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Arginine-PNAs
yield in this reaction. Moreover, since the byproduct had a retention time very similar to that
of the desired PNA molecule, the purification turned out to be very difficult, thus PNA 2 was
obtained as a mixture with this byproduct.
Figure 3-3: HPLC profiles and mass spectra of PNA1 and PNA 2
3.2.3 Selectivity of binding
In order to verify the affinity of the synthesized probes for complementary and mismatched
DNA sequences, melting temperatures were measured for the fullmatch hybrids and the
mismatch ones, the latter using an oligonucleotide with a guanine replacing the adenine
corresponding to the central modified PNA monomer. In the case of PNA 2, the presence of
the byproduct was taken into consideration for the calculation of the real concentration of the
molecule, assuming that the stability of the byproduct bound to DNA was too low to interfere
with the melting temperature of the complete probe. Melting temperatures were evaluated as
the first derivatives of the UV absorption curves at 260 nm in a temperature range from 20 to
90°C. The values obtained are shown in Table 3-1, together with those of the corresponding
achiral PNA and of the PNA containing a 2D,5L Lysine modified monomer in the middle,
which are reported in order to compare the results obtained with the new PNA probes 16 .
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