Approaches for making antibody-oligonucleotide conjugates
Antibody-oligonucleotide conjugates (AOCs) may be synthesized using a wide range of chemical approaches. These include stochastic methods where protein lysines provide a large number of potential sites of oligonucleotide attachment. A second category includes site-directed methods which allow the oligonucleotides to be attached at predetermined locations on the antibody molecule, or at least away from the antigen binding domains. Very precise control of the site of attachment generally requires genetic engineering of the heavy or light chains to insert motifs which can be acted upon by transferase enzymes. Some of the more popular approaches are outlined in the table, which will be expanded periodically.
| Conjugation approach | Oligo reactive group | Antibody reactive group | Oligo distribution | Original reference or Aboligo review |
|---|---|---|---|---|
| Disuccinimidyl substrate (homobifunctional) | NHS ester | Lysine | Stochastic | Smart application of an old conjugation reagent in AOC development |
| Click (strain-promoted) | DBCO | Azide (via STFP ester reaction with lysine) | Stochastic | Ref 1 |
| Click (strain-promoted) | Azide | DBCO (via DBCO-NHS ester reaction with lysine) | Stochastic | Ref 2 |
| Thiol-maleimide | Maleimide (via SH /bismaleimidohexane) | Thiol (via DTT reduction) | Site directed | Ref 3 |
| Thiol-maleimide | Protected thiol (SATA) + hydroxylamine | Maleimide (via lysine modification) | Stochastic | Ref 4 |
| mTransglutaminase/antibody engineering | DBCO | Azide introduced by mTGase at LLQG motif | Site directed | Site-directed antibody conjugation methods - article 1 |
| Sortase/antibody engineering | Gly-Gly-Gly | LPETGG motif | Site directed | Ref 5 |
| Glycan oxidation | Hydrazine | Aldehyde | Site directed | Ref 6 |
| Click (strain promoted) | TCO (trans-cyclooctene) | Tetrazine (via cleavable NHS ester) | Stochastic | Ref 7 |
References
1. Maerle AV et al. Development of the covalent antibody-DNA conjugates technology for detection of IgE and IgM antibodies by immuno-PCR. PLoS One. 2019 14(1):e0209860. doi:10.1371/journal.pone.0209860. PMID: 30608947
2. Gong H et al. Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells. Bioconjug Chem. 2016 27(1):217-25. doi: 10.1021/acs.bioconjchem.5b00613. PMID:26689321
3. Rudchenko S et al. Amplification of Signal on Cell Surfaces in Molecular Cascades. Cells. 2023 12(24):2858. doi: 10.3390/cells12242858. PMID:38132177
4. Hendrickson ER et al. High sensitivity multianalyte immunoassay using covalent DNA-labeled antibodies and polymerase chain reaction. Nucleic Acids Res. 1995 23(3):522-9. doi:10.1093/nar/23.3.522. PMID: 7885849
5. Al-Amin RA et al., Sensitive Protein Detection Using Site-Specifically Oligonucleotide-Conjugated Nanobodies. Anal Chem. 2022 94(28):10054-10061. doi: 10.1021/acs.analchem.2c00584. PMID:35786874
6. Kozlov IA et al. Efficient strategies for the conjugation of oligonucleotides to antibodies enabling highly sensitive protein detection. Biopolymers. 2004 73(5):621-30. doi:10.1002/bip.20009. PMID: 15048786