Simple, High‐Yield Syntheses of DNA Duplexes Containing Interstrand DNA‐DNA Cross‐Links Between an N4‐Aminocytidine Residue and an Abasic Site

Jacqueline Gamboa Varela1, Kent S. Gates2

1 Department of Chemistry, University of Missouri, Columbia, Missouri, 2 Department of Biochemistry, University of Missouri, Columbia, Missouri
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.16
DOI:  10.1002/cpnc.3
Online Posting Date:  June, 2016
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Abstract

The protocol describes the preparation and purification of interstrand DNA‐DNA cross‐links derived from the reaction of an N4‐aminocytidine residue with an abasic site in duplex DNA. The procedures employ inexpensive, commercially available chemicals and enzymes to carry out post‐synthetic modification of commercially available oligodeoxynucleotides. The yield of cross‐linked duplex is typically better than 90%. If purification is required, the cross‐linked duplex can be readily separated from single‐stranded DNA starting materials by denaturing gel electrophoresis. The resulting covalent hydrazone‐based cross‐links are stable under physiologically relevant conditions and may be useful for biophysical studies, structural analyses, DNA repair studies, and materials science applications. © 2016 by John Wiley & Sons, Inc.

Keywords: abasic; click chemistry; DNA cross‐link; DNA repair; hydrazone; nanomaterials; oligonucleotide

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of an Oligodeoxynucleotide Containing an N4‐Aminocytidine Residue (dC*)
  • Basic Protocol 2: Preparation of a 5′‐32P‐Labeled Oligodeoxynucleotide Duplex (5) Containing the Ap‐dC* Interstrand DNA‐DNA Cross‐link
  • Basic Protocol 3: Preparation of an Unlabeled DNA Duplex Containing the Interstrand Preparation of an Unlabeled Dna Duplex Containing the Interstrand Ap‐dC* Cross‐Link
  • Basic Protocol 4: Purification Cross‐Linked Duplex 8 Using PAGE
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of an Oligodeoxynucleotide Containing an N4‐Aminocytidine Residue (dC*)

  Materials
  • Water (HPLC grade, Sigma‐Aldrich)
  • 1 M Sodium Phosphate buffer, pH 5 (reagent grade, Fisher)
  • 3 M Sodium bisulfite (NaHSO 3), freshly prepared (ACS reagent grade, Sigma‐Aldrich)
  • 8 M Hydrazine monohydrochloride freshly prepared (reagent grade, Sigma‐Aldrich)
  • 0.1 mM Oligodeoxynucleotide 1 (Integrated DNA Technologies) in water
  • 0.5 M Tris base (ACS reagent grade, Sigma‐Aldrich)
  • 0.1 M Triethylamine, freshly prepared (TEA, reagent grade, Sigma‐Aldrich)
  • 0.001 M Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA; ACS reagent grade, Sigma‐Aldrich)
  • Acetonitrile (HPLC grade, Sigma‐Aldrich)
  • 0.01 M Ammonium acetate (BioXtra grade, Sigma‐Aldrich)
  • 8:2 (v:v) Methanol:water (HPLC grade, Sigma‐Aldrich)
  • Crushed dry ice
  • 1.5‐ and 2‐mL polypropylene microcentrifuge tubes (Eppendorf)
  • Vortex‐mixer (Fisher Vortex Genie 2)
  • Thermostat‐controlled aluminum heating block
  • C‐18 Sep‐Pak cartridges (Waters, 1 mL, 100 mg, cat. no. WAT023590)
  • 1‐mL syringes
  • Speed‐Vac Concentrator (SC 110, Savant)

Basic Protocol 2: Preparation of a 5′‐32P‐Labeled Oligodeoxynucleotide Duplex (5) Containing the Ap‐dC* Interstrand DNA‐DNA Cross‐link

