Abstract
AbstractMany bioconjugation strategies for DNA oligonucleotides and antibodies suffer limitations, such as site‐specificity, stoichiometry and hydrolytic instability of the conjugates, which makes them unsuitable for biological applications. Here, we report a new platform for the preparation of DNA‐antibody bioconjugates with a simple benzoylacrylic acid pentafluorophenyl ester reagent. Benzoylacrylic‐labelled oligonucleotides prepared with this reagent can be site‐specifically conjugated to a range of proteins and antibodies through accessible cysteine residues. The homogeneity of the prepared DNA‐antibody bioconjugates was confirmed by a new LC‐MS protocol and the bioconjugate probes were used in fluorescence or super‐resolution microscopy cell imaging experiments. This work demonstrates the versatility and robustness of our bioconjugation protocol that gives site‐specific, well‐defined and plasma‐stable DNA‐antibody bioconjugates for biological applications.
Highlights
Cross-linking of various types of molecules leading to chimeric constructs with combined functionalities has been gaining attention across the fields of chemical biology, biotechnology, and medicine.[1]
Additional steps to introduce the functional groups for the moiety to DNA ONs, we used bifunctional reagent 2, which is biorthogonal ligation, the latter cross-links the reactive amino prepared by activation of commercially available transacid residues on the protein surface with modified ONs 3-benzoylacrylic acid (1)
We have developed an improved protocol for the bioconjugation of DNA ONs to proteins and antibodies, and an optimized method for the LC-mass spectrometry (MS) characterization of prepared bioconjugates
Summary
Cross-linking of various types of molecules leading to chimeric constructs with combined functionalities has been gaining attention across the fields of chemical biology, biotechnology, and medicine.[1]. Numerous studies have been undertaken to extend the diversity of protein functionalities by introducing nonnatural labels or “tags” in the emerging field of chemical protein modification.[1a] Besides proteins, nucleic acids (NAs) are an extensively studied class of biomolecules. It is mainly the highly specific and predictable base pairing that makes DNA or RNA oligonucleotides (ONs) unique tools for the design of biomolecular hybrids with new structures and improved functions. Chemie we report a strategy that leads to chemically defined constructs by employing new benzoylacrylic acid pentafluorophenyl ester (BA-PFP) labelling reagent 2 (Figure 1 d) as an alternative to the cross-linking of ONs and proteins by conventional maleimide-NHS ester-based reagents. We use the DNA-antibody conjugates generated as probes in fluorescence microscopy and super-resolution DNA-PAINT imaging experiments
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