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 (HN-Lys-TCTCCTCCC-CONH) 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), 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 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 and ΔH. (iv) There was sequence and position dependence of the mismatch on ΔH and the activation energy (E), 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.

Fluorescent Trimethylated Naphthyridine Derivative with an Aminoalkyl Side Chain as the Tightest Non‐aminoglycoside Ligand for the Bacterial A‐site RNA

“…[49] Although ATMND-C 2 -NH 2 showedr ather selectiveb inding to the bacterial A-site ( Figure S13), better selectivity would be needed in order to make the ligand more suitable for clinical use. For this, fore xample, conjugation to ATMND-C 2 -NH 2 with triplex-forming peptide nucleic acids [50][51][52][53][54] for the double-stranded region near the A-site would be apossible approach. Such an improved ligand would be also useful for fluorescent indicator design suitable for FID assay.F or the application to high-throughput screening of al arge chemical library,i ti sh ighly desirable for the fluorescencei ndicator to have al ongere mission wavelengtht han that of ATMND-C 2 -NH 2 .…”

“…Although ATMND‐C 2 ‐NH 2 showed rather selective binding to the bacterial A‐site (Figure S13), better selectivity would be needed in order to make the ligand more suitable for clinical use. For this, for example, conjugation to ATMND‐C 2 ‐NH 2 with triplex‐forming peptide nucleic acids for the double‐stranded region near the A‐site would be a possible approach. Such an improved ligand would be also useful for fluorescent indicator design suitable for FID assay.…”

Enhanced Binding Affinity of siRNA Overhang‐Binding Fluorescent Probes by Conjugation with Cationic Oligopeptides for Improved Analysis of the siRNA Delivery Process

“…For example, it would be useful to integrate cyanine dyes with longer emission wavelengths than that of TO, such as TO3 derivatives, as the signaling unit in the probe design, which could reduce the background autofluorescence. As for the binding property, the use of triplex‐forming PNAs to target the RNA duplex near the overhanging structure would lead to improved binding affinity and selectivity of the probe to target siRNAs. We are now undertaking further studies in these directions.…”

Spectroscopic, thermodynamic and kinetic analysis of selective triplex formation by peptide nucleic acid with double‐stranded RNA over its DNA counterpart

“…We used a 9-mer PNA (cytosine content: 66%) which we reported bound to RR at pH 5.5 (Figure 2). [26][27][28]35] By means of UV melting, ITC and stopped-flow measurements, characteristics of the P*RR and P*DD F I G U R E 1 Comparison of the overall structures and the sugar puckering of dsRNA and dsDNA. (A) The dsDNA solution structure (PDB: 1LWA) (5 0 -GGG GTG ATT GTT CAG-3 0 /3 0 -CCC CAC TAA CAA GTC-5 0 ), solved by nuclear magnetic resonance.…”