# Investigation of the Characteristics of NLS-PNA: Influence of NLS Location on Invasion Efficiency
## 3 Results and Discussion  
### A Positive Effect on Invasion Efficiency by the Introduction of an NLS into Short PNAs  
In a previous report, we successfully demonstrated that conjugation of an NLS to pcRNAs results in enhanced invasion efficiency [37]. Specifically, an NLS peptide with the sequence PKKKRKV found in the SV40 virus, which has been widely studied, was conjugated at the C-terminus of a pentadecamer PNA. If the introduction of an NLS, even in short PNAs, exerts a positive effect upon invasion efficiency, then the properties of NLS-PNAs should be more easily elucidated by examining them with short PNAs, whose synthesis requires less effort than longer PNAs. Thus, we checked the effectiveness of the NLS in decamer PNAs and employed two types of PNA: (1) unmodified PNAs (U-PNAs), i.e., a pair of decamer pcRNAs without an NLS (with only a few lysine residues for solubility) and (2) C-PNAs, which are identical in sequence to U-PNAs but with an NLS at their C-termini. U-PNAs and C-PNAs were synthesised by standard _tert_-butoxycarbonyl (Boc)-based solid-phase peptide synthesis. The PNA sequences are listed in Table 1. PNA1 and PNA2 represent each strand of pcPNA that is complementary to 10 bp in the pBR322 plasmid (PNA1: GUUDCUGDUG and PNA2: CDUCDGUDDC). All PNAs have a free N-terminal amino group, with the C-terminal carboxylic acid being converted to an amide. The efficiency of invasion complex formation was evaluated by an electrophoresis mobility shift assay (EMSA). The formation of the invasion complex results in a lower electrophoretic mobility, mainly due to changes in the local structure of the dsDNA at the invasion site [43]. This allows the separation of the invasion complex from the free dsDNA. An EMSA of the invasion complex formation in the presence of 75 mM NaCl is shown in Figure 2.  
In lane 1, a single band corresponding to free dsDNA was observed between 100 and 150 bp. In the presence of U-PNA, the invasion complex was barely formed under the high-salt conditions employed, giving only a faint band with lower mobility between 180 and 200 bp (lane 2), which was assignable to the invasion complex. However, the C-PNAs with NLS at their C-termini gave an intense band corresponding to the invasion complex (lane 3). It is clear from these results that the invasion efficiency was improved by the conjugation of an NLS to the short PNAs, leading to the formation of a \begin{table}
\begin{tabular}{c c} \hline
**Name** & **Sequences [a] (N to C)** \\ \hline U-PNA1 & H-\(K\)**GUUDCUGDUG**\(KK\) -NH\({}_{2}\) \\ \hline U-PNA2 & H-\(K\)**CDUCDGUDDC**\(KK\) -NH\({}_{2}\) \\ \hline N-PNA1 & H-\(PKKKKV\)**GUUDCUGDUG**\(KK\) -NH\({}_{2}\) \\ \hline N-PNA2 & H-\(PKKKKV\)**CDUCDGUDDC**\(KK\) -NH\({}_{2}\) \\ \hline C-PNA1 & H-\(K\)**GUUDCUGDUG**\(PKKKKKV\) -NH\({}_{2}\) \\ \hline N-Pro-PNA1 & H-\(PKKKKV\)**GUUDCUGDUG**\(KK\) -NH\({}_{2}\) \\ \hline N-Pro-PNA2 & H-\(PKKKKV\)**CDUCDGUDDC**\(KK\) -NH\({}_{2}\) \\ \hline \end{tabular} \({}^{[a]}\) Bold: PNAs; Italic: amino acids; \(K\) = lysine; \(P\) = proline; \(R\) = arginine; \(V\) = valine; D = 2,6-diaminopurine; U = 2-thioouracil. \(\underline{P}\) = proline inserted between the PNA and valine of the NLS  
\end{table} 
Table 1: PNAs synthesised and used in this research. stable invasion complex even at elevated salt concentrations. As a result, it was confirmed that the NLS modifications in short PNAs showed a beneficial effect on invasion. Thus, we decided to investigate the properties of NLS-PNAs by using these short decamer PNAs. The effect of NLS modification appears to be mainly attributable to electrostatic interactions. However, the conjugation of PNA with repeating cationic peptides (e.g., polylysine) also increases non-specific interactions with the DNA backbone, resulting in a negative effect on the formation of the invasion complex [44]. Presumably, in biological motifs, unfavourable sequences (e.g., those provoking non-specific binding to DNA) have been evolutionarily excluded.