DNA-templated self-assembly of bradykinin into bioactive nanofibrils.

Bradykinin (BK) is a peptide hormone that plays a crucial role in blood pressure control, regulates inflammation in the human body, and has recently been implicated in the pathophysiology of COVID-19. In this study, we report a strategy for fabricating highly ordered 1D nanostructures of BK using DNA fragments as a template for self-assembly. We have combined synchrotron small-angle X-ray scattering and high-resolution microscopy to provide insights into the nanoscale structure of BK-DNA complexes, unveiling the formation of ordered nanofibrils. Fluorescence assays hint that BK is more efficient at displacing minor-groove binders in comparison with base-intercalant dyes, thus, suggesting that interaction with DNA strands is mediated by electrostatic attraction between cationic groups at BK and the high negative electron density of minor-grooves. Our data also revealed an intriguing finding that BK-DNA complexes can induce a limited uptake of nucleotides by HEK-293t cells, which is a feature that has not been previously reported for BK. Moreover, we observed that the complexes retained the native bioactivity of BK, including the ability to modulate Ca2+ response into endothelial HUVEC cells. Overall, the findings presented here demonstrate a promising strategy for the fabrication of fibrillar structures of BK using DNA as a template, which keep bioactivity features of the native peptide and may have implications in the development of nanotherapeutics for hypertension and related disorders.

Amyloid-like Self-Assembly of a Hydrophobic Cell-Penetrating Peptide and Its Use as a Carrier for Nucleic Acids.

Cell-penetrating peptides (CPPs) are a topical subject potentially exploitable for creating nanotherapeutics for the delivery of bioactive loads. These compounds are often classified into three major categories according to their physicochemical characteristics: cationic, amphiphilic, and hydrophobic. Among them, the group of hydrophobic CPPs has received increasing attention in recent years due to toxicity concerns posed by highly cationic CPPs. The hexapeptide PFVYLI (P, proline; F, phenylalanine; V, valine; Y, tyrosine; L, leucine; and I, isoleucine), a fragment derived from the C-terminal portion of α1-antitrypsin, is a prototypal example of hydrophobic CPP. This sequence shows reduced cytotoxicity and a capacity of nuclear localization, and its small size readily hints at its suitability as a building block to construct nanostructured materials. In this study, we examine the self-assembling properties of PFVYLI and investigate its ability to form noncovalent complexes with nucleic acids. By using a combination of biophysical tools including synchrotron small-angle X-ray scattering and atomic force microscopy-based infrared spectroscopy, we discovered that this CPP self-assembles into discrete nanofibrils with remarkable amyloidogenic features. Over the course of days, these fibrils coalesce into rodlike crystals that easily reach the micrometer range. Despite lacking cationic residues in the composition, PFVYLI forms noncovalent complexes with nucleic acids that retain ß-sheet pairing found in amyloid aggregates. In vitro vectorization experiments performed with double-stranded DNA fragments indicate that complexes promote the internalization of nucleic acids, revealing that tropism toward cell membranes is preserved upon complexation. On the other hand, transfection assays with splice-correction oligonucleotides (SCOs) for luciferase expression show limited bioactivity across a narrow concentration window, suggesting that the propensity to form amyloidogenic aggregates may trigger endosomal entrapment. We anticipate that the findings presented here open perspectives for using this archetypical hydrophobic CPP in the fabrication of nanostructured scaffolds, which potentially integrate properties of amyloids and translocation capabilities of CPPs.

Self-assembly and intracellular delivery of DNA by a truncated fragment derived from the Trojan peptide Penetratin.

Penetratin is a short Trojan peptide that attracts great interest in biomedical research for its capacity to translocate biological membranes. Herein, we study in detail both self-assembly and intracellular delivery of DNA by the heptamer KIWFQNR, a truncated peptide derived from Penetratin. This shortened sequence possesses a unique design with bolaamphiphilic characteristics that preserves the longest noncationic amino acid portion found in Penetratin. These features convey amphipathicity to assist self-assembly and make it a suitable model for exploring the role of hydrophobic residues for peptide interaction and cell uptake. We show that the fragment forms peptiplexes (i.e., peptide-DNA complexes), and aggregates into long nanofibers with clear ß-sheet signature. The supramolecular structure of nanofibers is likely composed of DNA cores surrounded by a peptide shell to which the double helix behaves as a template and induces fibrillization. A nucleation and growth mechanism proceeding through liquid-liquid phase separation of coacervates is proposed for describing the self-assembly of peptiplexes. We also demonstrate that peptiplexes deliver double-stranded 200 bp DNA into HeLa cells, indicating its potential for preparing non-viral vectors for oligonucleotides through noncovalent strategies. Since the main structural features of native Penetratin are conserved in this simpler fragment, our findings also highlight the role of uncharged amino acids for structuration, and thus for the ability of Penetratin to cross cell membranes.