Carboxyl polystyrene beads

To functionalize polystyrene microspheres with DNA, we mixed either single-stranded or double-stranded oligonucleotides containing a 5’ amino-modifier (Table s2) with 2.1 μm carboxyl-polystyrene beads (Spherotech). Coupling of the oligonucleotide to the bead surface was achieved by chemical crosslinking with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC; ThermoFisher) in the presence of N-hydroxysulfosuccinimide (Sulfo-NHS) in 100 mM MES-Na (pH 6) buffer. The reaction was quenched by addition of TE8 buffer (20 mM Tris-HCl, 1 mM EDTA, pH 8). Unreacted material was removed by washing the beads three times with TE8. Beads modified with single-stranded DNA (“ssDNA-beads”) were used to generate long DNA handles. Beads modified with double-stranded DNA (“dsDNA-beads”) were used to immobilize proteins and RNCs. In this case, the double-stranded oligonucleotides contained a 4-nucleotide single-stranded overhang with a sequence reverse-complementary to the overhang of the dsOligo-CoA DNA.

Carboxyl-polystyrene beads of 2.0-μm diameters (Spherotech, Lake Forest, IL) were covalently coupled with streptavidin (Invitrogen) as previously described.[15 (link), 17 (link)] Briefly, streptavidin-coated beads were incubated with a VWF domain fragment. SpyCatcher was coupled to biotin-DNA handle, and then incubated with streptavidin beads. For pulling experiments, one bead with an Avi-His-SpyTag VWF fragment on its surface was fixed by the micropipette, while the other bead coupled with SpyCatcher-DNA handle was trapped and controlled by the optical tweezers. The force measurement was performed at force-ramp mode, where the stretching force increases linearly with the pulling distance. The force-extension data were fitted to the WLC model [18 (link)] to determine its mechanical properties.