Schematic illustration of two-step purification of affinity binder-oligonucleotide conjugates.
(a) Antibody-DNA conjugates illustrated here as an example. Conjugation of antibodies and oligonucleotides yields a mixture of desired conjugates, along with unconjugated antibodies and unconjugated oligonucleotides. (A) Biotinylated capture DNA oligonucleotides are hybridized to the oligonucleotides in the mixture. (B) Streptavidin-coated Sepharose beads are used to capture the biotinylated capture oligonucleotides, both in the form of conjugates and free oligonucleotides. (C) The unconjugated antibodies are removed by washes. (D) The MlyI enzyme is used to cleave the captured oligonucleotide hybrids, allowing both conjugates and oligonucleotides to be eluted from the solid support. (E) The eluate is then incubated either with protein G beads (for antibody-DNA conjugates) or with Dynabeads His tag (for DARPin-DNA conjugates), while free oligonucleotides are removed by washes. (F) Finally, purified conjugates are eluted from the solid support. (b) Illustration of hybridization of Arm1_long and Arm1 Capture, and the subsequent MlyI cleavage.