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Efficient small-scale conjugation of DNA to primary antibodies for multiplexed cellular targeting

View ORCID ProfileGlenn A.O. Cremers, View ORCID ProfileBas J.H.M. Rosier, Roger Riera Brillas, Lorenzo Albertazzi, Tom F.A. de Greef
doi: https://doi.org/10.1101/662791
Glenn A.O. Cremers
aLaboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
bComputational Biology Group, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
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  • ORCID record for Glenn A.O. Cremers
Bas J.H.M. Rosier
aLaboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
bComputational Biology Group, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
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Roger Riera Brillas
aLaboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
cMolecular Biosensing for Medical Diagnostics, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
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Lorenzo Albertazzi
aLaboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
cMolecular Biosensing for Medical Diagnostics, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
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Tom F.A. de Greef
aLaboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
bComputational Biology Group, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
dInstitute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
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  • For correspondence: t.f.a.d.greef@tue.nl
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ABSTRACT

The combination of the specificity of antibodies and the programmability of DNA nanotechnology has provided the scientific community with a powerful tool to label and unambiguously distinguish a large number of subcellular targets using fluorescence-based read-out methods. While primary antibodies are commercially available for a large class of targets, a general stoichiometric site-specific DNA labeling strategy for this affinity reagent is lacking. Here, we present a universal, site-selective, conjugation method using a small photocrosslinkable protein G adaptor that allows labeling of antibodies of different host species with a controlled number of short oligonucleotides (ODNs). Importantly, we illustrate that this conjugation method can be directly performed on commercially-available primary antibodies, on a small scale and without cross-reactivity towards other proteins, such as bovine serum albumin. In addition, we present a general, benchtop-compatible strategy to purify DNA-labeled antibodies without loss of function. The application of protein G-ODN labeled primary antibodies is demonstrated by employing three well-known methods for detecting subcellular targets using fluorescent read-out, including flow cytometry, DNA-PAINT, and dSTORM. This work thus establishes a general and efficient platform for the synthesis of a library of unique ODN-antibody conjugates, facilitating the broader use of DNA-based programmable tags for multiplexed labeling to identify subcellular features with nanometer-precision, improving our understanding of cellular structure and function.

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Posted June 06, 2019.
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Efficient small-scale conjugation of DNA to primary antibodies for multiplexed cellular targeting
Glenn A.O. Cremers, Bas J.H.M. Rosier, Roger Riera Brillas, Lorenzo Albertazzi, Tom F.A. de Greef
bioRxiv 662791; doi: https://doi.org/10.1101/662791
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Efficient small-scale conjugation of DNA to primary antibodies for multiplexed cellular targeting
Glenn A.O. Cremers, Bas J.H.M. Rosier, Roger Riera Brillas, Lorenzo Albertazzi, Tom F.A. de Greef
bioRxiv 662791; doi: https://doi.org/10.1101/662791

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