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Development of DNA-based methods for analysis of protein interactions
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
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
Keywords [en]
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: urn:nbn:se:uu:diva-340078ISBN: 978-91-513-0222-5 (print)OAI: oai:DiVA.org:uu-340078DiVA, id: diva2:1177746
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
List of papers
1. Proximity-dependent initiation of hybridization chain reaction
Open this publication in new window or tab >>Proximity-dependent initiation of hybridization chain reaction
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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.

National Category
Medical and Health Sciences Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-255596 (URN)10.1038/ncomms8294 (DOI)000357171100008 ()26065580 (PubMedID)
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
2. Compaction of rolling circle amplification products increases signal integrity and signal–to–noise ratio
Open this publication in new window or tab >>Compaction of rolling circle amplification products increases signal integrity and signal–to–noise ratio
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, p. 12317:1-10, article id 12317Article in journal (Refereed) Published
National Category
Medical Image Processing
Research subject
Computerized Image Processing
Identifiers
urn:nbn:se:uu:diva-260286 (URN)10.1038/srep12317 (DOI)000358358900001 ()26202090 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568EU, FP7, Seventh Framework Programme, 259796Swedish Research Council
Available from: 2015-07-23 Created: 2015-08-18 Last updated: 2018-02-27Bibliographically approved
3. Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes
Open this publication in new window or tab >>Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 5400Article in journal (Refereed) Published
Abstract [en]

We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes -UnFold probes - where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic "unfolding" step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-340077 (URN)10.1038/s41598-018-23582-1 (DOI)000428618900043 ()29599435 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568 264737 294409Swedish Foundation for Strategic Research Swedish Research Council
Note

Ola Söderberg and Ulf Landegren jointly supervised this work

Available from: 2018-01-25 Created: 2018-01-25 Last updated: 2018-08-06Bibliographically approved
4. A Boolean analysis of protein interactions at a molecular level in single cells
Open this publication in new window or tab >>A Boolean analysis of protein interactions at a molecular level in single cells
(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)
Identifiers
urn:nbn:se:uu:diva-340076 (URN)
Available from: 2018-01-25 Created: 2018-01-25 Last updated: 2018-01-25

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