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

08:00 EDT 10th March 2020 | BioPortfolio

Summary of "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.

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This article was published in the following journal.

Name: Journal of biochemistry
ISSN: 1756-2651
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Medical and Biotech [MESH] Definitions

RNA consisting of two strands as opposed to the more prevalent single-stranded RNA. Most of the double-stranded segments are formed from transcription of DNA by intramolecular base-pairing of inverted complementary sequences separated by a single-stranded loop. Some double-stranded segments of RNA are normal in all organisms.

An endoribonuclease that is specific for double-stranded RNA. It plays a role in POST-TRANSCRIPTIONAL RNA PROCESSING of pre-RIBOSOMAL RNA and a variety of other RNA structures that contain double-stranded regions.

Enzyme systems containing three different subunits and requiring ATP, S-adenosylmethionine, and magnesium for endonucleolytic activity to give random double-stranded fragments with terminal 5'-phosphates. They function also as DNA-dependent ATPases and modification methylases, catalyzing the reactions of EC 2.1.1.72 and EC 2.1.1.73 with similar site-specificity. The systems recognize specific short DNA sequences and cleave at sites remote from the recognition sequence. Enzymes from different microorganisms with the same specificity are called isoschizomers. EC 3.1.21.3.

Enzyme systems containing a single subunit and requiring only magnesium for endonucleolytic activity. The corresponding modification methylases are separate enzymes. The systems recognize specific short DNA sequences and cleave either within, or at a short specific distance from, the recognition sequence to give specific double-stranded fragments with terminal 5'-phosphates. Enzymes from different microorganisms with the same specificity are called isoschizomers. EC 3.1.21.4.

Enzymes that catalyze the endonucleolytic cleavage of single-stranded regions of DNA or RNA molecules while leaving the double-stranded regions intact. They are particularly useful in the laboratory for producing "blunt-ended" DNA molecules from DNA with single-stranded ends and for sensitive GENETIC TECHNIQUES such as NUCLEASE PROTECTION ASSAYS that involve the detection of single-stranded DNA and RNA.

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