RNAs are emerging as important biomarkers and therapeutic targets. The strategy of directly targeting double-stranded RNA (dsRNA) by triplex-formation is relatively underexplored mainly due to the weak binding at physiological conditions for the traditional triplex-forming oligonucleotides (TFOs). Compared to DNA and RNA, peptide nucleic acids (PNAs) are chemically stable and have a neutral peptide-like backbone, and thus, they show significantly enhanced binding to natural nucleic acids. We have successfully developed nucleobase-modified dsRNA-binding PNAs (dbPNAs) to facilitate structure-specific and selective recognition of dsRNA over single-stranded RNA (ssRNA) and dsDNA regions at near-physiological conditions. The triplex formation strategy facilitates the targeting of not only the sequence but also the secondary structure of RNA. Here, we report the development of novel dbPNA-based fluorescent light-up probes through the incorporation of A-U pair-recognizing 5-benzothiophene uracil ( bt U). The incorporation of bt U into dbPNAs does not affect the binding affinity toward dsRNAs significantly, in most cases, as evidenced by our nondenaturing gel shift assay data. The blue fluorescence emission intensity of bt U-modified dbPNAs is sequence-and structure-specifically enhanced by dsRNAs, including the influenza viral RNA panhandle duplex and HIV-1−1 ribosomal frameshift-inducing RNA hairpin, but not ssRNAs or DNAs, at 200 mM NaCl, pH 7.5. Thus, dbPNAs incorporating bt U-modified and other further modified fluorescent nucleobases will be useful biochemical tools for probing and detecting RNA structures, interactions, and functions.

Recognition of RNAs at physiological conditions is important for developing chemical probes and therapeutic ligands. Nucleobase modified dsRNA-binding PNAs (dbPNAs) are promising for the recognition of dsRNAs in a sequence and structure specific manner at near-physiological conditions. Guanidinium is often present in proteins and small molecules for the recognition of G bases in nucleic acids, in cell-penetrating carriers, as well as in bioactive drug molecules, which might be due to the fact that guanidinium is amphiphilic with unique hydrogen bonding and stacking properties. We hypothesized that a simple guanidinium moiety can be directly incorporated into PNAs for facilitating enhanced molecular recognition of G-C pairs in dsRNAs and for improved bioactivity. We grafted guanidinium moiety directly into a PNA monomer (designated as R) using a two-carbon linker as guided by computational modeling studies. The synthetic scheme of PNA R monomer is relatively simple compared to that of previously reported L monomer. We incorporated the R residue into various dbPNAs for binding studies. dbPNAs incorporated with R residues are excellent in sequence specifically recognizing G-C pairs in dsRNAs over dsDNA and ssRNAs. We demonstrated that R residue is compatible with unmodified T and C and previously developed modified L and Q residues in dbPNAs for targeting model dsRNAs, influenza A viral panhandle duplex structure, and HIV-1 frameshift site RNA hairpin. Furthermore, R residues enhance the cellular uptake of PNAs.

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