TFOs recognize purine stretches inside the major groove of duplex DNA and bind them sequence-specifically via one of two hydrogen bonding schemes . The normal Hoogsteen (nHS) motif yields a parallel triplex structure stabilized by the T:AT and C + :GC base triads (bt) with two hydrogen bonds each (Fig. ). The stability of nHS triple-stranded (ts) DNA depends on TFO length among other factors and favours mildly acidic conditions due to required protonation of cytosine . Intermolecular parallel triplexes start forming at approximately 10 bt and are stable at room temperature from ≥14 bt (Supplementary Fig. ). A 20 nt TFO typically elevates the melting temperature of the original duplex by approximately 10 °C, while a non-peripheral mismatch negates most of this stabilization (Supplementary Fig. ). A "TFO staple" encoding two unique nHS recognition domains connected by a linker base (Figs. ) thus specifically co-localizes two duplex targets, thereby demonstrating that this noncanonical sequence-structure relationship can coordinate simple intermolecular assemblies (Supplementary Fig. ). ). An odd number of helical half-turns separates consecutive crossovers perpendicular to the helical axes (red to blue and blue to red); depicted are five triplex half-turns (30 bt). The dsDNA scaffold folds through the helices in a zigzag raster and creates scaffold loops wherever it reverses direction. h, Similar to g but with the scaffold folded into five antiparallel segments and DNA drawn as helices.