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research article

Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation

Gu, Gucci Jijuan
•
Friedman, Mikaela
•
Jost, Christian
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2013
New Biotechnology

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.

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Type
research article
DOI
10.1016/j.nbt.2012.05.005
Web of Science ID

WOS:000313786400007

Author(s)
Gu, Gucci Jijuan
Friedman, Mikaela
Jost, Christian
Johnsson, Kai  
Kamali-Moghaddam, Masood
Plueckthun, Andreas
Landegren, Ulf
Soderberg, Ola
Date Issued

2013

Publisher

Elsevier

Published in
New Biotechnology
Volume

30

Issue

2

Start page

144

End page

152

Editorial or Peer reviewed

REVIEWED

Written at

EPFL

EPFL units
LIP  
Available on Infoscience
March 28, 2013
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/90864
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