LAUSR

Significance Helicases are ubiquitous NTP-dependent enzymes that disrupt nucleic acid (NA) helices and NA–protein interactions. Despite the implication of helicases in many cellular processes and diseases, their target repertoires and determinants of functional specialization often remain uncertain. We developed Helicase-SELEX to combinatorically probe helicase substrate requirements and find natural or synthetic substrates in large NA sequence libraries. Using the transcription termination Rho helicase as prototype, we discovered ∼3,300 functional substrate sequences in Escherichia coli, thereby providing a detailed map of Rho utilization (Rut) sites at genome scale. We also evolved synthetic Rut switches eliciting Rho activity only in the presence of a selected inducer. Thus, Helicase-SELEX is a unique approach to characterize or exploit helicases for fundamental or biotechnology purposes.