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Chapter 3
Table 3-1. PNA sequences and melting temperatures of the PNA:DNA antiparallel
fullmatch/mismatch duplexes in phospate buffer (pH=7) at a 5µm concentration for each strand
Sequence
Tm (°C) Tm (°C) ΔTm (°C)
PNA/DNA
Fullmatch
PNA/DNA
Mismatch
H-GTAGATCACT-NH 2 50 40 10
H-GTAGA(T 2D,5L-Lys )CACT-NH 2 60 43 17
PNA 1: H-GTAGA(T 2D,5L-Arg )CACT-NH 2 60 41 19
PNA2: H-GTAG(A 2D-Arg )(T 2D,5L-Arg )(C 5L-Arg )ACT-NH 2 60 40 20
Oligonucleotide sequences: Fullmatch: 5’-AGTGATCTAC-3’ Mismatch: 5’-AGTGGTCTAC-3’
The melting temperatures in Table 3-1 confirm that the substitution of lysine side chains with
arginine side chains did not impair the binding abilities of PNAs. The stability of the
fullmatch PNA-DNA hybrids, indeed, was the same for both new PNAs, identical to those
measured for the analogous Lys-based PNA 11 and much better than that shown by the
unmodified PNA. Even the selectivity was not affected: no selectivity decrease was observed
for the Arg-PNAs, and even a small increase could be observed for these new probes, making
it even more attractive for diagnostic applications. Thus, these new molecules are perfectly
suitable for applications on surface, preserving all the good features of lysine-based PNAs
without the presence of reactive amino groups. Quite unexpectedly, PNA 2, albeit the
presence of an extended series of four arginine side chains flanking the nucleobase
corresponding to the mismatch, showed for fullmatch and mismatch hybrids stabilities fully
comparable to those obtained by using PNA 1, which only had two arginine side chains.
Although PNA 2 presents two more positive charges, the fullmatch hybrid stability did not
change, and the increased sterical hindrance did not significantly decrease the stability of the
mismatch hybrid. This can be explained according to previously published works 16 , by
supposing that most of the contribution to the stability of the fullmatch duplex is given by the
2D,5L monomer: both the positively charged arginine side chains and the correct
configurations imparting a right-handedness to the PNA strand increase the DNA affinity.
Increasing the number of arginine side chains probably does not further affect the preferred
handedness, and the increased number of positive charges, which should enhance electrostatic
attraction for the complementary DNA strand, is probably balanced by the increased
hindrance introduced in the middle of the PNA strand. The same counteracting effects
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