Pseudo‐Ligandless Click Chemistry for Oligonucleotide Conjugation

Stephanie Mack1, Munira F. Fouz1, Sourav K. Dey1, Subha R. Das2

1 These authors contributed equally to this work, 2 Department of Chemistry and Center for Nucleic Acids Science & Technology, Carnegie Mellon University, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.1
Online Posting Date:  June, 2016
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Abstract

Particularly for its use in bioconjugations, the copper‐catalyzed (or copper‐promoted) azide‐alkyne cycloaddition (CuAAC) reaction or ‘click chemistry’, has become an essential component of the modern chemical biologist's toolbox. Click chemistry has been applied to DNA, and more recently, RNA conjugations, and the protocols presented here can be used for either. The reaction can be carried out in aqueous buffer, and uses acetonitrile as a minor co‐solvent that serves as a ligand to stabilize the copper. The method also includes details on the analysis of the reaction product. © 2016 by John Wiley & Sons, Inc.

Keywords: alkyne; azide; bioconjugation; DNA; labeling; nucleic acids; RNA

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: General Pseudo‐Ligandless Click Reaction
  • Support Protocol 1: Degassing Of Solutions and Reactions
  • Support Protocol 2: HPLC Purification and Analysis of Pseudo‐Ligandless Click Reaction
  • Support Protocol 3: Purification and Analysis of Pseudo‐Ligandless Click Reaction by Denaturing (8 M Urea) Polyacrylamide Gel Electrophoresis
  • Alternate Protocol 1: Pseudo‐Ligandless Click Reaction for Conjugation of Alkyne DNA or RNA to Cy3 Azide Dye
  • Alternate Protocol 2: Pseudo‐Ligandless Click Reaction for Synthesis of Backbone Branched DNA or RNA
  • Reagents And Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: General Pseudo‐Ligandless Click Reaction

  Materials
  • Argon source (see protocol 2)
  • 10 mM aqueous copper (II) sulfate (CuSO 4) solution
  • 50 mM aqueous sodium ascorbate solution (prepared fresh)
  • 10× phosphate‐buffered saline (PBS; see recipe)
  • 20% acetonitrile (ACN) solution in distilled, deionized H 2O
  • Solution of alkyne DNA or RNA in distilled deionized H 2O (see Tables 15.2.2100 and 15.2.2100)
  • Solution of azide Cy3 dye or DNA or RNA in distilled, deionized H 2O (see Tables 15.2.2100 and 15.2.2100)
  • Gel loading solution (see recipe)
  • 0.65‐ and 1.7‐ml microcentrifuge tubes
  • Vortex Genie with foam insert for microcentrifuge tubes
  • Desalting column: Glen Pak RNA or DNA column (Glen Research)
  • Additional reagents and equipment for degassing ( protocol 2) and denaturing PAGE (Andrus and Kuimelis, )

Support Protocol 1: Degassing Of Solutions and Reactions

  Materials
  • Argon gas
  • Solutions to be gassed, in microcentrifuge tubes
  • 50‐ml Erlenmeyer flask
  • 19/22 rubber septum
  • 3‐ml plastic syringe
  • 7‐in. balloon
  • Rubber bands
  • 21 G 1.5‐in. needle
  • Vortex Genie with foam insert for microtubes

Support Protocol 2: HPLC Purification and Analysis of Pseudo‐Ligandless Click Reaction

  Materials
  • Reaction mixture from Basic Protocol 1
  • 0.1 M TEAA, pH 7 (see recipe)
  • 80/20 (v/v) ACN/aqueous 0.1 M TEAA (see recipe)
  • Lyophilizer
  • Waters HPLC running Breeze software or equivalent system for binary separations
  • PDA detector
  • Waters XBridge C 18 column 5 μm (4.6 × 150 mm)
  • Additional reagents and equipment for HPLC (Sinha and Jung, )

Support Protocol 3: Purification and Analysis of Pseudo‐Ligandless Click Reaction by Denaturing (8 M Urea) Polyacrylamide Gel Electrophoresis

  Materials
  • Ethanol
  • 20% gel mix with 8 M urea in 1× TBE (see recipe)
  • Tetramethylethylenediamine (TEMED)
  • 10% (v/v) aqueous ammonium persulfate (APS)
  • 0.5× TBE buffer (see recipe)
  • Gel loading solution for denaturing PAGE (see recipe)
  • Acetonitrile (ACN)
  • H 2O
  • 50/50 (v/v) ACN/H 2O
  • Glass plates for 14‐cm‐wide gel; 17 cm × 12 cm (width × height; one plate is 2 cm shorter)
  • 1‐mm‐thick spacers
  • Binder clips
  • 100‐ml beaker
  • 1 mm thick comb with at least 4 teeth of approximately 7 mm width for sample loading
  • Electrophoresis system
  • UV lamp (shortwave; 254 nm) transilluminator
  • UV fluorescent TLC plate (20 cm × 20 cm)
  • Whatman Elutrap Device
  • SepPak Classic C18 desalting cartridge (Waters)
  • Lyophilizer
  • Additional reagents and equipment for denaturing PAGE (Andrus and Kuimelis, )
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Figures

Videos

Literature Cited

Literature Cited
  Andrus, A. and Kuimelis, R.G. 2000. Polyacrylamide gel electrophoresis (PAGE) of synthetic nucleic acids. Curr. Protoc. Nucleic Acid Chem. 1:10.4:10.4.1–10.4.10.
  Averick, S., Paredes, E., Li, W., Matyjaszewski, K., and Das, S.R. 2011. Direct DNA conjugation to star polymers for controlled reversible assemblies. Bioconjugate Chem. 22:2030‐2037. doi: 10.1021/bc200240q.
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  Paredes, E., Zhang, X., Ghodke, H., Yadavalli, V.K., and Das, S.R. 2013. Backbone‐branched DNA building blocks for facile angular control in nanostructures. ACS Nano. 7:3953‐3961. doi: 10.1021/nn305787.
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