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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.


Related in: MedlinePlus

Kinetics of the oligonucleotide system.(a) The entire proxHCR process were broken down in four processes and evaluated using a SPR biosensor technology. Upper left corner: hybridization and invasion of the activator oligonucleotide to PH1 is reasonably fast and dissociation is not observed. Binding of this oligonucleotide complex to PH2 also shows high association and almost no reverse reaction (upper right corner). Binding of H1 to the initiator sequence (lower left corner) as well as binding of H2 to H1-initiator oligonucleotide complex is also strong (lower right corner). (b) In the Opera High Content Screening System, we further evaluated the properties of the used oligonucleotide system. Here, even after 5 min we could distinguish between 10 and 20 nM HCR oligonucleotides. The reaction slowed down after about 30 min with fluorescence only increasing marginally up to 120 min. These results could be confirmed using an epifluorescence microscope (c). After 10 min of amplification, a strong difference could be observed between positive and negative control. The difference continued to increase until 30 min, after which it stagnated, indicating depletion of HCR oligonucleotides. (d) Pictures of the positive beads at 10, 30, 60 and 150 min.
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f3: Kinetics of the oligonucleotide system.(a) The entire proxHCR process were broken down in four processes and evaluated using a SPR biosensor technology. Upper left corner: hybridization and invasion of the activator oligonucleotide to PH1 is reasonably fast and dissociation is not observed. Binding of this oligonucleotide complex to PH2 also shows high association and almost no reverse reaction (upper right corner). Binding of H1 to the initiator sequence (lower left corner) as well as binding of H2 to H1-initiator oligonucleotide complex is also strong (lower right corner). (b) In the Opera High Content Screening System, we further evaluated the properties of the used oligonucleotide system. Here, even after 5 min we could distinguish between 10 and 20 nM HCR oligonucleotides. The reaction slowed down after about 30 min with fluorescence only increasing marginally up to 120 min. These results could be confirmed using an epifluorescence microscope (c). After 10 min of amplification, a strong difference could be observed between positive and negative control. The difference continued to increase until 30 min, after which it stagnated, indicating depletion of HCR oligonucleotides. (d) Pictures of the positive beads at 10, 30, 60 and 150 min.

Mentions: The first round of experiments was performed using surface plasmon resonance biosensor technology (SPR), to evaluate the kinetics of the interacting oligonucleotides. The experiments show an efficient binding of the activator oligonucleotide to PH1 (Fig. 3a). Furthermore, the subsequent binding of the opened PH1 to the PH2 also occurs efficiently without any measurable dissociation. Owing to the very slow dissociation (kd<10−4 s−1), complex affinity or kinetic rate constants could not be quantified. Next, we switched to the initiator sequence (Table 1), which is identical to the part that sticks out of the activator–PH1–PH2 complex and acts as an initiating oligonucleotide for the HCR. The results show fast association of H1 with the initiator, while again almost no dissociation could be observed. The same holds true for the H1–H2 interaction. Again, for both reactions no quantitative data could be obtained. Control experiments show no visible association between PH2 alone and H1 or H2. In addition, the activator–PH1 complex alone does not interact with H1 or H2 (Supplementary 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)

Kinetics of the oligonucleotide system.(a) The entire proxHCR process were broken down in four processes and evaluated using a SPR biosensor technology. Upper left corner: hybridization and invasion of the activator oligonucleotide to PH1 is reasonably fast and dissociation is not observed. Binding of this oligonucleotide complex to PH2 also shows high association and almost no reverse reaction (upper right corner). Binding of H1 to the initiator sequence (lower left corner) as well as binding of H2 to H1-initiator oligonucleotide complex is also strong (lower right corner). (b) In the Opera High Content Screening System, we further evaluated the properties of the used oligonucleotide system. Here, even after 5 min we could distinguish between 10 and 20 nM HCR oligonucleotides. The reaction slowed down after about 30 min with fluorescence only increasing marginally up to 120 min. These results could be confirmed using an epifluorescence microscope (c). After 10 min of amplification, a strong difference could be observed between positive and negative control. The difference continued to increase until 30 min, after which it stagnated, indicating depletion of HCR oligonucleotides. (d) Pictures of the positive beads at 10, 30, 60 and 150 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Kinetics of the oligonucleotide system.(a) The entire proxHCR process were broken down in four processes and evaluated using a SPR biosensor technology. Upper left corner: hybridization and invasion of the activator oligonucleotide to PH1 is reasonably fast and dissociation is not observed. Binding of this oligonucleotide complex to PH2 also shows high association and almost no reverse reaction (upper right corner). Binding of H1 to the initiator sequence (lower left corner) as well as binding of H2 to H1-initiator oligonucleotide complex is also strong (lower right corner). (b) In the Opera High Content Screening System, we further evaluated the properties of the used oligonucleotide system. Here, even after 5 min we could distinguish between 10 and 20 nM HCR oligonucleotides. The reaction slowed down after about 30 min with fluorescence only increasing marginally up to 120 min. These results could be confirmed using an epifluorescence microscope (c). After 10 min of amplification, a strong difference could be observed between positive and negative control. The difference continued to increase until 30 min, after which it stagnated, indicating depletion of HCR oligonucleotides. (d) Pictures of the positive beads at 10, 30, 60 and 150 min.
Mentions: The first round of experiments was performed using surface plasmon resonance biosensor technology (SPR), to evaluate the kinetics of the interacting oligonucleotides. The experiments show an efficient binding of the activator oligonucleotide to PH1 (Fig. 3a). Furthermore, the subsequent binding of the opened PH1 to the PH2 also occurs efficiently without any measurable dissociation. Owing to the very slow dissociation (kd<10−4 s−1), complex affinity or kinetic rate constants could not be quantified. Next, we switched to the initiator sequence (Table 1), which is identical to the part that sticks out of the activator–PH1–PH2 complex and acts as an initiating oligonucleotide for the HCR. The results show fast association of H1 with the initiator, while again almost no dissociation could be observed. The same holds true for the H1–H2 interaction. Again, for both reactions no quantitative data could be obtained. Control experiments show no visible association between PH2 alone and H1 or H2. In addition, the activator–PH1 complex alone does not interact with H1 or H2 (Supplementary 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.


Related in: MedlinePlus