DeNAno selections were performed as previously described, with minor modifications (1 (link)). For the streptavidin and rituximab selections, 3 × 1010 nanoparticles were incubated with target beads for 20 m at RT. For streptavidin, the target was streptavidin-coated magnetic beads; for rituximab selections, the target was rituximab-coated beads (described above). Non-binding particles were removed by repeated washes. Bound DeNAno particles were amplified by Hemo KlenTaq (NEB) back to the 100-bp oligo, and the template strand was amplified by asymmetric PCR. The template strand was then re-circularized as above and the entire process repeated through 4–5 rounds of selection. 100-bp oligos were then cloned into pGEM T-easy vector (Promega, Madison, WI, USA), transformed into NEB 5-alpha high efficiency competent cells (NEB) and sequenced via colony PCR (Eton Bioscience Inc, San Diego, CA, USA).
Selection on bevacizumab-coated beads differed in these ways: round one selection was performed with 10 times more particles (3 × 1011 unique particles) and selection was performed overnight at 4°C. Subsequent selection rounds were performed for 2–4 h at RT, but with a standard number of DeNAno particles (3 × 1010). These conditions were employed because multiple selection attempts with the standard conditions failed.
Selection on bevacizumab-coated beads differed in these ways: round one selection was performed with 10 times more particles (3 × 1011 unique particles) and selection was performed overnight at 4°C. Subsequent selection rounds were performed for 2–4 h at RT, but with a standard number of DeNAno particles (3 × 1010). These conditions were employed because multiple selection attempts with the standard conditions failed.