Fabrication of Multicomponent Multivesicular Peptidoliposomes and Their Directed Cytoplasmic Delivery

A novel self-assembly strategy for the formation of multicomponent and multicompartment vesicles via the hierarchical assembly of the cyclic peptide and lipid building blocks is reported. The primary driving force underlying the formation of dual-component (i.e., peptide and lipid) heteromultivesicular vesicles (hMVVs) is the differential thermostability between the supramolecular building blocks. Furthermore, the combination of the differential thermostability and charge-based separation further enables the fabrication of the hMVVs that incorporate up to four different components (i.e., two different building blocks and two different encapsulated molecules). The quadruple-component hMVVs consist of cyclic peptides, lipids, negatively charged green fluorescent probes (GFPr), and positively charged red fluorescent probes (RFPr). Intracellular delivery study shows that cellular localization of hMVVs is directed by the function of hMVV envelopes, and the nuclear localization signal (NLS) of peptide vesicles appears to use different cellular pathways depending on the site of action (i.e., extracellular space or cytoplasm). This study provides the hierarchical peptide-based hMVVs with sophisticated architectures and cell delivery characteristics, thus making a step toward artificial cells or viruses

Macrocyclic Peptides Self-Assemble into Robust Vesicles with Molecular Recognition Capabilities

In this study, we developed macrocyclic peptide building blocks that formed self-assembled peptide vesicles with molecular recognition capabilities. Macrocyclic peptides were significantly different from conventional amphiphiles, in that they could self-assemble into vesicles at very high hydrophilic-to-total mass ratios. The flexibility of the hydrophobic self-assembly segment was critical for vesicle formation. The unique features of this peptide vesicle system include a homogeneous size distribution, unusually small size, and robust structural and thermal stability. The peptide vesicles successfully entrapped a hydrophilic model drug, released the payload very slowly, and were internalized by cells in a highly efficient manner. Moreover, the peptide vesicles exhibited molecular recognition capabilities, in that they selectively bound to target RNA through surface-displayed peptides. This study demonstrates that self-assembled peptide vesicles can be used as strong intracellular delivery vehicles that recognize specific biomacromolecular targets

Helix Stabilized, Thermostable, and Protease-Resistant Self-Assembled Peptide Nanostructures as Potential Inhibitors of Protein–Protein Interactions

Self-assembled peptide nanostructures with actively folded secondary structures have potential to mimic the function of proteins. We here show that α-helix-stabilized self-assembled peptide nanostructures (αSSPNs), whose sizes are comparable to those of proteins, have potential to be developed as protein–protein interaction (PPI) inhibitors along with several unprecedented properties. Using p53-MDM2 PPI as a model system, the molecular recognition and modulation of PPIs by αSSPN grafted with a p53 α-helix (p53 αSSPN) were investigated. The competition assay showed that the p53 αSSPN can inhibit the p53-MDM2 interaction. Interestingly, the p53 αSSPN was far more resistant to degradation by the protease chymotrypsin than the monomeric p53 peptide and had high thermal stability. These results suggest that the αSSPN scaffold holds great potential to be developed as a novel class of PPI inhibitors

Bioinspired Self-Assembled Peptide Nanofibers with Thermostable Multivalent α‑Helices

The stabilization of peptide’s active conformation is a critical determinant of its target binding efficiency. Here we present a structure-based self-assembly strategy for the design of nanostructures with multiple and thermostable α-helices using bioinspired peptide amphiphiles. The design principle was inspired by the oligomerization of the human immunodeficiency virus type-1 (HIV-1) Rev protein. Our goal was to find a strategy to modify the Rev protein into a chemically manageable self-assembling peptide while stabilizing its α-helical structure. Instead of using cyclic peptides for structure stabilization, this strategy utilizes the pseudocyclization for helix stabilization. The self-assembly induced stabilization of α-helical conformation could be observed, and the α-helices were found to be stable even at high temperature (at least up to 74 °C). Conjugation of a hydrophobic alkyl chain to the Rev peptide was crucial for forming the self-assembled nanostructures, and no nanostructures could be obtained without this modification. Because chemical modifications to the α-helical peptide domain can be avoided, potentially any α-helical peptide fragment can be grafted into this self-assembling peptide scaffold

Cyclic Peptide-Decorated Self-Assembled Nanohybrids for Selective Recognition and Detection of Multivalent RNAs

