Dangling Ends of Third Strand and Duplex Drive Nucleic Acid Triplex Stabilization through Bimodal Association

Nucleic acid triplexes are crucial structural motifs in gene regulation and biotechnology, yet the kinetic principles governing their formation remain poorly understood. While a stability hierarchy of RNA*DNA-DNA > DNA*DNA-DNA > RNA*RNA-RNA, with no DNA*RNA-RNA triplex forming, is known, the kinetic roles of terminal residues remain poorly understood. Here, we employ bio-layer interferometry (BLI) and circular dichroism (CD) spectroscopy to demonstrate that dangling ends from both the third strand (triplex-forming oligonucleotide, TFO) and the duplex dramatically enhance triplex stability. Kinetic analysis reveals this stabilization is primarily driven by a marked increase in the association rate (k{square}{square}). Crucially, creating a single-base-pair dangling end at either terminus of the duplex enhanced triplex stability more effectively than blunt ends. For example, DNA TFO dTFO5 binding to d(HP5+TA) was enhanced compared to dHP5, and similarly RNA TFO rTFO5 binding to RNA duplex r(HP5+UA) and DNA duplex d(HP5+TA) showed stronger affinity and faster association than to blunt-ended rHP5 and dHP5. Interestingly, removal of a terminal base pair from the blunt-end duplex, generating a TFO dangling end, also enhances binding affinity and association rate. This indicates that both duplex and TFO dangling ends provide critical nucleation platforms, while blunt-ended terminal triples are dynamic and contribute minimally to stability. Thus, our work establishes that optimal triplex formation requires strategic optimization of both TFO and duplex terminal structures through a fundamental kinetic principle (bimodal nucleation). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/686076v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1dc6950org.highwire.dtl.DTLVardef@1861387org.highwire.dtl.DTLVardef@16c24a5org.highwire.dtl.DTLVardef@da0bf0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDangling ends at both third strand and duplex termini enhance triplex stability by accelerating association C_LIO_LITriplex formation can be nucleated from either end with a bimodal association mechanism C_LIO_LITerminal blunt-end base triples are dynamic and contribute minimally to stability compared to tailored overhangs C_LI