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Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
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2013 (English)In: New Biotechnology, ISSN 1871-6784, Vol. 30, no 2, 144-152 p.Article in journal (Refereed) Published
Abstract [en]

While antibodies currently play a dominant role as affinity reagents in biological research and for diagnostics, a broad range of recombinant proteins are emerging as promising alternative affinity reagents in detection assays and quantification. DNA-mediated affinity-based assays, such as immuno-PCR and proximity ligation assays (PLA), use oligonucleotides attached to affinity reagents as reporter molecules. Conjugation of oligonucleotides to affinity reagents generally employs chemistries that target primary amines or cysteines. Because of the random nature of these processes neither the number of oligonucleotides conjugated per molecule nor their sites of attachment can be accurately controlled for affinity reagents with several available amines and cysteines. Here, we present a straightforward and convenient approach to functionalize recombinant affinity reagents for PLA by expressing the reagents as fusion partners with SNAP protein tags. This allowed us to conjugate oligonucleotides in a site-specific fashion, yielding precisely one oligonucleotide per affinity reagent. We demonstrate this method using designed ankyrin repeat proteins (DARPins) recognizing the tumor antigen HER2 and we apply the conjugates in different assay formats. We also show that SNAP or CLIP tags expressed as fusion partners of transfected genes, allow oligonucleotide conjugations to be performed in fixed cells, with no need for specific affinity reagents. The approach is used to demonstrate induced interactions between the fusion proteins FKBP and FRB by allowing the in situ conjugated oligonucleotides to direct the production of templates for localized rolling circle amplification reactions.

Place, publisher, year, edition, pages
2013. Vol. 30, no 2, 144-152 p.
Keyword [en]
genome reduction, SAR11, Alphaproteobacteria, mismatch repair system, ocean surface waters, Candidatus Pelagibacter ubique, recombination, gene loss
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-175988DOI: 10.1016/j.nbt.2012.05.005ISI: 000313786400007PubMedID: 22664266OAI: oai:DiVA.org:uu-175988DiVA: diva2:533815

De 2 första författarna delar förstaförfattarskapet.

Available from: 2012-06-14 Created: 2012-06-14 Last updated: 2013-03-08Bibliographically approved
In thesis
1. Proximity Ligation Assay for High Performance Protein Analysis in Medicine
Open this publication in new window or tab >>Proximity Ligation Assay for High Performance Protein Analysis in Medicine
2012 (English)Doctoral thesis, comprehensive summary (Other academic) [Artistic work]
Abstract [en]

High quality reagents are preconditions for high performance protein analyses. But despite progress in some techniques, e.g. mass spectrometry, there is still a lack of affinity-based detection techniques with enhanced precision, specificity, and sensitivity. Building on the concept of multiple affinity recognition reactions and signal amplification, a proximity ligation assay (PLA) was developed as a molecular tool for analyzing proteins and their post-translational modification and interactions. PLA enhanced the analysis of protein expression levels and post-translational modifications in western blotting (Paper I), which had elevated sensitivity and specificity, and an ability to investigate protein phosphorylation.

A general and straightforward method was established for the functionalization of affinity reagents through adding DNA strands to protein domains for protein analysis in medicine (Paper II). A method for protein domain-mediated conjugation was developed to simplify the use of recombinant affinity reagents, such as designed ankyrin repeat protein (DARPin), in DNA-mediated protein analyses.

Alzheimer’s disease (AD) is characterized by progressive cognitive decline and memory impairment, and amyloid-beta plaques and neurofibrillary tangles (NFT) in the brain are clinical hallmarks of the disease. In order to understand the mechanisms underlying the formation of NFT, in situ PLA was used to explore the role of microtubule affinity related kinase 2 (MARK2) in phosphorylating tau protein during the pathological progress of AD (Paper III). The analyses of roles of MARK proteins 1-4 in phosphorylating tau protein in cells and in post-mortem human brains were performed in Paper IV.

The focus of this thesis was the study of post-translational modifications and interactions of proteins in medicine. Procedures for high performance protein analysis in western blotting via proximity ligation were developed, and a functionalization method for recombinant affinity reagents in DNA-mediated protein analysis was established. These and other techniques were used to investigate the roles of tau-phosphorylating MARK family proteins in AD.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 49 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 801
protein, post-translational modification, interactions, protein domain, western blotting, MARK, tau, AD
National Category
Biological Sciences
Research subject
Biology with specialization in Molecular Biotechnology
urn:nbn:se:uu:diva-179827 (URN)978-91-554-8449-1 (ISBN)
Public defence
2012-10-05, Rudbeck hall, Rudbeck Laboratory, Dag Hammarskjölds väg20, Uppsala, 09:15 (English)
Available from: 2012-09-14 Created: 2012-08-23 Last updated: 2013-01-22Bibliographically approved

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