Engineered Ribonucleoprotein Granules Inhibit Translation in Protocells.

Liquid granules rich in intrinsically disordered proteins and RNA play key roles in critical cellular functions such as RNA processing and translation. Many details of the mechanism via which this occurs remain to be elucidated. Motivated by the lacuna in the field and by the prospects of developing de novo artificial granules that provide extrinsic control of translation, we report a bottom-up approach to engineer ribonucleoprotein granules composed of a recombinant RNA-binding IDP that exhibits phase behavior in water. We developed a kinetic model to illustrate that these granules inhibit translation through reversible or irreversible sequestration of mRNA. Within monodisperse droplets capable of transcription and translation, we experimentally demonstrate temporal inhibition of translation by using designer IDPs that exhibit tunable phase behavior. This work lays the foundation for developing artificial granules that promise to further our mechanistic understanding of their naturally occurring counterparts.

Negative lipid membranes enhance the adsorption of TAT-decorated elastin-like polypeptide micelles.

A cell-penetrating peptide (CPP) is a short amino-acid sequence capable of efficiently translocating across the cellular membrane of mammalian cells. However, the potential of CPPs as a delivery vector is hampered by the strong reduction of its translocation efficiency when it bears an attached molecular cargo. To overcome this problem, we used previously developed diblock copolymers of elastin-like polypeptides (ELPBCs), which we end functionalized with TAT (transactivator of transcription), an archetypal CPP built from a positively charged amino acid sequence of the HIV-1 virus. These ELPBCs self-assemble into micelles at a specific temperature and present the TAT peptide on their corona. These micelles can recover the lost membrane affinity of TAT and can trigger interactions with the membrane despite the presence of a molecular cargo. Herein, we study the influence of membrane surface charge on the adsorption of TAT-functionalized ELP micelles onto giant unilamellar vesicles (GUVs). We show that the TAT-ELPBC micelles show an increased binding constant toward negatively charged membranes compared to neutral membranes, but no translocation is observed. The affinity of the TAT-ELPBC micelles for the GUVs displays a stepwise dependence on the lipid charge of the GUV, which, to our knowledge, has not been reported previously for interactions between peptides and lipid membranes. By unveiling the key steps controlling the interaction of an archetypal CPP with lipid membranes, through regulation of the charge of the lipid bilayer, our results pave the way for a better design of delivery vectors based on CPPs.

Elastin-Like Polypeptides for Biomedical Applications.

Elastin-like polypeptides (ELPs) are stimulus-responsive biopolymers derived from human elastin. Their unique properties-including lower critical solution temperature phase behavior and minimal immunogenicity-make them attractive materials for a variety of biomedical applications. ELPs also benefit from recombinant synthesis and genetically encoded design; these enable control over the molecular weight and precise incorporation of peptides and pharmacological agents into the sequence. Because their size and sequence are defined, ELPs benefit from exquisite control over their structure and function, qualities that cannot be matched by synthetic polymers. As such, ELPs have been engineered to assemble into unique architectures and display bioactive agents for a variety of applications. This review discusses the design and representative biomedical applications of ELPs, focusing primarily on their use in tissue engineering and drug delivery.

Nanoscopic Dynamics Dictate the Phase Separation Behavior of Intrinsically Disordered Proteins.

Many intrinsically disordered proteins (IDPs) in nature may undergo liquid-liquid phase separation to assemble membraneless organelles with varied liquid-like properties and stability/dynamics. While solubility changes underlie these properties, little is known about hydration dynamics in phase-separating IDPs. Here, by studying IDP polymers of similar composition but distinct liquid-like dynamics and stability upon separation, namely, thermal hysteresis, we probe at a nanoscopic level hydration/dehydration dynamics in IDPs as they reversibly switch between phase separation states. Using continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy, we observe distinct backbone and amino acid side-chain hydration dynamics in these IDPs. This nanoscopic view reveals that side-chain rehydration creates a dynamic water shield around the main-chain backbone that effectively and counterintuitively prevents water penetration and governs IDP solubility. We find that the strength of this superficial water shell is a sequence feature of IDPs that encodes for the stability of their phase-separated assemblies. Our findings expose and offer an initial understanding of how the complexity of nanoscopic water-IDP interactions dictate their rich phase separation behavior.

