Supercharged coiled-coil protein with N-terminal decahistidine tag boosts siRNA complexation and delivery efficiency of a lipoproteoplex.

Short interfering RNA (siRNA) therapeutics have soared in popularity due to their highly selective and potent targeting of faulty genes, providing a non-palliative approach to address diseases. Despite their potential, effective transfection of siRNA into cells requires the assistance of an accompanying vector. Vectors constructed from non-viral materials, while offering safer and non-cytotoxic profiles, often grapple with lackluster loading and delivery efficiencies, necessitating substantial milligram quantities of expensive siRNA to confer the desired downstream effects. We detail the recombinant synthesis of a diverse series of coiled-coil supercharged protein (CSP) biomaterials systematically designed to investigate the impact of two arginine point mutations (Q39R and N61R) and decahistidine tags on liposomal siRNA delivery. The most efficacious variant, N8, exhibits a twofold increase in its affinity to siRNA and achieves a twofold enhancement in transfection activity with minimal cytotoxicity in vitro. Subsequent analysis unveils the destabilizing effect of the Q39R and N61R supercharging mutations and the incorporation of C-terminal decahistidine tags on α-helical secondary structure. Cross-correlational regression analyses reveal that the amount of helical character in these mutants is key in N8's enhanced siRNA complexation and downstream delivery efficiency.

Fluorescent azobenzene-confined coiled-coil mesofibers.

Fluorescent protein biomaterials have important applications such as bioimaging in pharmacological studies. Self-assembly of proteins, especially into fibrils, is known to produce fluorescence in the blue band. Capable of self-assembly into nanofibers, we have shown we can modulate its aggregation into mesofibers by encapsulation of a small hydrophobic molecule. Conversely, azobenzenes are hydrophobic small molecules that are virtually non-fluorescent in solution due to their highly efficient photoisomerization. However, they demonstrate fluorogenic properties upon confinement in nanoscale assemblies by reducing the non-radiative photoisomerization. Here, we report the fluorescence of a hybrid protein-small molecule system in which azobenzene is confined in our protein assembly leading to fiber thickening and increased fluorescence. We show our engineered protein Q encapsulates AzoCholine, bearing a photoswitchable azobenzene moiety, in the hydrophobic pore to produce fluorescent mesofibers. This study further investigates the photocontrol of protein conformation as well as fluorescence of an azobenze-containing biomaterial.

Engineered multivalent self-assembled binder protein against SARS-CoV-2 RBD.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic since December 2019, and with it, a push for innovations in rapid testing and neutralizing antibody treatments in an effort to solve the spread and fatality of the disease. One such solution to both of these prevailing issues is targeting the interaction of SARS-CoV-2 spike receptor binding domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2) receptor protein. Structural studies have shown that the N-terminal alpha-helix comprised of the first 23 residues of ACE2 plays an important role in this interaction. Where it is typical to design a binding domain to fit a target, we have engineered a protein that relies on multivalency rather than the sensitivity of a monomeric ligand to provide avidity to its target by fusing the N-terminal helix of ACE2 to the coiled-coil domain of the cartilage oligomeric matrix protein. The resulting ACE-MAP is able to bind to the SARS-CoV-2 RBD with improved binding affinity, is expressible in E. coli, and is thermally stable and relatively small (62 kDa). These properties suggest ACE-MAP and the MAP scaffold to be a promising route towards developing future diagnostics and therapeutics to SARS-CoV-2.

Engineered Protein-Iron Oxide Hybrid Biomaterial for MRI-traceable Drug Encapsulation.

Labeled protein-based biomaterials have become a popular for various biomedical applications such as tissue-engineered, therapeutic, or diagnostic scaffolds. Labeling of protein biomaterials, including with ultrasmall super-paramagnetic iron oxide (USPIO) nanoparticles, has enabled a wide variety of imaging techniques. These USPIO-based biomaterials are widely studied in magnetic resonance imaging (MRI), thermotherapy, and magnetically-driven drug delivery which provide a method for direct and non-invasive monitoring of implants or drug delivery agents. Where most developments have been made using polymers or collagen hydrogels, shown here is the use of a rationally designed protein as the building block for a meso-scale fiber. While USPIOs have been chemically conjugated to antibodies, glycoproteins, and tissue-engineered scaffolds for targeting or improved biocompatibility and stability, these constructs have predominantly served as diagnostic agents and often involve harsh conditions for USPIO synthesis. Here, we present an engineered protein-iron oxide hybrid material comprised of an azide-functionalized coiled-coil protein with small molecule binding capacity conjugated via bioorthogonal azide-alkyne cycloaddition to an alkyne-bearing iron oxide templating peptide, CMms6, for USPIO biomineralization under mild conditions. The coiled-coil protein, dubbed Q, has been previously shown to form nanofibers and, upon small molecule binding, further assembles into mesofibers via encapsulation and aggregation. The resulting hybrid material is capable of doxorubicin encapsulation as well as sensitive T2*-weighted MRI darkening for strong imaging capability that is uniquely derived from a coiled-coil protein.

Rediscovering Chemical Gardens: Self-Assembling Cytocompatible Protein-Intercalated Silicate-Phosphate Sponge-Mimetic Tubules.

The classic chemical garden experiment is reconstructed to produce protein-intercalated silicate-phosphate tubules that resemble tubular sponges. The constructs were synthesized by seeding calcium chloride into a solution of sodium silicate-potassium phosphate and gelatin. Sponge-mimetic tubules were fabricated with varying percentages of gelatin (0-15% w/v), in diameters ranging from 200 µm to 2 mm, characterized morphologically and compositionally, functionalized with biomolecules for cell adhesion, and evaluated for cytocompatibility. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy analysis (EDS) experiments showed that the external surface of the tubules was relatively more amorphous in texture and carbon/protein-rich in comparison to the interior surface. Transmission electron microscopy (TEM) images indicate a network composed of gelatin incorporated into the inorganic scaffold. The presence of gelatin in the constructs was confirmed by infrared spectroscopy. Powder X-ray diffraction (XRD) was used to identify inorganic crystalline phases in the scaffolds that are mainly composed of Ca(OH)2, NaCl, and Ca2SiO4 along with a band corresponding to amorphous gelatin. Bioconjugation and coating protocols were developed to program the scaffolds with cues for cell adhesion, and the resulting constructs were employed for 3D cell culture of marine (Pyrocystis lunula) and mammalian (HeLa and H9C2) cell lines. The cytocompatibility of the constructs was demonstrated by live cell assays. We have successfully shown that these biomimetic materials can indeed support life; they serve as scaffolds that facilitate the attachment and assembly of individual cells to form multicellular entities, thereby revisiting the 350-year-old effort to link chemical gardens with the origins of life. Hybrid chemical garden biomaterials are programmable, readily fabricated and could be employed in tissue engineering, biomolecular materials development, 3D mammalian cell culture and by researchers investigating the origins of multicellular life.