## 2 Double-Stranded DNA Recognition by Peptide Nucleic Acid (PNA)
### Invasion Complex Formation by PNA  
The invasion of PNA is achieved by its high DNA-binding affinity mentioned above. In other words, the energy loss of dissociating the DNA duplex is compensated by more stable PNA/DNA duplex formation. Consequently, by using PNAs with complementary sequences, the sequences in dsDNA can be directly and selectively recognized without a denaturing treatment, which is generally not possible with other artificial nucleic acids. The invasion complex shown in Figure 2 is more correctly called double-duplex invasion complex [42], and the following other invasion complexes have also been reported (Figure 4).  
Figure 4: Various recognition modes of target dsDNA by PNA: (**a**) triplex invasion; (**b**) duplex invasion; and (**c**) double-duplex invasion.  
Figure 3: Structures of PNA analogs with modified backbone.  
In the triplex invasion complex [52] (Figure 4a), two PNAs are bound to one strand of a dsDNA by both Watson-Crick and Hoogsteen base pairing, forming a triplex. The DNA recognized by this triplex invasion must be a homopyrimidine/homopurine sequence, which limits the target sequence. Homopyrimidine PNAs are commonly used for triplex invasion, which requires protonation of the PNA nucleobases to form Hoogsteen base pairs. Therefore, the complex is stabilized under acidic conditions. To stabilize the triplex invasion complex, bis-PNA, in which two PNAs are linked by a linker, has been developed [53; 54]. Recently, tail-cramp PNAs (tcRNAs), asymmetric bis-PNAs in which the PNA strand for the Watson-Crick side is designed to recognize mix sequences, have been developed and reported for intracellular applications [38; 55; 56; 57; 58].  
The duplex invasion complex (Figure 4b) is formed when one complementary PNA forms a PNA/DNA duplex in a dsDNA by Watson-Crick base pairing [59]. The formation of this invasion complex was first reported for specific sequences with homopyrimidine-homopurine sequences, but it differs from the triplex invasion in that there is no third strand to form the Hoogsteen base pair. The recognition sequences of duplex invasion and triplex invasion are generally restricted to homopyrimidine-homopurine. In 2007, it was revealed that duplex invasion of PNA occurs even in mixed sequences, and the invasion complex was first confirmed by electrophoresis [60]. Subsequently, reports of duplex invasion in mixed sequences have continued with the methodology of enhancing the binding strength of PNA [61; 62].  
Double-duplex invasion [42] (Figure 4c), mainly discussed in this paper, is a complex formation in which two PNAs with complementary sequences form a Watson-Crick base pair with the target sequences in dsDNA. This complex does not require homopyrimidine/homopurine sequences like triplex and duplex invasion complexes, making it more suitable for various applications. This invasion is unique because PNAs directly recognize (invade into) dsDNA, which is not seen in other artificial nucleic acids. Moreover, another attractive feature is the ability to induce local structural changes in the target DNA during the formation of the invasion complex.