Communication Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/bc
Closing the Loop: Constraining TAT Peptide by γPNA Hairpin for Enhanced Cellular Delivery of Biomolecules Xiaohong Tan,*,† Marcel P. Bruchez,*,†,‡ and Bruce A. Armitage*,† †
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Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States ‡ Department of Biological Sciences and Molecular Biosensor and Imaging Center, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States S Supporting Information *
ABSTRACT: 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. Inspired by the design of molecular beacons16 and our previous work using DNA hairpins to mask fluorescent reporter functionality or aptamer activity,17−19 we reasoned that Watson−Crick base pairing could provide a simple and generalizable mechanism for noncovalently constraining peptides into quasi-cyclic conformations. Thus, embedding a CPP within the loop of a hairpin-forming nucleic acid oligomer should effectively cyclize the CPP, while an overhanging singlestranded region can be used to pick up and deliver molecular cargo functionalized with a complementary nucleic acid. To the best of our knowledge, this study is the first to report how to manipulate peptide cyclization as well as assembly through Watson−Crick base pairing for enhancing biomolecular delivery into cells. The design of our hairpin-forming oligomers is shown in Figure 1 and consists of the cell-penetrating TAT peptide inserted between two complementary peptide nucleic acid (PNA) domains. The chemical compatibility of PNA and
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he efficient delivery of bioactive molecules into cells is a major challenge in biomedical research.1 Due to their inherent ability to cross biological membranes, cell-penetrating peptides (CPPs) offer one of the most frequently used means for efficient, intracellular delivery of various cargoes including nucleic acids2−4 or proteins.5−8 The first reported CPP, derived from the transactivator of transcription (TAT) of human immunodeficiency, is a linear, arginine-rich peptide.9,10 Interestingly, the transduction efficiency of the TAT peptide can be further enhanced by covalent cyclization, 11,12 presumably due to the reduced conformational freedom. Although cyclized peptides have been proven more efficient than their linear analogues for the delivery of biomolecules into cells,18,21−23 they are not widely used because they are often difficult to prepare using traditional synthetic methods.24 Current covalent cyclization methods require extra purification steps, and generating the final cyclized products, especially those rich in basic and polar residues, such as TAT, can exhibit low yields (
