VIPR systems employ a triplex-driven mechanism for noncontiguous RNA-guided DNA recognition in which Vipr proteins oligomerize along vrRNA into a helical filament that sequesters GGY motifs, enabling NN dinucleotides to pair with target DNA through a skip-base rule, while DNA unwinding proceeds through formation of an RNA-dsDNA triplex intermediate that undergoes local target strand handoff to generate a final R-loop triplex structure where the non-target strand threads through the RNA-DNA hybrid core.

Viral Interference Programmable Repeat (VIPR) systems use a noncontiguous code for RNA-guided transcriptional silencing. How the Vipr protein and a vrRNA comprising alternating GGY and NN segments achieve precise DNA targeting is unknown. Here we present 21 cryo-electron microscopy structures that span the VIPR assembly pathway. Vipr protomers oligomerize along the vrRNA to form a right-handed helical filament, sequestering each GGY motif and positioning the adjacent NN bases for target base pairing. DNA binding, in which every third nucleotide is skipped, results in target strand rotation to form a gapped vrRNA-DNA hybrid helix that wraps around the non-target DNA strand to form a structural triplex. These findings provide the structural basis of noncontiguous RNA-guided DNA binding in VIPR, establishing triplex-driven target-strand handoff as an elegant mechanism of programmable nucleic acid recognition.