Peptide nucleic acid (PNA) has become an extremely powerful tool in chemistry and biology. Although PNA recognizes single-stranded nucleic acids with exceptionally high affinity and sequence selectivity, there is considerable ongoing effort to further improve properties of PNA for both fundamental science and practical applications. The present paper discusses selected recent studies that improve on cellular uptake and binding of PNA to double-stranded DNA and RNA. The focus is on chemical modifications of PNA's backbone and heterocyclic nucleobases. The paper selects representative recent studies and does not attempt to provide comprehensive coverage of the broad and vibrant field of PNA modification. 1. Introduction Peptide nucleic acid (PNA) is a DNA analogue that has the entire sugar-phosphodiester backbone replaced by a pseudopeptide linkage built of 2-aminoethylglycine residues (Figure 1) [1]. PNA is highly stable chemically and, because of the unnatural backbone, highly resistant to enzymatic degradation, which makes it an excellent candidate for in vivo applications as an oligonucleotide analogue. The neutral pseudopeptide backbone eliminates electrostatic repulsion (a factor that negatively affects oligonucleotide binding) and PNA binds to DNA and RNA with excellent affinity. PNA binds to double helical DNA via two competing binding modes, triple helix (PNA？:？DNA, 1？:？1), and strand invasion, where PNA displaces one of the DNA strands, typically followed by a triplex formation (PNA？:？DNA, 2？:？1) [1]. PNA also forms exceptionally strong and sequence-specific Watson-Crick duplexes with single-stranded DNA and RNA [2]. Interestingly, the sequence specificity of duplexes involving PNA is substantially higher than that of unmodified nucleic acids. Because of these superior qualities, PNA has become a powerful tool in chemical biology and biotechnology [3–5]. The main applications of PNA are as hybridization probes and molecular diagnostics of high affinity and selectivity for single-stranded DNA and RNA. PNA also holds a promise of becoming a novel gene therapy agent for targeting specific RNA molecules [3, 4]. Figure 1: Structures of DNA and PNA repeating units. Although PNA binds single-stranded DNA and RNA with superior affinity and selectivity, there are other properties of PNA that can be further improved. Most importantly, in vivo applications of unmodified PNA are hindered by poor cellular uptake and endosomal entrapment [6]. Current methods to enhance the cellular uptake of PNA, such as conjugation with cell penetrating peptides (CPP) [7,
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