Genome-wide discovery of natural substrates for the Rho helicase of Escherichia coli

We have developed a new screening approach, called Helicase-SELEX, which allows the identification of helicase substrate sequences from large sequence libraries. In the present case, the starting library consisted of randomly PCR-amplified fragments (~90 to ~130 bp) from the genome of Escherichia coli MG1655. 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 E. coli 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 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. Two Helicase-SELEX experiments were performed in parallel, whereby the cofactor NusG was present or absent during reactions with the Rho helicase (R10plus and R10minus libraries, respectively).