Synthesis of Peptide‐Oligonucleotide Conjugates Using a Heterobifunctional Crosslinker

Berea A.R. Williams1, John C. Chaput1

1 The Biodesign Institute at Arizona State University, Tempe, Arizona
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.41
DOI:  10.1002/0471142700.nc0441s42
Online Posting Date:  September, 2010
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Abstract

Peptide‐oligonucleotide conjugates (POCs) are molecular chimeras composed of a nucleic acid moiety covalently attached to a polypeptide moiety. POCs have been used in numerous applications from therapeutics to nanotechnology, and most recently as combinatorial agents in the assembly of bivalent protein affinity reagents. This unit describes the synthesis and purification of POC molecules using the heterobifunctional crosslinking reagent succinimidyl‐4‐(N‐maleimidomethyl)cyclohexane‐1‐carboxylate (SMCC), which enables amine‐modified oligonucleotides to become covalently linked to cysteine‐modified polypeptides. This solution‐based protocol consists of a two‐step synthesis followed by a single purification step. Curr. Protoc. Nucleic Acid Chem. 42:4.41.1‐4.41.20. © 2010 by John Wiley & Sons, Inc.

Keywords: peptide‐oligonucleotide conjugate; succinimidyl‐4‐(N‐maleimidomethyl)cyclohexane‐1‐carboxylate (SMCC); fragment conjugation; antisense; DNA nanotechnology; synbodies; nanodisplay

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of Amine‐Modified Oligodeoxynucleotide
  • Basic Protocol 2: Conjugation of SMCC to the Amine‐Modified Oligonucleotide
  • Basic Protocol 3: Conjugation of SMCC‐Oligonucleotide to a Cysteine‐Containing Peptide
  • Basic Protocol 4: Purification of Peptide‐Oligonucleotide Conjugates Using native‐PAGE
  • Alternate Protocol 1: Purification of Peptide‐Oligonucleotide Conjugates Using Reversed‐Phase HPLC
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Purification of Amine‐Modified Oligodeoxynucleotide

  Materials
  • Acrylamide (Promega)
  • 8 M urea (BDH)
  • Bisacrylamide (Promega)
  • 10× TBE buffer (see recipe)
  • 10% (w/v) ammonium persulfate (APS, EMD Biosciences)
  • N′,N′,N′,N′, Tetraethylmethylenediamine (TEMED, Pierce)
  • Amine‐modified oligonucleotide
  • 50 mM ethylenediaminetetraacetic acid, pH 8.0 (EDTA, Pierce)
  • Running dye: 0.05% (w/v) bromophenol blue and 0.05% (w/v) xylene cyanol in 1× TBE buffer (see recipe)
  • Gel elution buffer (see recipe)
  • 3 M sodium acetate, pH 5.2 (Sigma)
  • Absolute ethanol (Sigma)
  • 70% ethanol
  • Gel plates (19.7 × 16– and 19.7 × 18.5–cm)
  • Spacers (1.5‐mm thick)
  • Comb (1 well with 2 marker lanes)
  • 100‐mL beaker
  • Magnetic stir bar and stir plate
  • Electrophoresis apparatus
  • 50‐mL plastic syringe
  • Power supply
  • Heating block
  • 1.5‐mL disposable tubes
  • Plastic transfer pipets (pulled capillary)
  • Spatula
  • Plastic wrap
  • UV‐active thin‐layer chromatography (TLC) plate
  • Handheld UV lamp (254 nm)
  • Black permanent marker
  • Razor blade or scalpel
  • Ultrafiltration spin filter tubes (0.45‐µm, Millipore)
  • Microcentrifuge
  • Vortex

Basic Protocol 2: Conjugation of SMCC to the Amine‐Modified Oligonucleotide

  Materials
  • Purified amine‐modified oligonucleotide (see protocol 1)
  • Ultrapure water
  • 100 mM KH 2PO 4 buffer, pH 7.2 (BDH)
  • Succinimidyl‐4‐(N‐maleimidomethyl)cyclohexane‐1‐carboxylate (SMCC; Pierce)
  • Acetonitrile (Sigma)
  • Absolute ethanol (Sigma)
  • 70% ethanol
  • 3 M sodium acetate, pH 5.2 (Sigma)
  • 1.5‐mL disposable tubes
  • Vortex
  • Microcentrifuge

Basic Protocol 3: Conjugation of SMCC‐Oligonucleotide to a Cysteine‐Containing Peptide

  Materials
  • Polypeptide (>98% purity)
  • SMCC‐oligonucleotide (see protocol 2)
  • 100 mM KH 2PO 4 buffer, pH 7.2 (BDH)
  • 1.5‐mL siliconized disposable tubes (Fisher)
  • Vortex
  • Microcentrifuge

Basic Protocol 4: Purification of Peptide‐Oligonucleotide Conjugates Using native‐PAGE

  Materials
  • 40% (19:1) acrylamide/bisacrylamide solution (BioRad)
  • 10× TAE‐Mg2+ (see recipe)
  • 10% ammonium persulfate (APS, EMD Biosciences)
  • N′,N′,N′,N′, Tetraethylmethylenediamine (TEMED, Pierce)
  • Glycerol (BDH)
  • Crude POC material (see protocol 3)
  • Native running dye: 0.05% (w/v) bromophenol blue, 0.05% (w/v) xylene cyanol, 80% (v/v) glycerol
  • SyberGold (Invitrogen), optional
  • Gel plates (19.7 × 16–cm and 19.7 × 18.5–cm)
  • Spacers (1.5‐mm thick)
  • Comb (10‐wells)
  • 100‐mL beaker
  • Stir bar and stir plate
  • Electrophoresis apparatus
  • Power supply
  • 50‐mL syringes
  • NAP‐10 (GE Lifesciences)
  • 1.5‐mL siliconized disposable tubes (Fisher)
  • Lyophilizer

Alternate Protocol 1: Purification of Peptide‐Oligonucleotide Conjugates Using Reversed‐Phase HPLC

  Materials
  • HPLC mobile phase B: acetonitrile (HPLC‐grade, Sigma)
  • HPLC mobile phase A: 0.1 M ammonium acetate, pH 7.0
  • Crude POC material (see protocol 3)
  • 0.1 M ammonium acetate, pH 7.0
  • Nanopure water
  • High‐performance liquid chromatography (HPLC) system with:
    • Injector (autosampler preferred), sample loop, and syringe (for manual injections)
    • Binary pumping system
    • UV/Vis detector with wavelength detection between 200 and 300 nm
    • Analytical column: reversed‐phase column (i.e., Source 5RPC ST 4.6/150, Amersham)
    • Semi‐preparative column: reversed‐phase column (i.e., Source 15RPC ST 4.6/100, Amersham)
    • Automatic fraction collector (optional)
  • Nap‐10 columns
  • Lyophilizer
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Figures

Videos

Literature Cited

Literature Cited
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