Helicase-SELEX mapping of Bacillus subtilis transcription terminator Rut sites

We have used a global screening approach, called Helicase-SELEX, to identify Rho helicase substrate sequences within the genome of Bacillus subtilis. The starting library consisted of randomly PCR-amplified fragments (~90 to ~130 bp) from the genome of B. subtilis strain 168. Each DNA fragment was framed by upstream and downstream constant regions for T7 transcription and biotinylated oligonucleotide hybridization, respectively (R0 library). This allowed conversion of the dsDNA fragment library into a library of biotinylated RNA:DNA duplexes (each containing a ~90 to ~130 nucleotide-long ssRNA sequence of genomic origin), which were immobilized on streptavidin-coated beads and then incubated with the Rho helicase from either E. coli or B. subtilis in the presence of ATP. In this way, only duplexes containing suitable substrate sequences were unwound by the Rho helicase and released in the supernatant. The corresponding ssRNA species were recovered from the supernatant, amplified by RT-PCR and, following the process described above, converted into a new library of RNA:DNA duplexes enriched in substrate sequences that was used in the next round of Helicase-SELEX. After 10 (E. coli's Rho) or 14 (B. subtilis's Rho) rounds of this iterative enrichment process, the activity of the library towards the Rho helicase became constant and the library composition was determined by NGS. For each Rho helicase, two Helicase-SELEX enrichment replicates were obtained (R10A and R10B for E. coli's Rho and R14A and R14B for B. subtilis's Rho).