Unlike conventional triplex-forming oligonucleotide (TFO), triplex-forming peptide nucleic acid (PNA) can tightly bind with double-stranded RNA (dsRNA) than double-stranded DNA (dsDNA). Here, we performed spectroscopic, thermodynamic and kinetic experiments for triplex formation by PNA to examine different binding behaviors between PNA − dsRNA and PNA − dsDNA triplexes. We found 9-mer PNA (cytosine content of 66%) formed the thermally stable triplex with dsRNA compared to dsDNA over a wide range of pH (5.5-8.0), salt concentration (50-500 mM NaCl). Both the calorimetric binding constant and the association rate constant for dsRNA were larger than those for dsDNA, indicating the favorable association process for the PNA − dsRNA triplex formation. Comparison with the DNA/RNA heteroduplexes revealed that the DNA strand was detrimental to the triplex stability for PNA, a contrasting result for conventional TFO. The keys underlying the difference in the triplex formation of PNA with different duplexes appear to be the conformational adoptability and the geometric compatibility of PNA to fit the deep, narrow major groove of dsRNA and the helical rigidity difference of the duplexes. Our results emphasize the importance of both the sugar puckering of the duplex and the appropriate conformational flexibility of PNA for the triplex formation.
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