Constructing Labeled DNA Constructs

For the fully ssDNA construct, an 8.1 kbp dsDNA construct with digoxigenin (DIG) and biotin labeled ends with a free 3′ end was constructed as previously described (Figure 1A) (Naufer et al., 2017 (link)). The dsDNA vector pBacgus11 (gift from Borja Ibarra) was linearized using high fidelity restriction enzymes BamHI and SacI (New England Biolabs). A dsDNA handle with digoxigenin (DIG) labeled bases with a complementary end to the BamHI sequence was PCR amplified (Ibarra et al., 2009 (link)). The DIG handle and a biotinylated oligonucleotide with a 3′ end complementary to the SacI sequence (Integrated DNA Technologies) were ligated to the linearized pBacgus11 using T4 ligase (NEB). For the dsDNA/ssDNA hybrid construct, a 6.5 kbp fragment contained in a plasmid (gift from Tom Perkins) containing a designed sequence (Okoniewski et al., 2017 (link)) was PCR amplified using KOD Hot Start DNA Polymerase (Novagen). One end of product contains a biotinylated primer, and the other end was cut and ligated to same DIG handle described above. The labeled construct was then nicked using restriction enzymes Nt.BspQI and Nb.BsmI (New England Biolabs).