Top: schematic of DNA covalently anchored onto a bead. See Figure S3C for the reaction pipeline and Mendeley data (doi:https://doi.org/10.17632/htskytjh6v.1) for a plasmid map detailing the modification sites. Bottom: JET cleavage of bead-anchored DNA. We adopted a gap-filling approach37to generate covalently closed 2.9 kb DNA circles containing one or two SNAP ligands, benzylguanine (BG) groups, attached to selected thymidine nucleobases (see also Figure S3A). The labeled DNA was then reacted with purified SNAP-tag protein (Figure S3B), and further anchored to Dynabeads through chemical cross-linking via amino-reactive epoxy chemistry. Uncoupled DNA was removed by washing before treatment with JET nuclease. For gel analysis, the DNA covalently coupled to the beads was eventually released by digesting the SNAP-tag protein using Proteinase K (Figure S3C). We note that a population of BG-DNA circles migrated slower than closed covalent circles (ccc); these open covalent circles (occ) were likely generated by DNA strand breakage during prolonged treatment for DNA coupling to beads.
