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Abstract
Antibody-oligonucleotide conjugates (AOCs) are a versatile class of chimeric biomolecules for therapeutics and biotechnological applications. Most widely employed chemical labeling methods for proteins are based on targeting of Lys or Cys residues that leads to mixed stoichiometry in the degree of conjugation and may interfere with antigen binding, thus, compromising the function of the antibody. A site-specific oligonucleotide conjugation technology providing full control over valency in mild reaction conditions would be an advancement to the state-of-the-art in bioconjugation. Herein, we demonstrate the production of single-chain variable fragment antibodies with fused SpyCatcher (scFv-SpyCatcher, monovalent) and alkaline phosphatase-SpyCatcher (scFv-AP-SpyCatcher, bivalent) on C-terminus and their conjugation to SpyTag002-oligonucleotide in phosphate-buffered saline (PBS). The formation of a covalent isopeptide bond between the protein and SpyTag002-oligonucleotide was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the functionality of the obtained AOCs was confirmed in immuno-polymerase chain reaction (PCR) assays for the detection of microcystin-LR and 17β-estradiol. Based on time-resolved fluorescence immunoassays with scFv-AP fusion constructs, we observed that the SpyCatcher and SpyCatcher-SpyTag002-oligonucleotide part lowered the absolute signal obtained from the assay by 27.6 and 48.4% at 2 nM and by 26.2 and 27.6% at 100 pM microcystin-LR and 17β-estradiol concentrations, respectively. Nevertheless, the overall sensitivity of the immuno-PCR assays was similar to the time-resolved fluorescence immunoassays performed with the same components. In this study, vectors for SpyCatcher-fusion construction were created for directional cloning with SfiI sites enabling the rapid generation of AOC constructs for site-specific SpyTag-oligonucleotide conjugation.
Highlights
Antibody-oligonucleotide conjugates (AOCs) are a versatile class of chimeric biomolecules, which combine the unique functions of two fundamentally different types of biopolymers
To facilitate the production of well-defined antibody-oligonucleotide conjugates, we report a convenient approach for the covalent site-specific attachment of a peptide-tagged oligonucleotide to a recombinantly produced scFv-SpyCatcher fusion protein in biocompatible conditions
Owing to the modular structure of an antibody, compact size of the SpyCatcherSpyTag-system, and the well-established expression procedures for these recombinant proteins, the approach can be adapted to the production of different types of the antibodyoligonucleotide conjugate constructs, e.g., with different binding valencies
Summary
Antibody-oligonucleotide conjugates (AOCs) are a versatile class of chimeric biomolecules, which combine the unique functions of two fundamentally different types of biopolymers. Biophysical properties of antibody-oligonucleotide conjugates intended for either therapeutic or bioaffinity assay applications The synthesis of such conjugates greatly benefits from a stable and site-specific labeling technique that can be achieved by incorporating peptide tags, e.g., Halo-tag, Sortase A, Avi biotin ligase recognition peptide, or modified amino acids.[6] Other widely used site-specific conjugation strategies, such as GlyClick technology, involves the Fc domain of an antibody, making the conjugation of smaller antibody formats (Fab, scFv, diabody) impossible. The method involves click chemistry-based synthesis of the SpyTagoligonucleotide as well as expression of recombinant singlechain (scFv) antibody fragment as a fusion protein with SpyCatcher Using this approach, we constructed antibodyoligonucleotide conjugates and analyzed their bifunctional properties, antigen recognition, and programmable specificity, with immuno-PCR and immunoassays in mono- and bivalent formats
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