PCR-based approach for site-specific conjugation of long double-stranded DNA to a single-domain VHH antibody.
J Biochem
; 168(1): 63-72, 2020 Jul 01.
Article
em En
| MEDLINE
| ID: mdl-32154894
ABSTRACT
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 (variable domain of a heavy chain antibody) antibody with arbitrarily sized double-stranded DNA by PCR. Cysteine in anti-human epidermal growth factor receptor (EGFR) VHH was replaced by alanine, and an unpaired cysteine was introduced at the carboxyl terminus. These modifications enabled site-specific labelling with a maleimide-modified DNA oligo via thioether bond formation; the ensuing product-single-stranded DNA conjugated at the carboxyl 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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
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Reação em Cadeia da Polimerase
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Cadeias Pesadas de Imunoglobulinas
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Imunoconjugados
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Anticorpos de Domínio Único
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article