Sequence selective recognition of double-stranded RNA by cationic peptide nucleic acids

Double-stranded RNA has become an attractive drug target because of the vital role it plays in gene expression and disease development. Peptide Nucleic Acids (PNA) are DNA analogues that can recognize single-stranded nucleic acids with high affinity and sequence selectivity. We recently showed that PNA also binds double-stranded RNA, such as the trans-activation responsive element (TAR) of HIV-1, with high affinity and sequence selectivity. Although PNA is promising as an antisense agent, it suffers from poor cellular uptake. Cellular uptake may be improved by cationic modifications either introduced directly in PNA's backbone (guanidine-modified GPNA) or attached to PNA ends. We showed that short PNAs carrying cationic nucleobase and conjugated with tetralysine had strongly enhanced binding affinity to double-stranded RNA without comprising sequence selectivity at physiological relevant conditions. Most importantly, attachment of short lysine peptides to PNA enhanced binding affinity for complementary double-stranded RNA while no binding could be detected for the complementary DNA hairpin. Confocal fluorescence microscopy showed that cationic modifications greatly enhanced cellular uptake of PNAs in HEK293 cells, whereas little uptake was observed for unmodified PNAs. Our results show that cationic modifications can be used to selectively optimize PNA's binding to double-stranded RNA. Cationic PNA holds promise as an antisense agent and as a tool to gain understanding of specific RNA functions in live cells.