## 4 Recent Invasion-Related Research  
### Development of New Backbone- or Nucleobase-Modified PNAs for DNA Recognition  
In addition to the \(\gamma\)PNA mentioned above, there have been significant modifications of the PNA backbone to enable dsDNA recognition. Prof. [PERSON] et al. synthesized various pyrrolinyl PNAs, which have a cyclic structure in the backbone [77; 78; 79]. Among them, a PNA having a (2\({}^{\prime}\)R,4\({}^{\prime}\)R)-pyrrolidine/(2S)-amino-cyclopentane-(1S)-carboxylic acids backbone (acpcPNA; Figure 10a) formed a very stable duplex with DNA. On the other hand, acpcPNAs composed of a bulky backbone cannot form stable acpcPNA/acpcPNA duplexes. In other words, the acpcPNA backbone is pseudo-complementary, suggesting acpcPNA is  
Figure 9: (**a**) Structure of cationic guanine PNA (G\({}^{+}\)-PNA). (**b**) Enhancement of DNA affinity and suppression of self-duplex formation through electrostatic interaction by using G\({}^{+}\)-PNAs. an alternative candidate to pcPNA for achieving double-duplex invasion. The stability of each duplex with DNA and acpcPNA is in the following order; PNA/DNA > PNA/PNA, which is completely opposite to that of aegPNA (PNA/PNA > PNA/DNA). Despite the absence of pseudo-complementary nucleobases, the fluorescence change indicated that fluorophore-labeled acpcPNAs recognized the short synthetic dsDNA (30 bp) through invasion (Figure 10b), and the invasion efficiency was around 15% [80]. In contrast, the fluorescence emission is negligible for the mismatch sequence, showing the sequence selectivity of acpcPNA. Furthermore, in polyacrylamide gel electrophoresis, a new band with slower mobility than dsDNA also appeared, indicating that complementary 10-mer acpcPNAs form an invasion complex with 30-bp dsDNA. Invasion-like structures in which a single PNA strand recognizes two DNA strands have also been reported. Prof. [PERSON] et al. introduced a second nucleobase into the \(\alpha\)-(C\(\alpha\)) or \(\gamma\)-position (C\(\gamma\)) of PNA in addition to the original nucleobase [81; 82; 83] (Figure 11a). This new type of PNA termed \"bimodal PNA (_bm_-PNA)\" can recognize two DNA strands simultaneously. Furthermore, by using multiple bimodal PNAs and combining duplex and triplex formation with complementary DNAs, fused duplexes, triplexes, and extended PNA/DNA assemblies were created [84] (Figure 11b). Besides, Prof. [PERSON] et al. employed bifacial nucleobases (namely, Janus nucleobases) and succeeded in recognizing dsDNA with one strand of PNA based on the \(\gamma\)PNA backbone (JBy\(\gamma\)NA) [85]. Bifacial nucleobases can selectively recognize A-T, T-A, G-C, and C-G base pairs by forming new base pairs on each side of bifacial nucleobases (Figure 12). Molecular dynamics (MD) simulations demonstrated that DNA/JByPNA on one side maintained a stable structure in antiparallel orientation, whereas JBy\(\gamma\)NA/DNA on the other side in parallel orientation collapsed its structure. The weaker parallel JBy\(\gamma\)NA/DNA interaction is attributed to less favorable binding orientation and fewer hydrogen bonds. The structure of JBy\(\gamma\)NA/JB\(\gamma\)PNA was collapsed due to the steric collision of its backbone. These results of MD simulations are consistent with circular dichroism (CD) and UV melting experiments. JBy\(\gamma\)PNA with six bifacial nucleobases can invade into not only 6-bp hairpin DNA but also internal binding sites in a stable 26-bp dsDNA under physiological conditions, and its complex formation was confirmed by gel electrophoresis. Figure 10: (**a**) Chemical structure of acpcPNA. (**b**) Invasion complex formation by fluorophore-labeled acpcPNAs due to steric hindrance between acpcPNAs. Figure 11: (**a**) Chemical structures of Ca and Cy-_bm_-PNAs. (**b**) _bm_-PNA recognizes two DNA strands forming a double duplex. Figure 12: (**a**) Structure of \(\gamma\)-miniPEG PNA. (**b**) Bifacial PNA (JByPNA) recognizes two strands of dsDNA simultaneously in both antiparallel and parallel orientations.