Conjugated and Side-Chain Cationic Peptide Nucleic Acids (PNAs): Synthesis and Enhanced Cell Uptake of Cationic PNAs and Their DNA Duplexes into Cells

Peptide nucleic acids are neutral analogues of DNA with an aminoethyl glycine polyamide backbone, which renders a higher binding affinity to complementary DNA but reduces cell permeability due to increased hydrophobicity. We have conjugated aeg-PNA with the cationic amino acids lysine (lys), arginine (arg), and the dipeptides lys-lys and arg-arg at the N/C-terminus and introduced cationic S-aminomethylene (S-am) and S-methylene guanidine (S-mgd) side chains at Cα in 8-mer PNAs. The thermal stability of cationic PNA:DNA duplexes was enhanced due to electrostatic interactions between the cationic PNA substitutions and the anionic DNA in the range of 6–11 °C and in the following order: PNA 12-S-am2 >PNA 9-S-mgd >PNA 10-lys2 >PNA 5-S-mgd >PNA 4 S-am)∼PNA 8-arg>PNA 11-arg2, and the rest were < 3.0 °C. The side-chain-modified substitutions were generally better than the end-conjugated amino acids. The single-stranded C-terminus cationic PNAs showed 1.4–1.7-fold higher cell uptake than aeg-PNA in SH-SY5Y cells, in the following order: S-am2>S-mgd >arg2>lys> lys2>arg>S-am>aeg-PNA. FRET experiments of cationic PNA hybridized with fluorescently labeled DNA within cells exhibited increased cellular uptake of PNA:DNA duplexes, with the CF-PNA 12-S-am2 up by 12.7-fold, suggesting significantly improved cell uptake of cationic PNA:DNA duplexes. FRET imaging indicated the uptake of intact cationic PNA:DNA duplexes into the cells, suggesting their application potential for transporting DNA into cells.