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Proximity-dependent initiation of hybridization chain reaction
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2015 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7294Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2015. Vol. 6, article id 7294
National Category
Medical and Health Sciences Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-255596DOI: 10.1038/ncomms8294ISI: 000357171100008PubMedID: 26065580OAI: oai:DiVA.org:uu-255596DiVA, id: diva2:822783
Funder
EU, FP7, Seventh Framework Programme, 278568, 316929, 259796Swedish Foundation for Strategic Research Swedish Research Council
Available from: 2015-06-17 Created: 2015-06-17 Last updated: 2018-01-25Bibliographically approved
In thesis
1. Applications of in situ proximity ligation assays for cancer research and diagnostics
Open this publication in new window or tab >>Applications of in situ proximity ligation assays for cancer research and diagnostics
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the field of cancer research and diagnostics it is crucial to have reliable methods for detecting molecules involved in the disease. New and better methods for diagnostics, prognostics and drug delivery therefore remain a permanent aim. In this thesis applications of the in situ proximity ligation assay (in situ PLA) were developed for diagnostics and research. Two new methods were developed, one more cost effective proximity assay without the use of enzymes and one method for loading pharmaceuticals in lipid rafts made from detergent resistant membranes (DRMs) to be used as a drug delivery platform.

In Paper I the aim was to develop a flow cytometric detection method of the fusion protein BCR-ABL that is the hallmark of chronic myeloid leukemia (CML). By using in situ PLA the malignant cells carrying the fusion protein could be detected in patients in a convenient workflow.

Paper II describes an application of multiplex in situ PLA, where extracellular vesicles (EVs) are detected and identified using flow cytometry. Up to five different antigens are targeted on the EVs, reflected in three different colors during detection in the flow cytometer. By using antibodies targeting proteins specific for prostasomes a population of prostasomes could be identified in human blood plasma.

In Paper III a new method is described for using lipid raft for drug delivery. In this method, lipid rafts, derived from prostasomes or erythrocytes, are loaded with pharmaceuticals. The vehicles were loaded with doxorubicin, added to cells and counted. Cells that received the vehicle with doxorubicin stopped proliferating and died, while controls that received the lipid raft vehicle without doxorubicin were not affected, suggesting that the vehicles are effectively loaded with the drug and that they are safe. This lipid raft vehicle could provide a safe drug delivery system.     

Paper IV investigates the crosstalk between the two major signal pathways Hippo and Wnt, and how these are affected in gastric cancer. When looking at different colon cancer tumor stages, we found that the cellular localization of TAZ/β-catenin interactions were different. We also found that protein complexes involved in the crosstalk increased in sparsely growing cells compared to more densely growing cells. On the basis of these results the protein E-cadherin, involved in maintenance of the epithelial integrity, was investigated and was found to have a probable role in regulating the crosstalk between Hippo and Wnt.    

A new method for localized protein detection is described in paper V. Here a proximity assay, based on the hybridization chain reaction (HCR), was developed. This assay, proxHCR, is more cost effective than in situ PLA because no enzymes are required. ProxHCR successfully detects protein interactions and can be used together with both fluorescence microscopy and flow cytometry. 

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. p. 56
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1241
Keyword
Cancer, Diagnostics, Research, Prognostics, Method development, Flow cytometry, Drug delivery
National Category
Medical and Health Sciences
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-300191 (URN)978-91-554-9638-8 (ISBN)
External cooperation:
Public defence
2016-09-23, B/B22, Husargatan 4, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2016-09-01 Created: 2016-08-04 Last updated: 2016-09-05
2. Development of DNA-based methods for analysis of protein interactions
Open this publication in new window or tab >>Development of DNA-based methods for analysis of protein interactions
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In situ proximity ligation assay (PLA) is a method for detection of protein interactions, post-translational modifications (PTMs) and individual proteins that allows information about their localization in a cell or tissue to be extracted. The method is based on oligonucleotide-conjugated antibodies (proximity probes) that upon binding of two epitopes in close proximity give rise to an amplifiable DNA circle. Rolling circle amplification (RCA) is used to create a DNA bundle of over a thousand repeats to which fluorescently labeled detection oligonucleotides are hybridized. This thesis is focused on improving the existing in situ PLA method and on developing new approaches for detection of proteins, protein-protein interactions and PTMs in situ in cells and tissues.

In paper I, a new enzyme-independent method capable of in situ detection of protein-protein interactions was developed. The method combined the proximity requirement of in situ PLA and the amplification of hybridization chain reaction (HCR) creating a proximity-dependent initiation of hybridization chain reaction (proxHCR). Circumventing the need for enzymes resulted in a cost-efficient method that is less sensitive to storing conditions.

Paper II addresses the problem of irregularly formed RCA products that can appear to be split into several fluorescent objects. A compaction oligonucleotide system was designed to crosslink the DNA bundle with itself and thereby reduce the size and increase the brightness of each individual RCA product.

In paper III, the conventional in situ PLA was redesigned to increase the detection efficiency of protein interactions and PTMs in situ. The new set of proximity probes was designed to have circularization oligonucleotides incorporated that were unfolded through enzymatic digestion. The UnFold in situ PLA was able to generate more signals and had a higher sensitivity than the conventional in situ PLA.

In paper IV, an oligonucleotide system able to generate signals for individual proteins (A or B) and their interaction (A and B) in a molecular Boolean (MolBoolean) protein analysis was designed. The MolBoolean design was able to generate signals detecting both individual proteins and their interaction in situ.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 47
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 245
Keyword
In situ proximity ligation assay (PLA), rolling circle amplification (RCA), Hybridization chain reaction (HCR), proxHCR, Oligonucleotide design, Protein-protein interactions, Post-translational modifications.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Cell and Molecular Biology
Research subject
Pharmaceutical Science; Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-340078 (URN)978-91-513-0222-5 (ISBN)
Public defence
2018-03-15, A1:107a, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2018-02-21 Created: 2018-01-25 Last updated: 2018-03-07

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Grannas, KarinLöf, LizaArngården, LindaHeldin, JohanKlaesson, Axelde Oliveira, Felipe Marques SouzaTalibov, Vladimir ODanielson, U HelenaKamali-Moghaddam, MasoodSöderberg, Ola

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Grannas, KarinLöf, LizaArngården, LindaHeldin, JohanKlaesson, Axelde Oliveira, Felipe Marques SouzaTalibov, Vladimir ODanielson, U HelenaKamali-Moghaddam, MasoodSöderberg, Ola
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