Discovery of serotonin-inducible regulatory switches governing the activity of the Rho helicase

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 synthetic dsDNA fragments containing a fully randomized 80 bp-long region framed by upstream and downstream constant regions for T7 transcription and biotinylated oligonucleotide hybridization, respectively. This allowed conversion of the dsDNA library into a library of biotinylated RNA:DNA duplexes, which were immobilized on streptavidin-coated beads and then incubated with the Rho helicase from E. coli in the presence of ATP and serotonin. 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. Positive Helicase-SELEX rounds (with serotonin) were alternated with negative selection rounds performed in absence of serotonin whereby unreactive, bead-bound RNA:DNA duplexes rather that ssRNA species were recovered and used for RT-PCR amplification and the next round of Helicase-SELEX. In this way, serotonin-inducible sequences were preferentially selected over constitutively active sequences. The compositions of the libraries obtained after 13 and 21 rounds of this iterative enrichment process were determined by NGS (sR13 and sR21 libraries, respectively).