Sequence- And Structure-Specific Probing of RNAs by Short Nucleobase-Modified dsRNA-Binding PNAs Incorporating a Fluorescent Light-up Uracil Analog

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 (U-bt). The incorporation of U-bt 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 U-bt-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 U-bt-modified and other further modified fluorescent nucleobases will be useful biochemical tools for probing and detecting RNA structures, interactions, and functions.