Fig. 2: Identification of IS621 bridge RNA binding loops with sequence-specific recognition of target and donor DNA.

a, Schematic of the computational approach to assess the base-pairing potential between the IS110 ncRNA and its cognate genomic target site or donor sequence. Covariation analysis between target–ncRNA or donor–ncRNA pairs yields a matrix in which diagonal stretches of red signal indicate ncRNA complementarity to the bottom strand of the DNA and blue stretches indicate complementarity to the top strand. b, Nucleotide covariation and base-pairing potential between the ncRNA and the target (left) and donor (right) sequences across 5,511 ncRNA–target pairs and 2,201 ncRNA–donor pairs. The IS621 ncRNA sequence is shown across the x axis, along with dot-bracket notation predictions of the secondary structure. Covariation scores are coloured according to strand complementarity, with −1 (blue) representing high covariation and a bias toward top-strand base-pairing, and 1 (red) representing high covariation and a bias toward bottom-strand base-pairing. Regions of notable covariation signal indicating base-pairing for IS621 are boxed. Complementary nucleotides within covarying regions are highlighted in bold. c, Schematic of the in vitro recombination (IVR) reaction with IS621. d,e, Gel electrophoresis of the IVR LD–RT PCR product (d) or LT–RD PCR product (e). Results are representative of three technical replicates. Rec, recombinase. f, Binding of target and donor DNA sequences by an IS621 RNP containing fluorescently labelled recombinase and ncRNA, using MST. Mean ± s.d. of three technical replicates. g, Schematic of the IS621 bridge RNA. The target-binding loop contains the LTG and RTG (blue), and the donor-binding loop contains the LDG and RDG (orange). h, Base-pairing model of the IS621 bridge RNA with cognate target and donor DNA.