Proximity-dependent initiation of hybridization chain reaction

Gaëlle Cane,Johan Heldin,Manfred Auer,Karin Grannas,Vladimir O. Talibov,Felipe M. S. de Oliveira,Axel Klaesson,M. Karoliina Hirvonen,Johannes Haybaeck,Ola Söderberg,Liza Löf,U. Helena Danielson,Carl-Magnus Clausson,Björn Koos,Masood Kamali-Moghaddam,Linda Arngården,Nhan T. Pham

doi:10.1038/ncomms8294

Abstract

Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

Highlights

Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes
As the activity of proteins in most cases is dependent on posttranslational modifications (PTMs) and protein–protein interactions (PPIs), analysis of these two events will provide information to determine the functional status of a cell or a signalling pathway
The first step would require proximal binding to make an oligonucleotide accessible that can in the second step facilitate the signal amplification brought about by hybridization chain reaction (HCR)

Summary

Introduction

Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. Here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. An enzyme-free signal amplification method employing oligonucleotides has recently been published[6] This method is called hybridization chain reaction (HCR) and is based on at least two different kinetically trapped hairpin structures. In this study we could show that proximity-dependent initiation of HCR (proxHCR) is a generally applicable enzymefree method to detect proteins, PTMs and PPIs

Methods

Results

Conclusion