Ultrabright Fluorescence Readout of an Inkjet-Printed Immunoassay Using Plasmonic Nanogap Cavities.

Fluorescence-based microarrays are promising diagnostic tools due to their high throughput, small sample volume requirements, and multiplexing capabilities. However, their low fluorescence output has limited their implementation for in vitro diagnostics applications in point-of-care (POC) settings. Here, by integration of a sandwich immunoassay microarray within a plasmonic nanogap cavity, we demonstrate strongly enhanced fluorescence which is critical for readout by inexpensive POC detectors. The immunoassay consists of inkjet-printed antibodies on a polymer brush which is grown on a gold film. Colloidally synthesized silver nanocubes are placed on top and interact with the underlying gold film creating high local electromagnetic field enhancements. By varying the thickness of the brush from 5 to 20 nm, up to a 151-fold increase in fluorescence and 14-fold improvement in the limit-of-detection is observed for the cardiac biomarker B-type natriuretic peptide (BNP) compared to the unenhanced assay, paving the way for a new generation of POC clinical diagnostics.

Inkjet-printed point-of-care immunoassay on a nanoscale polymer brush enables subpicomolar detection of analytes in blood.

The ELISA is the mainstay for sensitive and quantitative detection of protein analytes. Despite its utility, ELISA is time-consuming, resource-intensive, and infrastructure-dependent, limiting its availability in resource-limited regions. Here, we describe a self-contained immunoassay platform (the "D4 assay") that converts the sandwich immunoassay into a point-of-care test (POCT). The D4 assay is fabricated by inkjet printing assay reagents as microarrays on nanoscale polymer brushes on glass chips, so that all reagents are "on-chip," and these chips show durable storage stability without cold storage. The D4 assay can interrogate multiple analytes from a drop of blood, is compatible with a smartphone detector, and displays analytical figures of merit that are comparable to standard laboratory-based ELISA in whole blood. These attributes of the D4 POCT have the potential to democratize access to high-performance immunoassays in resource-limited settings without sacrificing their performance.

Convergence of Artificial Protein Polymers and Intrinsically Disordered Proteins.

A flurry of research in recent years has revealed the molecular origins of many membraneless organelles to be the liquid phase separation of intrinsically disordered proteins (IDPs). Consequently, protein disorder has emerged as an important driver of intracellular compartmentalization by providing specialized microenvironments chemically distinct from the surrounding medium. Though the importance of protein disorder and its relationship to intracellular phase behavior are clear, a detailed understanding of how such phase behavior can be predicted and controlled remains elusive. While research in IDPs has largely focused on the implications of structural disorder on cellular function and disease, another field, that of artificial protein polymers, has focused on the de novo design of protein polymers with controllable material properties. A subset of these polymers, specifically those derived from structural proteins such as elastin and resilin, are also disordered sequences that undergo liquid-liquid phase separation. This phase separation has been used in a variety of biomedical applications, and researchers studying these polymers have developed methods to precisely characterize and tune their phase behavior. Despite their disparate origins, both fields are complementary as they study the phase behavior of intrinsically disordered polypeptides. This Perspective hopes to stimulate collaborative efforts by highlighting the similarities between these two fields and by providing examples of how such collaboration could be mutually beneficial.

The Weak Link: Optimization of the Ligand-Nanoparticle Interface To Enhance Cancer Cell Targeting by Polymer Micelles.

Many promising targeting ligands are hydrophobic peptides, and these ligands often show limited accessibility to receptors, resulting in suboptimal targeting. A systematic study to elucidate the rules for the design of linkers that optimize their presentation on nanoparticles has not been carried out to date. In this study, we recombinantly synthesized an elastin-like polypeptide diblock copolymer (ELPBC) that self-assembles into monodisperse micelles. AHNP and EC1, two hydrophobic ErbB2-targeted peptide ligands, were incorporated at the C-terminus of the ELPBC with an intervening peptide linker. We tested more than 20 designs of peptide linkers, where the linker could be precisely engineered at the gene level to systematically investigate the molecular parameters-sequence, length, and charge-of the peptide linker that optimally assist ligands in targeting the ErbB2 receptor on cancer cells. We found that peptide linkers with a minimal length of 12 hydrophilic amino acids and an overall cationic charge-and that impart a zeta potential of the micelle that is close to neutral-were necessary to enhance the uptake of peptide-modified ELPBC micelles by cancer cells that overexpress the ErbB2 receptor. This work advances our understanding of the optimal presentation of hydrophobic ligands by nanoparticles and suggests design rules for peptide linkers for targeted delivery by polymer micelles, an emerging class of nanoparticle carriers for drugs and imaging agents.