  Materials
  • 0.1 mM Oligodeoxynucleotide 3 (Integrated DNA Technologies) in water
  • Water (HPLC grade, Sigma‐Aldrich)
  • Uracil‐DNA glycosylase reaction buffer 10× (UDG buffer; New England Biolabs)
  • Uracil‐DNA glycosylase enzyme (UDG enzyme; New England Biolabs)
  • 25:24:1 Phenol‐chloroform‐isoamyl alcohol (BioUltra for molecular biology, Sigma‐Aldrich)
  • 3 M Sodium acetate, pH 5.2 (ACS reagent grade, Sigma‐Aldrich)
  • Absolute ethanol (200 proof, Decon Labs)
  • Crushed dry ice
  • 8:2 (v:v) Ethanol:water (HPLC grade, Sigma‐Aldrich)
  • 1 M Sodium phosphate buffer, pH 5 (Fisher)
  • 1 M Sodium chloride (NaCl; Fisher)
  • dC*‐containing oligodeoxynucleotide 2 (from protocol 1)
  • 1.5‐mL microcentrifuge tubes
  • Vortex‐mixer (Fisher Vortex Genie 2)
  • Thermostat‐controlled oven‐incubator set at 37°C
  • Benchtop centrifuge (5424, Eppendorf)
  • Benchtop centrifuge in cold room 4°C (AccuSpin Micro 17, Fisher Scientific)
  • Speed‐Vac concentrator (SC 110, Savant)
  • Thermostat‐controlled heating block

Basic Protocol 3: Preparation of an Unlabeled DNA Duplex Containing the Interstrand Preparation of an Unlabeled Dna Duplex Containing the Interstrand Ap‐dC* Cross‐Link

  Materials
  • 0.1 mM Oligodeoxynucleotide 6 (Integrated DNA Technologies) in water
  • Water (HPLC grade, Sigma‐Aldrich)
  • Uracil‐DNA glycosylase “10×” reaction buffer (UDG buffer; New England Biolabs)
  • Uracil‐DNA glycosylase enzyme (UDG enzyme; New England Biolabs)
  • 25:24:1 Phenol‐chloroform‐isoamyl alcohol (BioUltra for molecular biology, Sigma‐Aldrich)
  • 1 M Sodium phosphate buffer, pH 5 (Fisher)
  • 1 M Sodium chloride (NaCl; Fisher)
  • dC*‐containing oligodeoxynucleotide 2 (from protocol 1)
  • 3 M Sodium acetate, pH 5.2 (Sigma‐Aldrich)
  • 1.5 mL microcentrifuge tubes
  • Vortex‐mixer (Fisher Vortex Genie 2)
  • Thermostat‐controlled oven‐incubator set at 37°C
  • Benchtop centrifuge (5424, Eppendorf)
  • Benchtop centrifuge in cold room 4°C (AccuSpin Micro 17, Fisher Scientific)
  • Speed‐vac Concentrator (SC 110, Savant)
  • Thermostat‐controlled heating block

Basic Protocol 4: Purification Cross‐Linked Duplex 8 Using PAGE

  Materials
  • 20% denaturing polyacrylamide solution (19:1 acrylamide/bis‐acrylamide, 8 M urea; Fisher)
  • N,N,N′,N′‐Tetramethylethylenediamine (TEMED; BioReagent, for molecular biology; Sigma‐Aldrich)
  • 10% (w/v) Aqueous ammonium persulfate (for molecular biology, for electrophoresis; Sigma‐Aldrich)
  • Tris‐borate‐EDTA buffer, 1× (TBE buffer; BioReagent, for molecular biology; Sigma‐Aldrich)
  • Formamide loading buffer (see recipe) (17.75 M formamide, deionized, Calbiochem), 0.01 M EDTA (Sigma‐Aldrich) with bromophenol blue dye (ACS reagent, Sigma‐Aldrich)
  • Elution buffer (0.2 M NaCl, 0.001 M EDTA, pH 8)
  • Crushed dry ice
  • Water (HPLC grade, Sigma‐Aldrich)
  • Glass gel plates (16 × 19.7 cm) with 2‐mm thick spacers and 12‐well comb
  • 1.5‐ and 2‐mL microcentrifuge tubes
  • 100‐mL beaker and magnetic stir bar
  • 20‐mL disposable syringe and 18‐G, 1.5‐in. needle
  • Electrophoresis power source (2060‐FBS, E‐C Apparatus Corporation)
  • Vortex‐mixer (Fisher Vortex Genie 2)
  • All‐Purpose Laboratory Wrap (Saran Wrap, Fisherbrand)
  • UV lamp and silica gel TLC plate impregnated with UV‐254 fluorophore (Sigma‐Aldrich)
  • Disposable razor blade
  • Glass rod
  • Poly‐Prep Chromatography column (spin column, Bio‐Rad Laboratories)
  • Clinical centrifuge (spin‐bucket, IEC)
  • C‐18 Sep‐Pak cartridges (1 mL, 100 mg, Waters, cat. no.WAT023590)
  • Speed‐vac Concentrator (SC 110, Savant)
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Figures

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Literature Cited

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