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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.


The different hairpin species.Proximity hairpin 1 (a) and proximity hairpin 2 (b) can be conjugated to the affinity reagents, to yield proximity probes. H1 (c) and H2 (d) are used for the amplification reagent and are labelled by a fluorophore or some other detection reagent. The reaction is started by the activator (e), which binds to PH1 and unlocks the bridging strand to bind at PH2. The different domains are colour coded to emphasize identical and reverse complementary regions in the different oligonucleotides.
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f2: The different hairpin species.Proximity hairpin 1 (a) and proximity hairpin 2 (b) can be conjugated to the affinity reagents, to yield proximity probes. H1 (c) and H2 (d) are used for the amplification reagent and are labelled by a fluorophore or some other detection reagent. The reaction is started by the activator (e), which binds to PH1 and unlocks the bridging strand to bind at PH2. The different domains are colour coded to emphasize identical and reverse complementary regions in the different oligonucleotides.

Mentions: Multiple optimization steps brought forward the sequences provided in Table 1. The optimization process is shown in Supplementary Table 1. The two arms adapt secondary structures with long stems (30 and 24 bp for PH1 and PH2, respectively) and relatively big loops (18 and 19 nt for the two proximity hairpins, respectively; Fig. 2a,b). The negative Gibbs free energy (–ΔG) for both secondary structures is estimated to be quite high (40 and 27 kcal mol−1 at 37 °C in 1 M NaCl). The HCR hairpins have similar structures to commonly used HCR oligonucleotides6. Namely a short sticky end (9 nt 5′ for H1 and 11 nt 3′ for H2), the stem is 15 bp for both oligonucleotides and the loops consist of 11 nt for H1 and 9 nt for H2 (Fig.2c,d). The two mismatches between the two proximity hairpins (position 16 and 17 of PH2) are worth mentioning. This mismatch is introduced to suppress generation of false-positive signal.


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)

The different hairpin species.Proximity hairpin 1 (a) and proximity hairpin 2 (b) can be conjugated to the affinity reagents, to yield proximity probes. H1 (c) and H2 (d) are used for the amplification reagent and are labelled by a fluorophore or some other detection reagent. The reaction is started by the activator (e), which binds to PH1 and unlocks the bridging strand to bind at PH2. The different domains are colour coded to emphasize identical and reverse complementary regions in the different oligonucleotides.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: The different hairpin species.Proximity hairpin 1 (a) and proximity hairpin 2 (b) can be conjugated to the affinity reagents, to yield proximity probes. H1 (c) and H2 (d) are used for the amplification reagent and are labelled by a fluorophore or some other detection reagent. The reaction is started by the activator (e), which binds to PH1 and unlocks the bridging strand to bind at PH2. The different domains are colour coded to emphasize identical and reverse complementary regions in the different oligonucleotides.
Mentions: Multiple optimization steps brought forward the sequences provided in Table 1. The optimization process is shown in Supplementary Table 1. The two arms adapt secondary structures with long stems (30 and 24 bp for PH1 and PH2, respectively) and relatively big loops (18 and 19 nt for the two proximity hairpins, respectively; Fig. 2a,b). The negative Gibbs free energy (–ΔG) for both secondary structures is estimated to be quite high (40 and 27 kcal mol−1 at 37 °C in 1 M NaCl). The HCR hairpins have similar structures to commonly used HCR oligonucleotides6. Namely a short sticky end (9 nt 5′ for H1 and 11 nt 3′ for H2), the stem is 15 bp for both oligonucleotides and the loops consist of 11 nt for H1 and 9 nt for H2 (Fig.2c,d). The two mismatches between the two proximity hairpins (position 16 and 17 of PH2) are worth mentioning. This mismatch is introduced to suppress generation of false-positive signal.

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.