PCR-based approach for site-specific conjugation of long double-stranded DNA to a single-domain VHH antibody.

Site-specific conjugation of double-stranded DNA using antibodies enables the development of unique applications for antibody drug conjugates utilizing recent advances in nucleic acid medicines. Here, we describe a novel method to conjugate a camelid-derived single domain VHH antibody with arbitrarily-sized double-stranded DNA by PCR. Cysteine in anti-human EGFR VHH were replaced by alanine, and an unpaired cysteine was introduced at the carboxyl-terminus. These modifications enabled site-specific labeling with a maleimide-modified DNA oligo via thioether bond formation; the ensuing product-single-stranded DNA conjugated at the C-terminus of VHH-retained its affinity for EGFR. To investigate whether this VHH-single-stranded DNA conjugate might be used as a forward primer, we subjected it to PCR, producing 100-500 bp DNA. We confirmed the amplification of the VHH-double-stranded DNA conjugate by examining its mobility on acrylamide gel; retention of the binding affinity of the conjugate for EGFR was identified by immuno-PCR.