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Unfolding proximity ligation probes for measuring and imaging individual nucleic acid and protein molecules
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. (Ulf Landegren)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. (Ulf Landegren)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. (Ola Söderberg)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. (Ola Söderberg)
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

We present a new class of probes for molecular detection reactions - probes for unfolding proximity ligation assays. These are secondarily structured nucleic acid reagents that can be induced to unfold in order to undergo proximity ligation reactions, followed by visualization via localized single molecule amplification. The probes enable highly specific targeting of individual nucleic acid or protein molecule. We demonstrated the performance of these new probes by detecting synthetic DNA and cancer-related transcripts in situ and multiplex probing of proteins in solution. 

Keyword [en]
unfolding, proximity ligation, single molecule, protein biomarker, cancer-related transcript
National Category
Biomedical Laboratory Science/Technology
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-158635OAI: oai:DiVA.org:uu-158635DiVA: diva2:441193
Available from: 2011-09-15 Created: 2011-09-12 Last updated: 2011-11-04
In thesis
1. Proximity Ligation Assays for Disease Biomarkers Analysis
Open this publication in new window or tab >>Proximity Ligation Assays for Disease Biomarkers Analysis
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

One of the pressing needs in the field of disease biomarker discovery is new technologies that could allow high performance protein analysis in different types of clinical material, such as blood and solid tissues. This thesis includes four approaches that address important limitations of current technologies, thus enabling highly sensitive, specific and parallel protein measurements.

Paper I describes a method for sensitive singleplex protein detection in complex biological samples, namely solid phase proximity ligation assay (SP-PLA). SP-PLA exhibited improved sensitivity compared to conventional sandwich immunoassays. We applied SP-PLA to validate the potential of GDF-15 as a biomarker for cardiovascular disease.  

Paper II describes ProteinSeq, a multiplexed immunoassay based on the principle of SP-PLA, for parallel detection of 36 proteins using next-generation sequencing as readout. ProteinSeq exhibited improved sensitivity compared to multiplexed sandwich immunoassays, and the potential to achieve even higher levels of multiplexing while preserving a high sensitivity and specificity. We applied ProteinSeq to analyze 36 proteins, including one internal control, in 5 μl of plasma samples in a cohort of patients with cardiovascular disease and healthy controls.

Paper III describes PLA-DTM, a strategy for recording all possible interactions between sets of proteins in clinical samples. Individual proteins and their interactions are first encoded to dual barcoded DNA by PLA, and the barcodes are interrogated by a method named dual tag microarray (DTM). We applied the method for studying interactions among protein members of the NFκB signaling pathway.

Paper IV describes a novel probing strategy for analyzing individual biomolecules in solution or in situ. The technique employs a new class of probes for unfolding proximity ligation assays - uPLA probes. The probes are designed so that each probe set is sufficient in forming and replicating circular DNA reporter, without interactions among themselves when incubated with the sample. The uPLA probing strategy provides ease in the design of multiple probe sets in parallelized assays while enhancing the specificity of detection. We used the uPLA probes to detect various targets, including synthetic DNA and cancer-related transcripts in situ.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 42 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 703
Keyword
proximity ligation assay, blood biomarkers, protein interactions, pathway analysis, single molecule, next-generation sequencing
National Category
Biomedical Laboratory Science/Technology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-158634 (URN)978-91-554-8158-2 (ISBN)
Public defence
2011-10-28, Rudbecksalen, Rudbecklaboratory, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Supervisors
Available from: 2011-10-07 Created: 2011-09-12 Last updated: 2015-08-10Bibliographically approved

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