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Proximity-dependent initiation of hybridization chain reaction.

Koos B, Cane G, Grannas K, Löf L, Arngården L, Heldin J, Clausson CM, Klaesson A, Hirvonen MK, de Oliveira FM, Talibov VO, Pham NT, Auer M, Danielson UH, Haybaeck J, Kamali-Moghaddam M, Söderberg O - Nat Commun (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Uppsala University, Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Biomedical center, Husargatan 3, Box 815, SE-75108 Uppsala, Sweden.

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.

No MeSH data available.


Principle of proxHCR.(a) Primary antibodies of two different species detect their respective targets and get bound by proximity probes (secondary antibodies conjugated to proximity hairpins). The activator is introduced. (b) Activator oligonucleotide binds in the loop of PH1 and invades the stem, thereby releasing the 3′-end of the oligonucleotide. (c) PH1 binds the loop of PH2 and invades it as well. The 3′-end of PH2 sticks out as a fishing rod, which serves as an initiator for the hybridization chain reaction. (d) One fluorescently labelled HCR hairpin (H1) molecule gets bound and invaded by this initiator and in turn binds and invades one molecule of H2 (e). (f) The HCR continues until there are no more HCR hairpin molecules left to hybridize.
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f1: Principle of proxHCR.(a) Primary antibodies of two different species detect their respective targets and get bound by proximity probes (secondary antibodies conjugated to proximity hairpins). The activator is introduced. (b) Activator oligonucleotide binds in the loop of PH1 and invades the stem, thereby releasing the 3′-end of the oligonucleotide. (c) PH1 binds the loop of PH2 and invades it as well. The 3′-end of PH2 sticks out as a fishing rod, which serves as an initiator for the hybridization chain reaction. (d) One fluorescently labelled HCR hairpin (H1) molecule gets bound and invaded by this initiator and in turn binds and invades one molecule of H2 (e). (f) The HCR continues until there are no more HCR hairpin molecules left to hybridize.

Mentions: To obtain a system where proximal binding of two probes would start an HCR, we had to design a two-step reaction. 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 HCR. In contrast to regular HCR6, this will require four hairpin species and an activator instead of just two hairpin species (Table 1). Activation of proximity sensing is brought about by the addition of an activator oligonucleotide that will invade the first proximity hairpin (PH1). This will liberate a bridging sequence in PH1 that will invade the second proximity hairpin (PH2) only if this is in close proximity. The bridged PH1–PH2 will exhibit the initiator sequence for HCR amplification, which previously was hidden in the stem of PH2 (Fig. 1).


Proximity-dependent initiation of hybridization chain reaction.

Koos B, Cane G, Grannas K, Löf L, Arngården L, Heldin J, Clausson CM, Klaesson A, Hirvonen MK, de Oliveira FM, Talibov VO, Pham NT, Auer M, Danielson UH, Haybaeck J, Kamali-Moghaddam M, Söderberg O - Nat Commun (2015)

Principle of proxHCR.(a) Primary antibodies of two different species detect their respective targets and get bound by proximity probes (secondary antibodies conjugated to proximity hairpins). The activator is introduced. (b) Activator oligonucleotide binds in the loop of PH1 and invades the stem, thereby releasing the 3′-end of the oligonucleotide. (c) PH1 binds the loop of PH2 and invades it as well. The 3′-end of PH2 sticks out as a fishing rod, which serves as an initiator for the hybridization chain reaction. (d) One fluorescently labelled HCR hairpin (H1) molecule gets bound and invaded by this initiator and in turn binds and invades one molecule of H2 (e). (f) The HCR continues until there are no more HCR hairpin molecules left to hybridize.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490387&req=5

f1: Principle of proxHCR.(a) Primary antibodies of two different species detect their respective targets and get bound by proximity probes (secondary antibodies conjugated to proximity hairpins). The activator is introduced. (b) Activator oligonucleotide binds in the loop of PH1 and invades the stem, thereby releasing the 3′-end of the oligonucleotide. (c) PH1 binds the loop of PH2 and invades it as well. The 3′-end of PH2 sticks out as a fishing rod, which serves as an initiator for the hybridization chain reaction. (d) One fluorescently labelled HCR hairpin (H1) molecule gets bound and invaded by this initiator and in turn binds and invades one molecule of H2 (e). (f) The HCR continues until there are no more HCR hairpin molecules left to hybridize.
Mentions: To obtain a system where proximal binding of two probes would start an HCR, we had to design a two-step reaction. 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 HCR. In contrast to regular HCR6, this will require four hairpin species and an activator instead of just two hairpin species (Table 1). Activation of proximity sensing is brought about by the addition of an activator oligonucleotide that will invade the first proximity hairpin (PH1). This will liberate a bridging sequence in PH1 that will invade the second proximity hairpin (PH2) only if this is in close proximity. The bridged PH1–PH2 will exhibit the initiator sequence for HCR amplification, which previously was hidden in the stem of PH2 (Fig. 1).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Uppsala University, Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Biomedical center, Husargatan 3, Box 815, SE-75108 Uppsala, Sweden.

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.

No MeSH data available.