# Investigation of the Characteristics of NLS-PNA: Influence of NLS Location on Invasion Efficiency
## 1 Introduction  
The sequence-specific recognition of double-stranded DNA (dsDNA) has been an important research subject owing to its wide range of potential applications [1; 2; 3; 4; 5; 6; 7]. For this purpose, various methods have been developed, including those that make use of DNA-binding proteins [1; 3; 4; 5; 8; 9; 10; 11; 12], small molecules such as minor groove binders [13; 14; 15; 16], and artificial DNA [17; 18; 19; 20; 21; 22; 23; 24]. In addition, the recognition of dsDNA by peptide nucleic acid (PNA), an artificial nucleic acid mimic, has been reported by [PERSON] et al. [7; 21; 24; 25; 26; 27; 28; 29; 30; 31]. In 1991, PNA was first designed and synthesised as an analogue of natural nucleic acids, where the negatively charged sugar-phosphate backbone of DNA was substituted with an electrostatically neutral artificial \(N\)-(2-aminoethyl)glycine backbone (Figure 1a) [25]. Consequently, electrostatic repulsion between PNA and DNA is absent, enabling PNA to form more stable duplexes with complementary DNA via Watson-Crick base pairing than those between complementary DNA strands [31]. Furthermore, when pseudo-complementary PNAs (pcPNAs: see Figure S1)--where conventional adenine (A) and thymine (T) have been replaced by 2,6-diaminopurine (D) and 2-thiouracil (U)--are used, an effective invasion into dsDNA becomes possible, with two strands of pcPNA forming a double-duplex invasion complex (or, simply, invasion complex, Figure 1b) with dsDNA [26; 32]. The introduction of D and U into PNA destabilises the formation of PNA/PNA duplexes through steric repulsions between the amino group of D and the thioketone group of U. The thermal stability of the PNA/DNA duplex is enhanced by the formation of stable base pairs between D/U and the natural nucleobases. PNA's unique DNA-recognition mode has been used in many applications, including site-directed mutagenesis [2; 7], inhibition of enzymatic activity [26; 33; 34], and the development of artificial DNA cutters [35], with all of these depending on the sequence specificity of the invasion complex. The pseudopeptide backbone of PNA facilitates easy oligomer synthesis and modification for further improvements and imparts enhanced resistance to enzymatic degradation. Thus, double-duplex invasion is a promising method for the targeting of dsDNA in cellula.  
However, to realise such applications, an important issue must be overcome--namely, the high salt concentrations encountered in the cellular environment. High salt concentrations increase the stability of the DNA/DNA duplexes whilst slightly destabilising the PNA/DNA duplexes [36], which results in an overall decrease in invasion efficiency [37]. Several approaches have been attempted to enhance invasion efficiency in physiological salt concentrations, such as chemical modification of PNA and conjugation with functional molecules [37; 38; 39; 40; 41]. Our group developed conjugates of PNA with Ru-complexes and PNAs modified with a nuclear localisation signal (NLS) peptide to increase their applicability for DNA recognition at high salt concentrations (Figure 1b). These two modified PNAs displayed higher DNA affinity than corresponding unmodified pcRNAs and effectively formed an invasion complex even at physiological salt concentrations, whereunder unmodified PNAs stop functioning efficiently. It is important to understand the properties and limitations of these modified PNAs in order to further improve PNAs. Of these two methods, NLS modification is the simpler PNA modification method as the resulting NLS-modified PNAs (NLS-PNAs) can be procured by simply conjugating PNA with the functional peptide, and can be readily introduced during the synthesis of PNAs. Thus, in this study, we focused on NLS-PNAs and investigated their characteristics to obtain insight into how they can be improved.