## 3 Promotion of Double-Duplex Invasion by Modified PNAs
### Chiral PNAs  
Unlike DNA and other conventional artificial nucleic acids, aegPNA, composed of an \(N\)-(2-aminoethyl)glycine backbone, has an achiral structure. It has been reported that aegPNA itself has no preference for helicity and its helical structure changes depending on the chirality of the C-terminal amino acid [64]. The duplex of PNA decamers with \({}_{L}\)-lysine at the C-termini gave the CD spectrum similar to that of DNA duplex in the nucleobase absorption region, and the mirror-image spectrum was obtained when \({}_{L}\)-lysine was substituted with \({}_{D}\)-lysine. On the other hand, since natural DNA is a chiral molecule and forms a right-handed helical structure, PNAs with chirality introduced into the backbone have been developed to improve the DNA recognition ability. Looking carefully at the structure of PNA, we can imagine that the chirality can be easily introduced by replacing glycine in its backbone with other amino acids. Thus, lysine-incorporated \({}^{D}\)K\({}_{\alpha}\)-PNA derived from \(N\)-(2-aminoethyl)-\({}_{D}\)-lysine was developed in 1996 [65; 66] (Figure 6a). This chiral PNA strongly interacts with DNA, whereas the \({}_{L}\)-isomer has the opposite effect, suggesting that \({}_{D}\)-chirality is very important for high DNA affinity. In addition, the \({}^{D}\)K\({}_{\alpha}\)-PNA has an amino group introduced as a side chain to the PNA backbone, leading to the addition of a positive charge. The resulting conformational control of PNA and electrostatic attraction of PNA to negatively charged DNA enhance the DNA recognition ability of PNA (Figure 6b). It has also been reported that chiral PNAs containing pseudo-complementary nucleobases show a higher efficiency of invasion complex formation than achiral pcPNA [67]. This result is ex  
Figure 5: (**a**) The structure of pseudo-complementary nucleobases D and U\({}_{s}\). (**b**) D/T base pair has an additional hydrogen bond, and A/U\({}_{s}\) pairs can avoid steric repulsion, unlike D/U\({}_{s}\) base pair, resulting in a stable duplex between pcPNA and DNA. (**c**) D/U\({}_{s}\) base pair has steric repulsion and destabilizes the duplex between two pcPNAs. plained by the fact that in addition to improving the DNA binding strength of pcPNAs, the electrostatic repulsion also causes more significant destabilization of the pcPNA/pcPNA duplex (Figure 6c). This function of chiral PNA assists the effect of pseudo-complementary nucleobases D and U\({}_{\text{s}}\), and even if the percentage of D and U\({}_{\text{s}}\) in PNA strands is low, high invasion efficiency was maintained by using chiral pcPNAs. Moreover, these chiral pcPNAs show mismatch discrimination ability, although the modification methods that introduce positive charges are often accompanied by reduced selectivity to the target due to nonspecific interactions with negatively charged DNA. The backbone-modified PNA introduced so far has a side chain at the \(\alpha\)-position (Figure 6a), and it is categorized as \(\alpha\)-PNA. Chiral \(\alpha\)-PNAs using other amino acids instead of lysine have also been reported [48; 65]. Furthermore, in addition to these \(\alpha\)-PNAs, PNAs with various functional side chains introduced into the PNA backbone at the \(\gamma\)-position (\(\gamma\)-PNA) were developed (Figure 6a) and have been widely studied for their high DNA-binding ability [61; 68]. Although \(\gamma\)-PNAs with lysine side chains (\({}^{L}\)K\({}_{\text{V}}\)-PNAs) also exhibit high DNA-binding ability like \({}^{D}\)K\({}_{\alpha}\)-PNA, interestingly, the optical isomerism of lysine suitable for DNA recognition is reversed from that of the aforementioned \(\alpha\)-PNAs. When the \(\alpha\)-lysine framework is used for \(\gamma\)-PNA, its DNA binding ability is reduced. \(\gamma\)-PNAs possessing a methyl group or ethylene glycol have been extensively studied, as they have a significant effect on preorganizing the structure in favor of double-strand formation with DNA.