Helicase-SELEX mapping of Mycobacterium tuberculosis transcription terminator Rho utilization (Rut) sites
We have used a global screening approach called Helicase-SELEX (see Delaleau et al., 2022, PMID: 36095194) to identify Rho helicase substrate sequences (aka Rut sequences) within the genome of Mycobacterium tuberculosis. The starting library consisted of randomly PCR-amplified gDNA fragments (~90 to ~130 bp) from the genome of M. tuberculosis reference strain H37Rv. Each DNA fragment was framed by upstream and downstream constant regions for T7 transcription and biotinylated oligonucleotide hybridization, respectively. Two distinct starting libraries of gDNA fragments (R0A and R0B) were prepared. Each gDNA fragment library was converted 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, in the presence of ATP, with either the full-length (FL) Rho helicase from M. tuberculosis or a truncated variant lacking the taxon-specific N-terminal insertion domain (DNID variant). In this way, only duplexes containing suitable substrate sequences were unwound by the FL or DNID 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 each library towards the Rho helicase became constant and its composition was determined by NGS. For each Rho variant, two Helicase-SELEX enrichment replicates were obtained (R10-FLA and R10-FLB for FL Rho and  R10-DNIDA and R10-DNIDB for the DNID Rho variant).
We have used a global screening approach called Helicase-SELEX (see Delaleau et al., 2022, PMID: 36095194) to identify Rho helicase substrate sequences (aka Rut sequences) within the genome of Mycobacterium tuberculosis. The starting library consisted of randomly PCR-amplified gDNA fragments (~90 to ~130 bp) from the genome of M. tuberculosis reference strain H37Rv. Each DNA fragment was framed by upstream and downstream constant regions for T7 transcription and biotinylated oligonucleotide hybridization, respectively. Two distinct starting libraries of gDNA fragments (R0A and R0B) were prepared. Each gDNA fragment library was converted 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, in the presence of ATP, with either the full-length (FL) Rho helicase from M. tuberculosis or a truncated variant lacking the taxon-specific N-terminal insertion domain (DNID variant). In this way, only duplexes containing suitable substrate sequences were unwound by the FL or DNID 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 each library towards the Rho helicase became constant and its composition was determined by NGS. For each Rho variant, two Helicase-SELEX enrichment replicates were obtained (R10-FLA and R10-FLB for FL Rho and  R10-DNIDA and R10-DNIDB for the DNID Rho variant).
Centre National de la Recherche Scientifique