Sequence-selective recognition of complex RNAs in live cells could find broad applications in biology, biomedical research, and biotechnology. However, specific recognition of structured RNA is challenging, and generally applicable and effective methods are lacking. Recently, we found that peptide nucleic acids (PNAs) were unusually well-suited ligands for recognition of double-stranded RNAs. Herein, we report that 2-aminopyridine (M) modified PNAs and their conjugates with lysine and arginine tripeptides form strong (K = 9.4 to 17 × 10 M) and sequence-selective triple helices with RNA hairpins at physiological pH and salt concentration. The affinity of PNA-peptide conjugates for the matched RNA hairpins was unusually high compared to the much lower affinity for DNA hairpins of the same sequence (K = 0.05 to 1.1 × 10 M). The binding of double-stranded RNA by M-modified PNA-peptide conjugates was a relatively fast process (k = 2.9 × 10 M sec) compared to the notoriously slow triple helix formation by oligodeoxynucleotides (k ∼ 10 M sec). M-modified PNA-peptide conjugates were not cytotoxic and were efficiently delivered in the cytosol of HEK293 cells at 10 µM. Surprisingly, M-modified PNAs without peptide conjugation were also taken up by HEK293 cells, which, to the best of our knowledge, is the first example of heterocyclic base modification that enhances the cellular uptake of PNA. Our results suggest that M-modified PNA-peptide conjugates are promising probes for sequence-selective recognition of double-stranded RNA in live cells and other biological systems.

Conjugation of short peptide nucleic acids (PNA) with tetralysine peptides strongly enhanced triple helical binding to RNA at physiologically relevant conditions. The PNA hexamers and heptamers carrying cationic nucleobase and tetralysine modifications displayed high binding affinity for complementary double-stranded RNA without compromising sequence selectivity. The PNA-peptide conjugates had unique preference for binding double-stranded RNA, while having little, if any, affinity for double-stranded DNA. The cationic PNAs were efficiently taken up by HEK293 cells, while little uptake was observed for unmodified PNA.

Compounds that bind specifically to double-stranded regions of RNA have potential as regulators of structure-based RNA function; however, sequence-selective recognition of double-stranded RNA is challenging. The modification of peptide nucleic acid (PNA) with unnatural nucleobases enables the formation of PNA-RNA triplexes. Herein, we demonstrate that a 9-mer PNA forms a sequence-specific PNA-RNA triplex with a dissociation constant of less than 1 nm at physiological pH. The triplex formed within the 5' untranslated region of an mRNA reduces the protein expression levels both in vitro and in cells. A single triplet mismatch destabilizes the complex, and in this case, no translation suppression is observed. The triplex-forming PNAs are unique and potent compounds that hold promise as inhibitors of cellular functions that are controlled by double-stranded RNAs, such as RNA interference, RNA editing, and RNA localization mediated by protein-RNA interactions.

Non-coding RNAs play important roles in regulation of gene expression. Specific recognition and inhibition of these biologically important RNAs that form complex double-helical structures will be highly useful for fundamental studies in biology and practical applications in medicine. This protocol describes a strategy developed in our laboratory for sequence-selective recognition of double-stranded RNA (dsRNA) using triple helix forming peptide nucleic acids (PNAs) that bind in the major grove of RNA helix. The strategy developed uses chemically modified nucleobases, such as 2-aminopyridine (M) that enables strong triple helical binding at physiologically relevant conditions, and 2-pyrimidinone (P) and 3-oxo-2,3-dihydropyridazine (E) that enable recognition of isolated pyrimidines in the purine rich strand of the RNA duplex. Detailed protocols for preparation of modified PNA monomers, solid-phase synthesis and HPLC purification of PNA oligomers, and measuring dsRNA binding affinity using isothermal titration calorimetry are included.

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