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, and Tom F. A. de Greef

Published: 18/09/2019


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. Whereas primary antibodies are commercially available for a large class of targets, a general stoichiometric site-selective DNA labeling strategy for this affinity reagent is lacking. Here we present a universal, site-selective conjugation method using a small photo-cross-linkable 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 bovine serum albumin. In addition, we present a general benchtop-compatible strategy to purify DNA-labeled antibodies without a 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 fluorescence 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 and improving our understanding of cellular structure and function.

Full Access Link: Bioconjugate Chemistry