4.2 Article

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

期刊

JOURNAL OF BIOCHEMISTRY
卷 168, 期 1, 页码 63-72

出版社

OXFORD UNIV PRESS
DOI: 10.1093/jb/mvaa029

关键词

disulphide; DNA conjugation; heat resistance; PCR; VHH antibody

资金

  1. Japan Society for the Promotion of Science (KAKENHI) [16K17939, 16H03295]
  2. Grants-in-Aid for Scientific Research [16K17939, 16H03295] Funding Source: KAKEN

向作者/读者索取更多资源

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 100-500bp 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.

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