Site-Specific Linking of an Oligonucleotide to Mono- and Bivalent Recombinant Antibodies with SpyCatcher-SpyTag System for Immuno-PCR

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 Sfi I sites enabling the rapid generation of AOC constructs for site-specific SpyTag-oligonucleotide conjugation.

Expanding the Scope of the Cleavable N-(Methoxy)oxazolidine Linker for the Synthesis of Oligonucleotide Conjugates

“…SpyTag peptide binds through irreversible isopeptide bond to a SpyCatcher protein domain [ 43 ]. This autocatalytic process has been utilized, e.g., for the preparation of antibody-ON-conjugates [ 50 ], but immunogenicity issues should be resolved prior to drug delivery applications. THR and its peptidase-resistant retro inverso version [ 44 ] binds to the transferrin receptor, and it has been applied to deliver RNA nanoparticles through the blood brain barrier [ 45 , 46 ].…”

Site-Specific Antibody Conjugation with Payloads beyond Cytotoxins

“…The lysine residue close to the site was subsequently site-selectively conjugated. SpyCatcher-SpyTag system was applied for site-specific coupling of oligonucleotide to antibody [ 118 ]. SpyTag-oligonucleotide was shown to be conjugated to scFv fused with SpyCatcher on C-terminus through the formation of a covalent isopeptide bond.…”

Antibodies as Biosensors’ Key Components: State-of-the-Art in Russia 2020–2021

“…The choice of the antibody/DNA conjugation strategy is of critical importance because it determines both the efficiency of the conjugation reaction itself and the activity of the obtained product and, as a consequence, the sensitivity of the assay (Figure 2a). For this reason, various conjugation techniques have been developed and optimized [26,27]. Alternatively, phage-display-mediated, real-time immuno-PCR enables the conjugation problem to be worked around.…”