A novel conformationally constrained pyridazinone E ag -base PNA-monomer 2 capable of binding thymine in a triplex motif was designed and synthesised. A bis-PNA with the E ag -base incorporated in the Hoogsteen strand was hybridised with a complementary DNA. Thermal stability studies revealed an increase in T m (4.3°C per mod.) compared to a no-base unit, but showed no improvement over a previously described unconstrained analogue (E, 1). Surprisingly, no significant difference was found in the thermodynamic parameters (ΔH°, ΔS° and ΔG°) for PNA–DNA triplex formation involving 2 or the unconstrained analogue 1.