Kinetic and thermodynamic analysis of triplex formation between peptide nucleic acid and double-stranded RNA

Abstract

Kinetics and thermodynamics of triplex formation between 9-mer homopyrimidine PNA (H₂N-Lys-TCTCCTCCC-CONH₂) and double-stranded RNA (dsRNA, 5′-AGAGGAGGG-3′/3′-UCUCCUCCC-5′) at acidic pH were studied by means of a stopped-flow technique and isothermal titration calorimetry (ITC). These results revealed the following main findings: (i) the stable PNA–dsRNA triplex formation mostly originated from the large association rate constant (k_on), which was dominated by both the charge neutral PNA backbone and the protonation level of the PNA cytosine. (ii) The temperature dependence of the enthalpy change (ΔH) and k_on suggested that the association phase of the PNA–dsRNA triplex formation comprised a non-directional nucleation–zipping mechanism that was coupled with the conformational transition of the unbound PNA. (iii) The destabilization by a mismatch in the dsRNA sequence mainly resulted from the decreased magnitude of both k_on and ΔH. (iv) There was sequence and position dependence of the mismatch on ΔH and the activation energy (E_on), which illustrated the importance of base pairing in the middle of the sequence. Our results for the first time revealed an association mechanism for the PNA–dsRNA triplex formation. A set of the kinetic and thermodynamic data we reported here will also expand the scope of understanding for nucleic acid recognition by PNA.