Although there has been substantial advancement in the development of nanostructures, the development of self-assembled nanostructures that can selectively recognize multivalent targets has been very difficult. Here we show the proof of concept that topology-controlled peptide nanoassemblies can selectively recognize and detect a multivalent RNA target. We compared the differential behaviors of peptides in a linear or cyclic topology in terms of peptide–gold nanoparticle hybrid nanostructure formation, conformational stabilization, monovalent and multivalent RNA binding in vitro, and multivalent RNA recognition in live cells. When the topology-dependent selectivity amplification of the cyclic peptide hybrids is combined with the noninvasive nature of dark-field microscopy, the cellular localization of the viral Rev response element (RRE) RNA can be monitored in situ. Because intracellular interactions are often mediated by overlapping binding partners with weak affinity, the topology-controlled peptide assemblies can provide a versatile means to convert weak ligands into multivalent ligands with high affinity and selectivity

Chameleon-like Self-Assembling Peptides for Adaptable Biorecognition Nanohybrids

We present here the development of adaptable hybrid materials in which self-assembling peptides can sense the diameter/curvature of carbon nanotubes and then adjust their overall structures from disordered states to α-helices, and <i>vice versa</i>. The peptides within the hybrid materials show exceptionally high thermal-induced conformational stability and molecular recognition capability for target RNA. This study shows that the context-dependent protein-folding effects can be realized in artificial nanosystems and provides a proof of principle that nanohybrid materials decorated with structured and adjustable peptide units can be fabricated using our strategy, from which smart and responsive organic/inorganic hybrid materials capable of sensing and controlling diverse biological molecular recognition events can be developed

Macromolecular Sensing of RNAs by Exploiting Conformational Changes in Supramolecular Nanostructures

Here, we report on a ratiometric fluorescence biosensor based on self-assembled peptide nanostructures (SPN), which can respond to conformational changes induced by RNA ligand binding. The design of the SPN biosensor was inspired by the conformational stabilization and multimerization behaviors of the HIV-1 Rev protein induced by cooperative protein–protein and protein–RNA interactions. Because conformation-sensitive SPN biosensors can orchestrate binding and signal transduction events, they can be developed as highly sophisticated and smart nanomaterials for biosensing

Tuning Oligovalent Biomacromolecular Interfaces Using Double-Layered α‑Helical Coiled-Coil Nanoassemblies from Lariat-Type Building Blocks

The target affinity and selectivity of many biomacromolecules depend on the three-dimensional (3D) distribution of multiple ligands on their surfaces. Here, we devised a self-assembly strategy to control the target-tailored 3D distribution of multiple α-helical ligands on a coiled-coil core scaffold using novel lariat-type supramolecular building blocks. Depending on the coiled-coil composition and ligand grafting sites in the lariat building blocks, the structural and functional features of the self-assembled peptide nanostructures (SPNs) could be variably fine-tuned. Using oligovalent protein–RNA (Rev-RRE) interactions as a model system, we demonstrate that longer grafting reinforces the helicity of the peptide ligands, whereas shorter grafting strengthens the target binding affinity of the SPNs in both monovalent and oligovalent interactions. This supramolecular approach should be useful in developing precisely controllable multivalent ligands for biomacromolecular interactions

Multiplexing Natural Orientation: Oppositely Directed Self-Assembling Peptides

We explore here the possibility that polypeptide chains with directional multiplicity might provide for the control of peptide self-assembly processes. We tested this new possibility using an oppositely directed peptide (ODP) supramolecular system. The ODP could make it possible to form a βαβ motif with antiparallel β-sheets, which does not exist in nature. Furthermore, the designed ODPs were able to self-assemble into discrete, homogeneous, and structured protein-like controlled nano-objects. ODPs represent a simple but powerful unnatural self-assembling peptide system that can become a basic scaffold for fabricating more complex and elaborate artificial nanostructures

Nanomorphological Diversity of Self-Assembled Cyclopeptisomes Investigated via Thermodynamic and Kinetic Controls

The physicochemical and biological characteristics of vesicles are dependent on the type of self-assembly building blocks and methods of preparation. In this report, we designed a vesicle-forming linear and cyclic peptide building blocks and investigated the effect of molecular topology and thermodynamic and kinetic controls on the stability and morphological features of the self-assembled vesicles. Comparison of topological effect on self-assembly revealed that the strong association of the aromatic hydrophobic segments is observed only in the cyclic peptide, which is most likely the results of constrained structure along with the restriction in the molecular degree of freedom. Consequently, the formation of stable vesicles could be observed only with the cyclic peptide. Further investigation with cyclic peptide building blocks revealed that depending on the control methods, vesicles with a variety of structural features, such as polygonal, wrinkled, round, round-patched, and round-fused vesicles, could be fabricated. Our results demonstrate that existing vesicle structures constitute only a fraction of the possible structural diversity and that macrocyclic peptides can provide a wealth of opportunities in vesicle engineering