Protocol to generate, purify, and analyze antibody-oligonucleotide conjugates from off-the-shelf antibodies.

Antibody-oligonucleotide conjugates (AOCs) are a fast-expanding modality for targeted delivery of therapeutic oligonucleotides to tissues. Here, we present a protocol to generate, purify, and analyze AOCs from off-the-shelf antibodies. We describe steps to conjugate single/double-stranded oligonucleotides bearing amine handles to linkers and, then, to antibodies using well-established chemistry. In addition, we provide details regarding the purification techniques and analytical methods suitable for AOC. This protocol can be applied for several purposes where AOC is a modality of interest. For complete details on the use and execution of this protocol, please refer to Rady et al.1.

Reinvestigation of the Automated Synthesis of Stoichiometrically Conjugated Antibodies to Access High Molecular Weight Payloads and Multiplexed Conjugation via an In-Solution Trans-Tagging Process.

Protein conjugates have found applications in a wide variety of fields, ranging from therapeutics to imaging and detection. However, robust control over the parameters of the conjugation process (such as sites and degree of conjugation) remains challenging. Previously, our group introduced Equimolar NAtive Chemical Tagging (ENACT), a method which allows for the monofunctionalization of proteins by combining an iterative low-conversion bioconjugation, an automated process, and a bioorthogonal trans-tagging reaction. However, while the automated ENACT was dimensioned to achieve monoconjugation at the mg scale, in early stage research, because of the rarity and cost of the starting materials, it is often necessary to prepare conjugates at the lower, µg, scale. Here, we introduce modified ENACT protocols, as well as a new ENACT conjugation reagent, which allow for the monofunctionalization of proteins on the micrograms scale, using minimal quantities of payload.