Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

Abstract We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circle...

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Autores principales:	Axel Klaesson, Karin Grannas, Tonge Ebai, Johan Heldin, Björn Koos, Mattias Leino, Doroteya Raykova, Johan Oelrich, Linda Arngården, Ola Söderberg, Ulf Landegren
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/75ad5875c3544fa48fd0da457da9bb4f
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spelling	oai:doaj.org-article:75ad5875c3544fa48fd0da457da9bb4f2021-12-02T15:09:02ZImproved efficiency of in situ protein analysis by proximity ligation using UnFold probes10.1038/s41598-018-23582-12045-2322https://doaj.org/article/75ad5875c3544fa48fd0da457da9bb4f2018-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-23582-1https://doaj.org/toc/2045-2322Abstract We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes –UnFold probes – where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic “unfolding” step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.Axel KlaessonKarin GrannasTonge EbaiJohan HeldinBjörn KoosMattias LeinoDoroteya RaykovaJohan OelrichLinda ArngårdenOla SöderbergUlf LandegrenNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-13 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Axel Klaesson Karin Grannas Tonge Ebai Johan Heldin Björn Koos Mattias Leino Doroteya Raykova Johan Oelrich Linda Arngården Ola Söderberg Ulf Landegren Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes
description	Abstract We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes –UnFold probes – where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic “unfolding” step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.
format	article
author	Axel Klaesson Karin Grannas Tonge Ebai Johan Heldin Björn Koos Mattias Leino Doroteya Raykova Johan Oelrich Linda Arngården Ola Söderberg Ulf Landegren
author_facet	Axel Klaesson Karin Grannas Tonge Ebai Johan Heldin Björn Koos Mattias Leino Doroteya Raykova Johan Oelrich Linda Arngården Ola Söderberg Ulf Landegren
author_sort	Axel Klaesson
title	Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes
title_short	Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes
title_full	Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes
title_fullStr	Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes
title_full_unstemmed	Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes
title_sort	improved efficiency of in situ protein analysis by proximity ligation using unfold probes
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/75ad5875c3544fa48fd0da457da9bb4f
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Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

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