## 3 Promotion of Double-Duplex Invasion by Modified PNAs
### PNA Modified with Nuclear Localization Signal (NLS) Peptide (NLS-PNA)  
Although the chiral PNAs have improved invasion efficiency, their effect in high-salt conditions like in vivo has not been fully investigated. On the other hand, some modification methods of pcPNA have been reported to improve invasion efficiency at high salt concentrations, and one of them is PNA modified with a nuclear localization signal (NLS) peptide (Figure 7a). The NLS peptide is a functional peptide involved in the nuclear transport of proteins. Since this peptide is rich in basic amino acids (lysine and arginine), it is positively charged and is expected to interact strongly with DNA under neutral pH conditions. PNA, which has a peptide-based backbone, is synthesized by the standard solid-phase peptide synthesis [33], so the modification of peptides to PNAs is much easier than to other artificial nucleic acids, and the introduction of cell-penetrating peptides (CPPs) has been reported [69; 70]. Moreover, compared to other PNA modification methods, NLS-PNA is a very simple strategy as it only requires the introduction of additional peptides into the PNA.  
The paper reported in 2013 suggested that NLS-PNAs showed the formation of duplex invasion (not double-duplex invasion) [71]. These NLS-PNAs have been applied to dsDNA recognition because the PNA modified with an NLS peptide has shown antigen effects, and fluorescence spectroscopic studies have shown complex formation with NLS-PNAs and plasmid DNA. Given that it is challenging to form duplex invasion with unmodified PNA in the mix sequence, the contribution of NLS to improved DNA binding is unquestionable. Interestingly, this NLS-PNA also achieved dsDNA recognition in a consecutive 14 bp GC sequence, which is difficult to be targeted by unmodified pcPNAs.  
Inspired by the work described above, pcPNAs modified with an NLS peptide (NLS-pcPNAs) were developed in 2015, and high invasion efficiency was reported with NLS-pcPNAs, enabling the full recognition of target dsDNA with a lower concentration of PNAs than unmodified pcPNAs [72] (Figure 7b). These NLS-PNAs are effective at higher salt concentrations and retain the high invasion efficiency even at 100 mM NaCl, where the invasion efficiency of unmodified pcPNAs is significantly reduced. Furthermore, NLS-pcPNAs show overwhelmingly high invasion efficiency, even under physiological conditions where the invasion efficiency is further reduced with unmodified pcPNAs. The high mismatch discrimination ability was also retained in NLS-pcPNA, despite the fact that it utilizes electrostatic interactions. More interestingly, the NLS peptide used in this study shows its biological function even when conjugated with PNAs, and the NLS-PNA was confirmed to be present in the nucleus upon introduction into the cell [71].  
In the NLS-PNAs mentioned above, NLS was introduced at C-termini, but the design of NLS-PNA was also studied in detail, and the invasion efficiency of N-terminal modified NLS-PNAs was examined [73]. The results showed no drastic difference between C- and  
Figure 7: (**a**) Structure of NLS-PNA conjugate. The NLS peptide was directly attached to the C-terminus of pcPNAs. (**b**) Promotion of double-duplex invasion by NLS-pcPNAs.  
N-terminal modified NLS-PNAs, and NLS was effective for invasion in both modifications. N-terminal modification was shown to be slightly more effective at higher salt concentrations, indicating that the amino acids connecting the NLS to PNA may affect invasion.