Site-Specific and Stoichiometric Stealth Polymer Conjugates of Therapeutic Peptides and Proteins.

As potent and selective therapeutic agents, peptides and proteins are an important class of drugs, but they typically have suboptimal pharmacokinetic profiles. One approach to solve this problem is their conjugation with "stealth" polymers. Conventional methods for conjugation of this class of polymers to peptides and proteins are typically carried out by reactions that have poor yield and provide limited control over the site of conjugation and the stoichiometry of the conjugate. To address these limitations, new chemical and biological approaches have been developed that provide new molecular tools in the bioconjugation toolbox to create stealth polymer conjugates of peptides and proteins with exquisite control over their properties. This review article highlights these recent advances in the synthesis of therapeutic peptide- and protein-stealth polymer conjugates.

Phase Behavior and Self-Assembly of Perfectly Sequence-Defined and Monodisperse Multiblock Copolypeptides.

This paper investigates how the properties of multiblock copolypeptides can be tuned by their block architecture, defined by the size and distribution of blocks along the polymer chain. These parameters were explored by the precise, genetically encoded synthesis of recombinant elastin-like polypeptides (ELPs). A family of ELPs was synthesized in which the composition and length were conserved while the block length and distribution were varied, thus creating 11 ELPs with unique block architectures. To our knowledge, these polymers are unprecedented in their intricately and precisely varied architectures. ELPs exhibit lower critical solution temperature (LCST) behavior and micellar self-assembly, both of which impart easily measured physicochemical properties to the copolymers, providing insight into polymer hydrophobicity and self-assembly into higher order structures, as a function of solution temperature. Even subtle variation in block architecture changed the LCST phase behavior and morphology of these ELPs, measured by their temperature-triggered phase transition and nanoscale self-assembly. Size and morphology of polypeptide micelles could be tuned solely by controlling the block architecture, thus demonstrating that when sequence can be precisely controlled, nanoscale self-assembly of polypeptides can be modulated by block architecture.

Recombinant Synthesis of Hybrid Lipid-Peptide Polymer Fusions that Self-Assemble and Encapsulate Hydrophobic Drugs.

Inspired by biohybrid molecules that are synthesized in Nature through post-translational modification (PTM), we have exploited a eukaryotic PTM to recombinantly synthesize lipid-polypeptide hybrid materials. By co-expressing yeast N-myristoyltransferase with an elastin-like polypeptide (ELP) fused to a short recognition sequence in E. coli, we show robust and high-yield modification of the ELP with myristic acid. The ELP's reversible phase behavior is retained upon myristoylation and can be tuned to span a 30-60 °C. Myristoylated ELPs provide a versatile platform for genetically pre-programming self-assembly into micelles of varied size and shape. Their lipid cores can be loaded with hydrophobic small molecules by passive diffusion. Encapsulated doxorubicin and paclitaxel exhibit cytotoxic effects on 4T1 and PC3-luc cells, respectively, with potencies similar to chemically conjugated counterparts, and longer plasma circulation than free drug upon intravenous injection in mice.

A Modular Method for the High-Yield Synthesis of Site-Specific Protein-Polymer Therapeutics.

A versatile method is described to engineer precisely defined protein/peptide-polymer therapeutics by a modular approach that consists of three steps: 1) fusion of a protein/peptide of interest with an elastin-like polypeptide that enables facile purification and high yields; 2) installation of a clickable group at the C terminus of the recombinant protein/peptide with almost complete conversion by enzyme-mediated ligation; and 3) attachment of a polymer by a click reaction with near-quantitative conversion. We demonstrate that this modular approach is applicable to various protein/peptide drugs and used it to conjugate them to structurally diverse water-soluble polymers that prolong the plasma circulation duration of these proteins. The protein/peptide-polymer conjugates exhibited significantly improved pharmacokinetics and therapeutic effects over the native protein/peptide upon administration to mice. The studies reported here provide a facile method for the synthesis of protein/peptide-polymer conjugates for therapeutic use and other applications.

