## 4 Recent Invasion-Related Research
### Double-Stranded DNA Recognition by Non-PNA-Type Artificial Nucleic Acids  
Several artificial nucleic acids targeting dsDNA have been reported in a recent study. Prof. [PERSON] et al. designed chemically modified oligonucleotides with intercalator-conjugated nucleotides (invader probes; Figure 13a) [86; 87; 88; 89]. These invader probes form a stable duplex with DNA owing to stacking interactions between base pairs and intercalators. In contrast, the intercalator plays a role in inducing local perturbation and destabilizing the duplex between one probe strand and a complementary probe strand. The stability difference between target DNA/invader probe and invader probe/invader probe duplex provides the driving force for dsDNA recognition via double-duplex invasion. The structure of the invader probe has been optimized, and 2\({}^{\prime}\)-O-(pyrene-1-yl)methyl-RNA is mainly used as the monomer for intercalator introduction. dsDNA-targeting properties of invader probes were evaluated by using hairpin DNA as a model target, resulting in successful DNA recognition. In the latest research, the authors have successfully improved dsDNA recognition of invader probes with unique strategies, including toehold and nicked invader designs [90; 91]. The authors have been using this system to work on FISH for targeting chromosomal DNA and have detected complex formation between invader probes and chromosomal DNA by fluorescence microscopy. Prof. [PERSON] et al. achieved dsDNA recognition and sequence-specific fluorescent labeling by combining unmodified PNA with a linear probe [92] (Figure 13b). The linear probe has multiple fluorophores on a \({}_{D}\)-threoninel scaffold in an oligonucleotide [93; 94]. In the single-stranded state, the linear probe does not emit fluorescence due to the self-quenching of fluorophores, but when the linear probe hybridizes with target single-stranded DNA, fluorophores intercalate between the base pairs, triggering a strong fluorescence. In contrast, hybridization between the linear probe and PNA is suppressed since PNA/DNA duplex is known to be inflexible to accommodate intercalators and the fluorophores in the linear probe exhibit inhibitory effect for hybridization with the complementary PNA. Melting temperature analysis demonstrated that the \(T_{\text{m}}\) values followed the order of DNA/PNA > linear probe/DNA >> linear probe/PNA, satisfying the requirements for effective invasion. An electrophoresis mobility shift assay demonstrated that linear probe and PNA invaded and fluorescently labeled the target dsDNA. Heat shock treatment enabled double-duplex invasion in the central region of dsDNA. Even without a heat shock, an invasion complex was observed at the ends of 50-bp dsDNA at 40 \({}^{\circ}\)C and 100 mM NaCl. Cross-linkable artificial nucleic acids have also been developed for the formation of a double-duplex invasion complex. Prof. [PERSON] et al. succeeded in performing photo-induced double-duplex invasion using ultrafast photo-cross-linking [95]. Artificial nucleobase cyanovinylcarbazole (\({}^{\text{CNV}}\)K; Figure 14a) was developed as the class of photoreactive moiety for nucleic acids, enabling covalent bonds between \({}^{\text{CNV}}\)K in oligonucleotide  
Figure 13: Sequence-selective recognition of dsDNA with (**a**) invader probes and (**b**) a combination of linear probe and unmodified PNA. and a pyrimidine base at the \(-1\) position in complementary DNA or RNA [96,97]. To expand this system to invasion, uracil analog 5-cyanouracil (\({}^{\text{CN}}\)U) was also used to inhibit photo-cross-linking between each invader strand [95,98]. The reactivity of photo-cross-linking between \({}^{\text{CNV}}\)K and \({}^{\text{CN}}\)U was lower than \({}^{\text{CNV}}\)K and T (Figure 14b,c).