The preparation of new metal-containing Peptide Nucleic Acids (PNAs) is currently a field of research intensively studied for various purposes, e.g. DNA biosensors. The role played by the metal centre, notably on the stability of the PNA.DNA hybrid, is obviously crucial, but has not yet been fully investigated. In this work, UV-Vis spectroscopic measurements of solutions of DNA.PNA hybrids, whose 11/12-mer PNA oligomers contained either one or two alkyne- (1) or ferrocene-containing (2) PNA monomers, were carried out to determine the effect of these monomers on the thermal stability of the hybrids (PNA: H-Gly-X-gggtc-Y-agctt-X-Lys-NH2 with X = 1 or and Y = 1 or 2 or blank position). Supplementary CD spectroscopic measurements were performed to gain insight into the structures of the PNA.DNA duplexes formed. The effect of both modified monomers was found to depend on their actual positions within the PNA sequences. Insertions at the N- or C-termini of a PNA oligomer did not change the melting temperatures (T(m) values of about 72 degrees C) of the DNA.PNA hybrids significantly. Insertion of monomers 1 or 2 in the middle of a PNA sequence induced a substantial decrease in the T(m) of the hybrids (by about 23 degrees C) when bound to the same DNA oligomer. Interestingly, it was found that the type of modification, namely alkyne or ferrocene, did not significantly influence the T(m) values in these cases. However, the thermal stability of hybrids with the DNA oligomers containing one to four additional thymines and the PNA oligomers containing the ferrocene moiety in its middle, varied significantly with the number of thymines added compared to its alkyne analogues (DeltaT(m) up to -13 degrees C). The presence of the ferrocene moiety induced a significant decrease in thermal stability of the hybrids, probably due to its bulkiness. In order to assess the effect of PNA backbone rigidity on the stability of DNA.PNA hybrids, PNA oligomers with an internal amino acid, propargylglycine (Pgl) or the dipeptide glycine-propargylglycine (Gly-Pgl), were synthesised. It was assumed that the orientation of the alkyne moiety in the Pgl-containing PNA sequence is not identical to an alkyne-containing PNA sequence, as a significantly higher T(m) value (DeltaT(m) = +10 degrees C) was measured. It is anticipated that the alkyne moiety in Pgl is not facing the DNA base and therefore does not disturb as much the neighbouring nucleobases and base-stacking of the complementary DNA, in contrast to the alkyne moiety of 1. Interestingly, no significant differences in the thermal stability of the hybrids was observed between Pgl-containing and dipeptide-containing PNA oligomers, although the former contracts the PNA backbone by three atoms.