Multifunctional Enzyme Packaging and Catalysis in the Qß Protein Nanoparticle.

The simultaneous expression in Escherichia coli cells of the Qß virus-like particle (VLP) capsid protein and protein "cargo" tagged with a positively charged Rev peptide sequence leads to the spontaneous self-assembly of VLPs with multiple copies of the cargo inside. We report the packaging of four new enzymes with potential applications in medicine and chemical manufacturing. The captured enzymes are active while inside the nanoparticle shell and are protected from environmental conditions that lead to free-enzyme destruction. We also describe genetic modifications to the packaging scheme that shed light on the self-assembly mechanism of this system and allow indirect control over the internal packaging density of cargo. The technology was extended to create, via self-assembly, VLPs that simultaneously display protein ligands on the exterior and contain enzymes within. Inverse relationships were observed between the size of both the packaged and externally displayed protein or domains and nanoparticle yield. These results provide a general method for the rapid creation of robust protein nanoparticles with desired catalytic and targeting functionalities.

Therapeutic peptides: Historical perspectives, current development trends, and future directions.

Peptide therapeutics have played a notable role in medical practice since the advent of insulin therapy in the 1920s. Over 60 peptide drugs are approved in the United States and other major markets, and peptides continue to enter clinical development at a steady pace. Peptide drug discovery has diversified beyond its traditional focus on endogenous human peptides to include a broader range of structures identified from other natural sources or through medicinal chemistry efforts. We maintain a comprehensive dataset on peptides that have entered human clinical studies that includes over 150 peptides in active development today. Here we provide an overview of the peptide therapeutic landscape, including historical perspectives, molecular characteristics, regulatory benchmarks, and a therapeutic area breakdown.

Using biomimetic polymers in place of noncollagenous proteins to achieve functional remineralization of dentin tissues.

In calcified tissues such as bones and teeth, mineralization is regulated by an extracellular matrix, which includes non-collagenous proteins (NCP). This natural process has been adapted or mimicked to restore tissues following physical damage or demineralization by using polyanionic acids in place of NCPs, but the remineralized tissues fail to fully recover their mechanical properties. Here we show that pre-treatment with certain amphiphilic peptoids, a class of peptide-like polymers consisting of N-substituted glycines that have defined monomer sequences, enhances ordering and mineralization of collagen and induces functional remineralization of dentin lesions in vitro. In the vicinity of dentin tubules, the newly formed apatite nano-crystals are co-aligned with the c-axis parallel to the tubular periphery and recovery of tissue ultrastructure is accompanied by development of high mechanical strength. The observed effects are highly sequence-dependent with alternating polar and non-polar groups leading to positive outcomes while diblock sequences have no effect. The observations suggest aromatic groups interact with the collagen while the hydrophilic side chains bind the mineralizing constituents and highlight the potential of synthetic sequence-defined biomimetic polymers to serve as NCP mimics in tissue remineralization.

Direct observation of mineral-organic composite formation reveals occlusion mechanism.

Manipulation of inorganic materials with organic macromolecules enables organisms to create biominerals such as bones and seashells, where occlusion of biomacromolecules within individual crystals generates superior mechanical properties. Current understanding of this process largely comes from studying the entrapment of micron-size particles in cooling melts. Here, by investigating micelle incorporation in calcite with atomic force microscopy and micromechanical simulations, we show that different mechanisms govern nanoscale occlusion. By simultaneously visualizing the micelles and propagating step edges, we demonstrate that the micelles experience significant compression during occlusion, which is accompanied by cavity formation. This generates local lattice strain, leading to enhanced mechanical properties. These results give new insight into the formation of occlusions in natural and synthetic crystals, and will facilitate the synthesis of multifunctional nanocomposite crystals.

Encapsidated atom-transfer radical polymerization in Qß virus-like nanoparticles.

Virus-like particles (VLPs) are unique macromolecular structures that hold great promise in biomedical and biomaterial applications. The interior of the 30 nm-diameter Qß VLP was functionalized by a three-step process: (1) hydrolytic removal of endogenously packaged RNA, (2) covalent attachment of initiator molecules to unnatural amino acid residues located on the interior capsid surface, and (3) atom-transfer radical polymerization of tertiary amine-bearing methacrylate monomers. The resulting polymer-containing particles were moderately expanded in size; however, biotin-derivatized polymer strands were only very weakly accessible to avidin, suggesting that most of the polymer was confined within the protein shell. The polymer-containing particles were also found to exhibit physical and chemical properties characteristic of positively charged nanostructures, including the ability to easily enter mammalian cells and deliver functional small interfering RNA.

Guiding plant virus particles to integrin-displaying cells.

Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.

Evolution and protein packaging of small-molecule RNA aptamers.

A high-affinity RNA aptamer (K(d) = 50 nM) was efficiently identified by SELEX against a heteroaryldihydropyrimidine structure, chosen as a representative drug-like molecule with no cross reactivity with mammalian or bacterial cells. This aptamer, its weaker-binding variants, and a known aptamer against theophylline were each embedded in a longer RNA sequence that was encapsidated inside a virus-like particle by a convenient expression technique. These nucleoprotein particles were shown by backscattering interferometry to bind to the small-molecule ligands with affinities similar to those of the free (nonencapsidated) aptamers. The system therefore comprises a general approach to the production and sequestration of functional RNA molecules, characterized by a convenient label-free analytical technique.