Ultra-Stable RNA-Based Monomolecular Triplexes

The formation of stable nucleic acid triplexes requires an underlying homopurine:homopyrimidine duplex and a third strand that is bound to the homopurine strand through Hoogsteen interactions. Depending on its nucleotide composition, the triplex-forming oligonucleotide (TFO) can align in either a parallel or antiparallel orientation relative to the purine strand of the duplex. Conventionally, monomolecular triplexes are formed by three equal-length segments connected by two U₄ (U-shaped) loops, and the TFO does not contribute to the stability of the underlying duplex. However, many noncanonical monomolecular quadruplex structures with high stability are known to form with single-nucleotide propeller (or Z-shaped) loops connecting G-rich segments. Whether Z-shaped loops could be used to increase triplex stability is unknown. We report here a novel folding principle: two adjacent GGG segments separated by a single nucleotide that can align in parallel orientation by employing the intervening nucleotide as a Z-shaped loop. We also designed DNA and RNA triplexes incorporating one U-shaped loop and one Z-shaped loop. Our results demonstrate that in such triplexes, the TFO increases the stability of an underlying RNA duplex, but not in the case of a DNA duplex. Moreover, the stabilized triplex unfolds as a single cooperative unit at temperatures exceeding the melting point of the RNA duplex itself. These findings reveal that ultrastable Z-looped intramolecular triplexes can indeed form in vitro, highlighting a previously unrecognized structural motif that may serve as a novel therapeutic target against pathogenic RNAs.