosphoramidite S.20 ( protocol 3)
  • 40% (w/v) methylamine (aqueous)
  • 50% (v/v) ethanol
  • Triethylamine⋅HF (Aldrich)
  • Methyl‐2‐pyrrolidinone (NMP; Aldrich)
  • Triethylamine (TEA) dried over CaH 2
  • Isopropoxytrimethylsilane (Aldrich)
  • Ethyl ether, anhydrous
  • 65°C water bath
  • Large rubber septum
  • Vent needle connected to bubbler
  • Tabletop centrifuge
  • Spatula cleaned with nitric acid and rinsed well

Basic Protocol 5: Procedures for Converting the N2‐Propyl‐tert‐Butyl Disulfide to the Ethylamine Disulfide, 5‐Thio‐2‐Nitrobenzoic Acid (TNB) Disulfide, or Free Thiol

  Materials
  • Dithiothreitol (DTT; Aldrich)
  • Concentrated aqueous ammonia
  • Purified S.21( protocol 4 or protocol 5)
  • 5,5′‐Dithiobis(2‐nitrobenzoic acid) (Aldrich)
  • 0.1 M potassium phosphate buffer, pH 8.0 ( appendix 2A)
  • Cysteamine hydrochloride (2‐aminoethanethiol hydrochloride; Aldrich)
  • Cystamine dihydrochloride (2,2′‐diaminodiethyl disulfide dihydrochloride; Aldrich)
  • 40°C water bath
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Literature Cited

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