Closing the Loop: Constraining TAT Peptide by γPNA Hairpin for Enhanced Cellular Delivery of Biomolecules

Based on the exceptionally high stability of γPNA duplexes, we designed a peptide/γPNA chimera in which a cell-penetrating TAT peptide is flanked by two short complementary γPNA segments. Intramolecular hybridization of the γPNA segments results in a stable hairpin conformation in which the TAT peptide is constrained to form the loop. The TAT/γPNA hairpin (self-cyclized TAT peptide) enters cells at least 10-fold more efficiently than its nonhairpin analog in which the two γPNA segments are noncomplementary. Extending one of the γPNA segments in the hairpin results in an overhang that can be used for binding and delivering a variety of nucleic acid-conjugated molecules into cells via hybridization to the overhang. We demonstrated efficient cellular delivery of a protein (as low as 10 nM) and a DNA tetrahedron by a TAT/γPNA hairpin.