Site-Specific Zwitterionic Polymer Conjugates of a Protein Have Long Plasma Circulation.

Many proteins suffer from suboptimal pharmacokinetics (PK) that limit their utility as drugs. The efficient synthesis of polymer conjugates of protein drugs with tunable PK to optimize their in vivo efficacy is hence critical. We report here the first study of the in vivo behavior of a site-specific conjugate of a zwitterionic polymer and a protein. To synthesize the conjugate, we first installed an initiator for atom-transfer radical polymerization (ATRP) at the N terminus of myoglobin (Mb-N-Br). Subsequently, in situ ATRP was carried out in aqueous buffer to grow an amine-functionalized polymer from Mb-N-Br. The cationic polymer was further derivatized to two zwitterionic polymers by treating the amine groups of the cationic polymer with iodoacetic acid to obtain poly(carboxybetaine methacrylate) with a one-carbon spacer (PCBMA; C1 ), and sequentially with 3-iodopropionic acid and iodoacetic acid to obtain PCBMA(mix) with a mixture of C1 and C2 spacers. The Mb-N-PCBMA polymer conjugates had a longer in vivo plasma half-life than a PEG-like comb polymer conjugate of similar molecular weights (MW). The structure of the zwitterion plays a role in controlling the in vivo behavior of the conjugate, as the PCBMA conjugate with a C1 spacer had significantly longer plasma circulation than the conjugate with a mixture of C1 and C2 spacers.

Prediction of solvent-induced morphological changes of polyelectrolyte diblock copolymer micelles.

Self-assembly processes of polyelectrolyte block copolymers are ubiquitous in industrial and biological processes; understanding their physical properties can also provide insights into the design of polyelectrolyte materials with novel and tailored properties. Here, we report systematic analysis on how the ionic strength of the solvent and the length of the polyelectrolyte block affect the self-assembly and morphology of the polyelectrolyte block copolymer materials by constructing a salt-dependent morphological phase diagram using an implicit solvent ionic strength (ISIS) method for dissipative particle dynamics (DPD) simulations. This diagram permits the determination of the conditions for the morphological transition into a specific shape, namely vesicles or lamellar aggregates, wormlike/cylindrical micelles, and spherical micelles. The scaling behavior for the size of spherical micelles is predicted, in terms of radius of gyration (R(g,m)) and thickness of corona (Hcorona), as a function of solvent ionic strength (c(s)) and polyelectrolyte length (NA), which are R(g,m) ∼ c(s)(-0.06)N(A)(0.54) and Hcorona ∼ c(s)(-0.11)N(A)(0.75). The simulation results were corroborated through AFM and static light scattering measurements on the example of the self-assembly of monodisperse, single-stranded DNA block-copolynucleotides (polyT50-b-F-dUTP). Overall, we were able to predict the salt-responsive morphology of polyelectrolyte materials in aqueous solution and show that a spherical-cylindrical-lamellar change in morphology can be obtained through an increase in solvent ionic strength or a decrease of polyelectrolyte length.

"Smart" DNA interfaces.

This review focuses on surface-grafted DNA, and its use as a molecular building block that exploits its unique properties as a directional (poly)anion that exhibits molecular recognition. The selected examples highlight innovative applications of DNA at surfaces and interfaces ranging from molecular diagnostics and sequencing to biosensing.

Noncanonical self-assembly of highly asymmetric genetically encoded polypeptide amphiphiles into cylindrical micelles.

