ISPpu10 is a structure-gated bridge RNA recombinase that drives safe, large-scale genomic plasticity

Na, X.-M.; Chen, B.-T.; Liu, H.; Lin, Y.-T.; Yuan, Y.; Chen, T.-J.; Xiong, Y.-N.; Zhang, Y.-H.; Yang, N.; Zhu, F.; Zhang, Y.; Chi, X.; Zong, Z.; Ling, C.

doi:10.64898/2026.03.19.712850

bioRxiv · 10.64898/2026.03.19.712850

ISPpu10 is a structure-gated bridge RNA recombinase that drives safe, large-scale genomic plasticity

Na, X.-M.·Chen, B.-T.·Liu, H.·Lin, Y.-T.·Yuan, Y.·Chen, T.-J.·Xiong, Y.-N.·Zhang, Y.-H.·Yang, N.·Zhu, F.·Zhang, Y.·Chi, X.·Zong, Z.·Ling, C.

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Abstract

IS110 elements employ bridge RNA to direct target site recognition, yet whether additional layers of specificity exist beyond RNA-DNA base pairing is unknown. Here we identified ISPpu10, an IS110 element in Pseudomonas putida that is associated with >100-kb segmental amplifications during adaptive laboratory evolution. All insertions mapped to multiple identical copies of a single conserved intergenic sequence harboring a GC-rich 5' extension that forms a stem-loop. Systematic mismatch and compensatory substitution scanning revealed a sequence-structure dual-match logic: base pairing between the bridge RNA and target DNA serves as an RNA-guided match, whereas the target-encoded stem-loop provides an additional specificity determinant. Transplanting this stem-loop into Escherichia coli targets improved on-target integration and reduced genome-wide off-target insertion, establishing the stem-loop as a portable structural gate. These findings reveal a structural recognition layer beyond base-pairing, suggesting how bacteria reconcile transposition with genome integrity and offering a strategy for precise genome editing.

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Na, X.-M., Chen, B.-T., Liu, H., Lin, Y.-T., Yuan, Y., Chen, T.-J., Xiong, Y.-N., Zhang, Y.-H., Yang, N., Zhu, F., Zhang, Y., Chi, X., Zong, Z., Ling, C.. 2026-03-20. ISPpu10 is a structure-gated bridge RNA recombinase that drives safe, large-scale genomic plasticity. https://doi.org/10.64898/2026.03.19.712850

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