Designing and Applying Proximity‐Dependent Hybridization Chain Reaction

Björn Koos1, Ola Söderberg2

1 Max Planck Institute for Molecular Physiology, Department of Systemic Cell Biology, Dortmund, 2 Department of Immunology, Genetics and Pathology, Uppsala University, Science for Life Laboratory, Uppsala
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.28
DOI:  10.1002/cpps.9
Online Posting Date:  August, 2016
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Abstract

Proximity‐dependent hybridization chain reaction (proxHCR) is a novel technique for detection of protein interaction, post‐translational modifications (PTMs), or protein expression. The method is based upon antibodies targeting the proteins of interest that are covalently conjugated to DNA oligonucleotides, which enables the induction of a hybridization chain reaction (HCR) to generate a fluorescent signal visible under a microscope. In contrast to the in situ proximity ligation assay (in situ PLA), which is another method that utilizes antibody‐DNA conjugates to detect protein interactions, proxHCR does not require enzymatic steps. This makes proxHCR an inexpensive alternative to in situ PLA. Another potential advantage might be that proxHCR could more readily be adapted for use in automated staining procedures and in point‐of‐care devices, as all reagents can be stored at room temperature. This unit describes how the oligonucleotide system for proxHCR can be designed and a protocol for how to perform proxHCR is presented. © 2016 by John Wiley & Sons, Inc.

Keywords: hybridization chain reaction; protein interaction; post‐translational modification; proxHCR; proximity ligation assay

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Proximity‐Dependent Hybridization Chain Reaction (proxHCR)
  • Support Protocol 1: Preparation of Oligonucleotide‐Conjugated Antibodies
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Proximity‐Dependent Hybridization Chain Reaction (proxHCR)

  Materials
  • Fixed cells on slides
  • Blocking buffer of choice (e.g., Duolink blocking reagent, Olink Bioscience)
  • Primary antibodies and proximity probes (from conjugation as described in protocol 2Support Protocol)
  • TBS or TBS supplemented with Tween
  • HCR buffer: 1 M NaCl, 50 mM Na 2HPO 4, pH 7.4
  • Activator oligonucleotide (IDT)
  • Amplification oligonucleotides H1 and H2 modified with either fluorophores or other reporter molecules (IDT)
  • Hoechst, optional
  • 1× PBS
  • Ethanol
  • Mounting medium of choice
  • 37º and 95ºC incubators
  • Beakers

Support Protocol 1: Preparation of Oligonucleotide‐Conjugated Antibodies

  Materials
  • Antibodies of choice
  • SANH powder (Solulink)
  • DMSO
  • PBS
  • Zeba desalting columns (Thermo scientific)
  • PH1 and PH2 oligonucleotides with 5′ aldehyde modification (TriLink Biotechnologies)
  • Aniline (Sigma)
  • Polyacrylamide gel, optional
  • Dialysis cups (Thermo Scientific)
  • Amicon Ultra 0.5‐ml 30 K concentration columns (Merck Millipore)
  • 95ºC heating block
  • Sephadex 200 columns, optional
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Figures

Videos

Literature Cited

Literature Cited
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  Dirks, R.M. and Pierce, N.A. 2004. Triggered amplification by hybridization chain reaction. Proc. Nat. Acad. Sci. U.S.A. 101:15275‐15278. doi: 10.1073/pnas.0407024101.
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  Koos, B., Andersson, L., Clausson, C.M., Grannas, K., Klaesson, A., Cane, G., and Soderberg, O. 2014. Analysis of protein interactions in situ by proximity ligation assays. Curr. Top. Microbiol. Immun. 377:111‐126. doi: 10.1007/82_2013_334.
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  Zadeh, J.N., Steenberg, C.D., Bois, J.S., Wolfe, B.R., Pierce, M.B., Khan, A.R., Dirks, R.M., and Pierce, N.A. 2011. NUPACK: Analysis and design of nucleic acid systems. J. Comput. Chem. 32:170‐173. doi: 10.1002/jcc.21596.
  Zieba, A., Pardali, K., Soderberg, O., Lindbom, L., Nystrom, E., Moustakas, A., Heldin, C.H., and Landegren, U. 2012. Intercellular variation in signaling through the TGF‐beta pathway and its relation to cell density and cell cycle phase. Mol. Cell. Proteom. MCP 11:M111 013482. doi: 10.1074/mcp.M111.013482.
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