PCR-based approach for site-specific conjugation of long double-stranded DNA to a single-domain VHH antibody
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Abstract
<jats:title>Abstract</jats:title><jats:p>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.</jats:p>
Journal
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- The journal of biochemistry
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The journal of biochemistry 168 (1), 63-72, 2020-07
Tokyo : Japanese Biochemical Society
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Details 詳細情報について
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- CRID
- 1520291853579821824
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- NII Article ID
- 40022279169
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- NII Book ID
- AA00694073
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- ISSN
- 0021924X
- 17562651
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- NDL BIB ID
- 030502573
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- Text Lang
- en
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- NDL Source Classification
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- ZR2(科学技術--生物学--生化学)
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- Data Source
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- NDL
- Crossref
- CiNii Articles
- KAKEN