uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Proximity Depended Initiation of Hybridization Chain Reaction
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Show others and affiliations
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: 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 should be cheap and robust.

Results: Building on hybridization chain reaction, we designed a four hairpin system which is metastable in solution at 37°C for several hours and undergoes rapid signal amplification upon introduction of an initiator oligonucleotide. When the proximity hairpins are conjugated to antibodies these proximity probes in combination with the HCR hairpins and the initiator oligonucleotide provide a specific, enzyme free method to detect HIF-1α/HIF-1β and potentially other protein interactions and PTMs in situ. Furthermore it was possible to detect single proteins in the different compartments of the cell, further proving the specificity of this technique.

Conclusion: In this study we present proximity dependent HCR, which is a cheap and robust method to detect protein interactions and post-translational modifications. Because of its independence from enzymes the technique has only low demands on storage and handling which makes it interesting for point of care devices.

National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-218249OAI: oai:DiVA.org:uu-218249DiVA: diva2:695200
Available from: 2014-02-10 Created: 2014-02-10 Last updated: 2015-07-07Bibliographically approved
In thesis
1. Making Visible the Proximity Between Proteins
Open this publication in new window or tab >>Making Visible the Proximity Between Proteins
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Genomic DNA is the template of life - the entity which is characterized by a self-sustaining anatomical development, regulated signaling processes, the ability to reproduce and to respond to stimuli. Through what is classically known as the central dogma, the genome is transcribed into mRNA, which in turn is translated into proteins. The proteins take part in most, if not all, cellular processes, and it is by unraveling these processes that we can begin to understand life and disease-causing mechanisms.

In vitro and in vivo assays are two levels at which protein communication may be studied, and which permit manipulation and control over the proteins under investigation. But in order to retrieve a representation of the processes as close to reality as possible, in situ analysis may instead be applied as a complement to the other two levels of study. In situ PLA offers the ability to survey protein activity in tissue samples and primary cell lines, at a single cell level, detecting single targets in their natural unperturbed environment.  

In this thesis new developments of the in situ PLA are described, along with a new technique offering in situ enzyme-free detection of proximity between biomolecules.

The dynamic range of in situ PLA has now been increased by several orders of magnitude to cover analogous ranges of protein expression; the output signals have been modified to offer a greater signal-to-noise ratio and to limit false-positive-rates while also extending the dynamic range further; simultaneous detection of multiple protein complexes is now possible; proximity-HCR is presented as a robust and inexpensive enzyme-free assay for protein complex detection.

The thesis also covers descriptions on how the techniques may be simultaneously applied, also together with other techniques, for the multiple data-point acquisition required by the emerging realm of systems biology. A future perspective is presented for how much more information may be simultaneously acquired from tissue samples to describe biomolecular interactions in a new manner. This will allow new types of biomarkers and drugs to be discovered, and a new holistic understanding of life.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 973
Keyword
Proximity ligation assay, In situ PLA, rolling circle amplification, protein interaction, protein-protein interaction, in situ, single cell, single molecule, protein complex, antibody, cancer, tissue section, microscopy, image analysis, system biology, multiplex, dynamic range, methods development, systems biology
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biomedical Laboratory Science/Technology Biochemistry and Molecular Biology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-217772 (URN)978-91-554-8878-9 (ISBN)
Public defence
2014-03-28, B8, Biomedicinskt centrum, Husargatan 3, SE-75123 Uppsala, 09:00 (English)
Opponent
Supervisors
Funder
Knut and Alice Wallenberg FoundationEU, FP7, Seventh Framework Programme, 259796EU, FP7, Seventh Framework Programme, 278568
Available from: 2014-03-06 Created: 2014-02-04 Last updated: 2014-04-29Bibliographically approved
2. Improvements and Applications of in situ Proximity Ligation Assays
Open this publication in new window or tab >>Improvements and Applications of in situ Proximity Ligation Assays
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cells building up the human body is in constant communication with each other. This communication is done through large complex networks of signaling pathways for inter- and intracellular signal transduction. The signaling activity regulates many important processes, for example cell death, proliferation and differentiation. Information within the signaling networks is communicated over the cell membrane, through the cytoplasm and entering the nucleus by protein activities such as protein-protein interactions (PPIs) and post translation modifications (PTMs). The cells adapts to their own environment, responding to multiple stimuli from their surroundings. This in combination with memory of previous responses, difference in cell cycles stages and sometimes altered genetic background generates heterogeneous cell populations in which every cell is slightly different from its neighbor. This calls for methods to study the activity of endogenous proteins in individual cells within a population.

In situ proximity ligation assay (in situ PLA) was originally developed to visualize interaction between endogenous proteins in fixed cells and tissue and can also be applied to detect PTMs. This thesis describe the application of in situ PLA to study PPIs in signaling pathways and the work to further develop and improve techniques for proximity dependent detection. 

In paper I in situ PLA is used to study cross talk between the Hippo and the TGFβ signaling pathways. The study shows the complex formation by the transcription co-factors of the Hippo pathway, Yap and Taz, and the main effectors of the TGFβ pathway Smad2/3. Furthermore the density dependent localization of the interaction is described.

Paper II presents a new version of the in situ PLA probes for simultaneous detection of multiple complexes. Visualization of various complexes involving EGFR, Her2 and Her3 is presented as a proof of concept.

The efficiency of in situ PLA is limited by several factors, one being the design of PLA probes and oligonucleotide systems. Even upon proximal binding of the probes there is a risk of formation of non-circular ligation products, which cannot be amplified and detected. In Paper III two new PLA probes are presented aiming to reduce the formation of non-circular ligation product and hence increase the detection efficiency of in situ PLA.

Paper IV presents a new method for detection of protein complexes and phosphorylation; proxHCR. ProxHCR combines signal amplification by enzyme free hybridization chain reaction (HCR) with the requirement of proximal binding of two affinity probes. As a proof of principle the method is used to detect multiple complexes and protein phosphorylation in fixed cells and tissue.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1099
National Category
Cell and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biomedical Laboratory Science/Technology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-248876 (URN)978-91-554-9233-5 (ISBN)
Public defence
2015-05-29, Biomedicinskt centrum (BMC), B/B42, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Foundation for Strategic Research
Available from: 2015-05-07 Created: 2015-04-08 Last updated: 2015-07-07

Open Access in DiVA

No full text

Authority records BETA

Koos, BjörnGrannas, KarinClausson, Carl-MagnusArngården, LindaKlaesson, AxelSöderberg, Ola

Search in DiVA

By author/editor
Koos, BjörnGrannas, KarinClausson, Carl-MagnusArngården, LindaKlaesson, AxelSöderberg, Ola
By organisation
Department of Immunology, Genetics and Pathology
Cell and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 551 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf