W-box DNA Binding Affinity Assay

Complementary single-stranded DNA fragments encoding the W-box DNA sequence were synthesized by IDT. For SPR assays, the forward strand encoded the W-box DNA sequence (5′-CGCCTTTGACCAGCGC-3′) while the complementary reverse strand added an extra 20-bp ReDCaT sequence (5′-CCTACCCTACGTCCTCCTGC-3′) to complement the linker DNA added to the SA chip. The double-stranded DNA was then diluted to a working concentration of 1 μM. SPR measurements were performed at 25 °C using the reusable DNA capture technique (ReDCaT) as described (59 (link), 60 (link)) and using a Biacore 8K System (Cytiva). Further details are provided in SI Appendix, Materials and Methods.