# Investigation of the Characteristics of NLS-PNA: Influence of NLS Location on Invasion Efficiency
## 3 Results and Discussion
### Changing the Location of the NLS from the C-Terminus to the N-Terminus of the PNAs  
As mentioned above, in the previous report on NLS-PNAs, the NLS peptide was conjugated to the C-termini of the PNAs (C-PNAs). The synthesis of NLS-PNA was carried out from the C-terminus to the N-terminus by solid-phase peptide synthesis, and the NLS peptide was introduced to the resin first, followed by the PNA. When examining various functional peptides as alternatives to NLS for future research, N-terminally modified PNA conjugates, rather than C-terminally modified PNA conjugates, would significantly reduce the time and effort required for investigation. This is because it is possible to synthesise a large number of peptide-modified PNAs at once by elongating the PNAs first and then splitting the resin followed by peptide conjugation. Therefore, we evaluated whether changing the location of the NLS from the C-terminus to the N-terminus of the PNA (N-PNA) would have a detrimental effect upon invasion efficiency. PNAs modified with NLS peptide at the N-termini (N-PNAs) were synthesised using the same method as C-PNAs, and the invasion experiments with a series of PNAs were carried out under a broad range of salt concentrations, ranging from 0 to 125 mM NaCl.  
As a control, the invasion efficiency of U-PNAs was also examined at increasing salt concentrations. Figure 3 shows that the band intensity of the invasion complex with U-PNA decreased with increasing salt concentration and almost disappeared when the NaCl concentration exceeded 50 mM. This result reaffirms the aforementioned phenomenon, wherein elevated salt concentrations stabilise dsDNA whilst weakening PNA/DNA interaction, resulting in a lower invasion efficiency. When the same experiment was conducted using C-PNAs, however, a high invasion efficiency beyond 50 mM NaCl was observed. Unlike in our previous report with pentadecamer NLS-PNAs, in the present study, a decrease in invasion efficiency was observed above 75 mM NaCl. This is due to the lower concentration of PNA, which we selected herein to amplify any effect. The introduction of NLS, even in shorter PNAs, was once more confirmed to be beneficial for invasion at high salt concentrations. Next, the effect of NLS position on invasion efficiency was investigated using N-PNAs. At first glance, both C-PNAs and N-PNAs exhibited similar behaviour in terms of invasion efficiency. The invasion efficiencies of both NLS-PNAs remained high, up to about 75 mM NaCl, although this value gradually decreased with  
Figure 2: Comparison of U-PNA and C-PNA invasion efficiency in the presence of 75 mM NaCl. M: 20-bp DNA Ladder (TaKaRa); lane 1: 130-bp DNA only; lane2 U-PNA; lane 3: C-PNA. Invasion conditions: [DNA] = [each PNA] = 50 nM, [HEPES (pH 7.0)] = 5 mM, and [NaCl] = 75 mM at 50 \({}^{\circ}\)C for 1 h.  
increasing salt concentration. This comparison confirms that, regardless of the location of the NLS, PNA-NLS conjugates display similar performance. However, N-PNAs were slightly better in terms of resistance to higher salt concentrations (over 75 m\(>\)M) than C-PNAs, with a more pronounced effect at very high salt concentrations of 125 mM.