Resolving the DNA-binding specificities of transcription factors (TFs) is of 
critical value for understanding gene regulation. Here, we present a novel, 
semiautomated protein-DNA interaction characterization technology, selective 
microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq). 
SMiLE-seq is neither limited by DNA bait length nor biased toward strong 
affinity binders; it probes the DNA-binding properties of TFs over a wide 
affinity range in a fast and cost-effective fashion. We validated SMiLE-seq by 
analyzing 58 full-length human, mouse, and Drosophila TFs from distinct 
structural classes. All tested TFs yielded DNA-binding models with predictive 
power comparable to or greater than that of other in vitro assays. De novo motif 
discovery on all JUN-FOS heterodimers and several nuclear receptor-TF complexes 
provided novel insights into partner-specific heterodimer DNA-binding 
preferences. We also successfully analyzed the DNA-binding properties of 
uncharacterized human C2H2 zinc-finger proteins and validated several using 
ChIP-exo.