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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.


Comparison proxHCR with in situ PLA.Interaction between E-cadherin and β-catenin in DLD1 cells (a: proxHCR; b: in situ PLA). The inlay panels in the upper left corner of a and b show a 150% magnification for better evaluation of signal size and number. The same interaction in frozen colon tissue also resulted in comparable results between proxHCR (c) and in situ PLA (d). White scale bars, 20 nm.
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f5: Comparison proxHCR with in situ PLA.Interaction between E-cadherin and β-catenin in DLD1 cells (a: proxHCR; b: in situ PLA). The inlay panels in the upper left corner of a and b show a 150% magnification for better evaluation of signal size and number. The same interaction in frozen colon tissue also resulted in comparable results between proxHCR (c) and in situ PLA (d). White scale bars, 20 nm.

Mentions: To test the feasibility of proxHCR to record PPIs and PTMs in situ, we established a number of assays against known interactions and PTMs in a multitude of different cell lines. Figure 4 shows the results of these assays. The E-cadherin/β-catenin shows a strong membranous staining in HT29 cells when both primary antibodies are applied (Fig.4a), whereas omitting either one or both of the primary antibodies results in no detectable signal (Fig. 4b–d). We can further show that a variety of PPIs and PTMs can be visualized using proxHCR (Fig. 4e–o). Among them are membrane receptors such as phosphoplatelet-derived growth factor receptor-β (PDGFR-β) (Fig. 4e,f), indicators of autophagy (that is, BCL2/BNIP3 (Fig. 4g,h) and LC3/STQM3 (Fig. 4i,j)) and members of prominent receptor tyrosine kinase pathways (MEK/ERK interaction (Fig. 4k,l) and phosphorylation of Akt (Fig. 4m,n)). Phosphorylation of Syk in HG3 cells is also very nicely shown (Fig. 4o,p). The biological controls of the induced interactions still show low basal activity (Fig. 4e,g,i,k,m), whereas the technical controls (omission of primary antibody) do not show visible signal (Supplementary Fig. 3). Even single protein detection is possible using proxHCR (Fig. 4q,r). Here, Her2 is visualized using two primary antibodies and two proximity probes. Furthermore, we can show the feasibility of proxHCR for formalin-fixed paraffin-embedded (FFPE) skin tissue sections, staining for E-cadherin and β-catenin (Fig. 4s,t). We used the interaction between E-cadherin and β-catenin in DLD1 cells and in fresh-frozen colon tissue as a model system to compare proxHCR with in situ PLA (Fig. 5a–d). The results show the same specific pattern of signal localization for in situ PLA and proxHCR in cultured cells (Fig. 5 a,b) and in fresh-frozen colon tissue (Fig. 5c,d).


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)

Comparison proxHCR with in situ PLA.Interaction between E-cadherin and β-catenin in DLD1 cells (a: proxHCR; b: in situ PLA). The inlay panels in the upper left corner of a and b show a 150% magnification for better evaluation of signal size and number. The same interaction in frozen colon tissue also resulted in comparable results between proxHCR (c) and in situ PLA (d). White scale bars, 20 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Comparison proxHCR with in situ PLA.Interaction between E-cadherin and β-catenin in DLD1 cells (a: proxHCR; b: in situ PLA). The inlay panels in the upper left corner of a and b show a 150% magnification for better evaluation of signal size and number. The same interaction in frozen colon tissue also resulted in comparable results between proxHCR (c) and in situ PLA (d). White scale bars, 20 nm.
Mentions: To test the feasibility of proxHCR to record PPIs and PTMs in situ, we established a number of assays against known interactions and PTMs in a multitude of different cell lines. Figure 4 shows the results of these assays. The E-cadherin/β-catenin shows a strong membranous staining in HT29 cells when both primary antibodies are applied (Fig.4a), whereas omitting either one or both of the primary antibodies results in no detectable signal (Fig. 4b–d). We can further show that a variety of PPIs and PTMs can be visualized using proxHCR (Fig. 4e–o). Among them are membrane receptors such as phosphoplatelet-derived growth factor receptor-β (PDGFR-β) (Fig. 4e,f), indicators of autophagy (that is, BCL2/BNIP3 (Fig. 4g,h) and LC3/STQM3 (Fig. 4i,j)) and members of prominent receptor tyrosine kinase pathways (MEK/ERK interaction (Fig. 4k,l) and phosphorylation of Akt (Fig. 4m,n)). Phosphorylation of Syk in HG3 cells is also very nicely shown (Fig. 4o,p). The biological controls of the induced interactions still show low basal activity (Fig. 4e,g,i,k,m), whereas the technical controls (omission of primary antibody) do not show visible signal (Supplementary Fig. 3). Even single protein detection is possible using proxHCR (Fig. 4q,r). Here, Her2 is visualized using two primary antibodies and two proximity probes. Furthermore, we can show the feasibility of proxHCR for formalin-fixed paraffin-embedded (FFPE) skin tissue sections, staining for E-cadherin and β-catenin (Fig. 4s,t). We used the interaction between E-cadherin and β-catenin in DLD1 cells and in fresh-frozen colon tissue as a model system to compare proxHCR with in situ PLA (Fig. 5a–d). The results show the same specific pattern of signal localization for in situ PLA and proxHCR in cultured cells (Fig. 5 a,b) and in fresh-frozen colon tissue (Fig. 5c,d).

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.