Programmable Edge-to-Edge Assembly of RNA Nanostructures

Building complex RNA nanostructures requires precise molecular connectors for controlled self-assembly. Existing connectors, such as kissing loops, are restricted to coaxial joining, limiting accessible geometries. Here, we introduce the alpha kissing loop (alphaKL), a compact, sequence-programmable RNA connector enabling edge-to-edge helix association. The alphaKL combines a four-nucleotide kissing loop with minor- and major-groove triplex interactions, preorganizing an α-shaped conformation for cotranscriptional folding. Incorporated into RNA origami tiles, alphaKLs drive multivalent assembly into filaments and lattices, characterized by atomic force microscopy. Molecular dynamics simulations reveal that triplex occupancy at each sequence position controls the preferred interhelical angle. Cooperative backbone contacts progressively rigidify multi-alphaKL interfaces beyond individual motifs. By linking helices edge-to-edge rather than end-to-end, the alphaKL expands the RNA nanostructure design space, enabling previously inaccessible architectures.