PNA-based antisense oligonucleotides for micrornas inhibition in the absence of a transfection reagent.

MicroRNAs (miRNAs) are noncoding RNAs approximately 22 nucleotides in length that play a major role in the regulation of important biological processes, including cellular development, differentiation, and apoptosis. Antisense oligonucleotides against miRNAs are useful tools for studying the biological mechanisms and therapeutic targets of miRNAs. Various antisense oligonucleotides chemistries, including peptide nucleic acids (PNAs), have been developed to enhance nuclease-resistance and affinity and specificity for miRNA targets. PNAs have a greater specificity and affinity for DNA and RNA than do natural nucleic acids, and they are resistant to nucleases-an essential property of an miRNA inhibitor that will be exposed to cellular nucleases. However, the main limiting factor in the use of PNAs is their reduced penetration into cells. Recently, several cell-penetrating peptides (CPPs) have been investigated as a means to overcome the limited penetration of PNAs. Here, we evaluated the ability of 11 CPPs to transport PNAs inside cells in the absence of transfection reagents and then investigated the ability of these CPPs to inhibit miRNAs. Of the 11 CPPs tested, Tat-modified-conjugated PNA showed the most effective penetration into cells in the absence of transfection reagents and most effectively inhibited miRNAs. Our data demonstrate that Tat-modified-conjugated CPP is the most suitable for supporting PNA-mediated miRNA inhibition.

A highly effective and long-lasting inhibition of miRNAs with PNA-based antisense oligonucleotides.

MiRNAs are non-coding RNAs that play a role in the regulation of major processes. The inhibition of miRNAs using antisense oligonucleotides (ASOs) is a unique and effective technique for the characterization and subsequent therapeutic targeting of miRNA function. Recent advances in ASO chemistry have been used to increase both the resistance to nucleases and the target affinity and specificity of these ASOs.Peptide nucleic acids (PNAs) are artificial oligonucleotides constructed on a peptide-like backbone. PNAs have a stronger affinity and greater specificity to DNA or RNA than natural nucleic acids and are resistant to nucleases, which is an essential characteristic for a miRNA inhibitor that will be exposed to serum and cellular nucleases.For increasing cell penetration, PNAs were conjugated with cell penetrating peptides (CPPs) at N-terminal. Among the tested CPPs, Tat-modified peptide-conjugated PNAs have most effective function for miRNA inhibition. PNA-based ASO was more effective miRNA inhibitor than other DNA-based ASOs and did not show cytotoxicity at concentration up to 1,000 nM. The effects of PNA-based ASOs were shown to persist for 9 days. Also, PNA-based ASOs showed considerable stability at storage temperature. These results suggest that PNA-based ASOs are more effective ASOs of miRNA than DNA-based ASOs and PNA-based ASO technology, compared with other technologies used to inhibit miRNA activity can be an effective tool for investigating miRNA functions.