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DNA-Mediated Detection and Profiling of Protein Complexes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules.

Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 43 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 923
Keyword [en]
Proximity ligation assay, Protein complexes, Protein interactions, Biomarkers, Prions, Antibodies
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-204861ISBN: 978-91-554-8718-8 (print)OAI: oai:DiVA.org:uu-204861DiVA: diva2:640552
Public defence
2013-09-27, Rudbecksalen, Rudbeck Laboratory, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2013-09-06 Created: 2013-08-12 Last updated: 2014-01-08
List of papers
1. Sensitive plasma protein analysis by microparticle-based proximity ligation assays
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2010 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 2, 327-335 p.Article in journal (Refereed) Published
Abstract [en]

Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-123198 (URN)10.1074/mcp.M900248-MCP200 (DOI)000275506200010 ()19955079 (PubMedID)
Available from: 2010-04-26 Created: 2010-04-26 Last updated: 2017-12-12Bibliographically approved
2. Sensitive detection of aggregated prion protein by proximity ligation assay
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-204809 (URN)
Available from: 2013-08-13 Created: 2013-08-12 Last updated: 2014-01-08
3. Profiling cellular protein complexes by proximity ligation with dual tag microarray readout
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 7, e40405- p.Article in journal (Refereed) Published
Abstract [en]

Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.

Keyword
proximity ligation assay, protein interaction, dual tag microarray
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-177984 (URN)10.1371/journal.pone.0040405 (DOI)000306355500039 ()22808155 (PubMedID)
Funder
Swedish Research CouncilNIH (National Institute of Health), 1R21CA126727-01A1
Note

De 2 första författarna delar förstaförfattarskapet.

Correction in: PLoS ONE 10(3): e0119890. doi: 10.1371/journal.pone.0119890ISI: 000351880000047

Available from: 2012-07-23 Created: 2012-07-23 Last updated: 2017-12-07Bibliographically approved
4. A DNA-mediated search for optimal combinations of protein binders
Open this publication in new window or tab >>A DNA-mediated search for optimal combinations of protein binders
(English)Manuscript (preprint) (Other academic)
National Category
Other Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-204810 (URN)
Available from: 2013-08-13 Created: 2013-08-12 Last updated: 2014-01-08

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