Elastin-like polypeptides (ELPs) are a class of biopolymers consisting of the pentameric repeat (VPGαG)n based on the sequence of mammalian tropoelastin that display a thermally induced soluble-to-insoluble phase transition in aqueous solution. We have discovered a remarkably simple approach to driving the spontaneous self-assembly of high molecular weight ELPs into nanostructures by genetically fusing a short 1.5 kDa (XGy)z assembly domain to one end of the ELP. Classical theories of self-assembly based on the geometric mass balance of hydrophilic and hydrophobic block copolymers suggest that these highly asymmetric polypeptides should form spherical micelles. Surprisingly, when sufficiently hydrophobic amino acids (X) are presented in a periodic sequence such as (FGG)8 or (YG)8, these highly asymmetric polypeptides self-assemble into cylindrical micelles whose length can be tuned by the sequence of the morphogenic tag. These nanostructures were characterized by light scattering, tunable resistive pulse sensing, fluorescence spectrophotometry, and thermal turbidimetry, as well as by cryogenic transmission electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS). These short assembly domains provide a facile strategy to control the size, shape, and stability of stimuli responsive polypeptide nanostructures.

Ring-opening polymerization of prodrugs: a versatile approach to prepare well-defined drug-loaded nanoparticles.

The synthesis of polymer-drug conjugates from prodrug monomers consisting of a cyclic polymerizable group that is appended to a drug through a cleavable linker is achieved by organocatalyzed ring-opening polymerization. The monomers polymerize into well-defined polymer prodrugs that are designed to self-assemble into nanoparticles and release the drug in response to a physiologically relevant stimulus. This method is compatible with structurally diverse drugs and allows different drugs to be copolymerized with quantitative conversion of the monomers. The drug loading can be controlled by adjusting the monomer(s)/initiator feed ratio and drug release can be encoded into the polymer by the choice of linker. Initiating these monomers from a poly(ethylene glycol) macroinitiator results in amphiphilic diblock copolymers that spontaneously self-assemble into micelles with a long plasma circulation, which is useful for systemic therapy.

Calcium binding peptide motifs from calmodulin confer divalent ion selectivity to elastin-like polypeptides.

Calcium-sensitive elastin-like polypeptides (CELPs) were synthesized by periodically interspersing a calcium-binding peptide sequence from calmodulin within an elastin-like polypeptide (ELP) with the goal of creating thermal and calcium responsive peptide polymers. The CELPs exhibit high sensitivity to calcium compared to monovalent cations but do not exhibit the exquisite selectivity for calcium over other divalent cations, such as magnesium, that is displayed by calmodulin. The CELPs were further used as a building block for the synthesis of calcium-sensitive nanoparticles by fusing a hydrophilic, noncalcium-sensitive ELP block with a CELP block that becomes more hydrophobic upon calcium binding. We show that addition of calcium at concentrations between 50 and 500 mM imparts sufficient amphiphilicity to the diblock polypeptide between 33 and 46 °C to trigger its self-assembly into monodisperse spherical micelles with a hydrodynamic radius of ∼50 nm.

Sortase-catalyzed initiator attachment enables high yield growth of a stealth polymer from the C terminus of a protein.

Conventional methods for synthesizing protein/peptide-polymer conjugates, as a means to improve the pharmacological properties of therapeutic biomolecules, typically have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of site- specificity, which results in heterogeneous products with significantly compromised bioactivity. To address these limitations, the use of sortase A from Staphylococcus aureus is demonstrated to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein (GFP), followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeds with high specificity and near-complete (≈95%) product conversion. Subsequent in situ ATRP in aqueous buffer produces 1:1 stoichiometric conjugates with >90% yield, low dispersity, and no denaturation of the protein. This approach introduces a simple and useful method for high yield synthesis of protein/peptide-polymer conjugates.

In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation.

This paper reports a general in situ method to grow a polymer conjugate solely from the C terminus of a recombinant protein. GFP was fused at its C terminus with an intein; cleavage of the intein provided a unique thioester moiety at the C terminus of GFP that was used to install an atom transfer radical polymerization (ATRP) initiator. Subsequent in situ ATRP of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) yielded a site-specific (C-terminal) and stoichiometric conjugate with high yield and good retention of protein activity. A GFP-C-poly(OEGMA) conjugate (hydrodynamic radius (R(h)): 21 nm) showed a 15-fold increase in its blood exposure compared to the protein (R(h): 3.0 nm) after intravenous administration to mice. This conjugate also showed a 50-fold increase in tumor accumulation, 24 h after intravenous administration to tumor-bearing mice, compared to the unmodified protein. This approach for in situ C-terminal polymer modification of a recombinant protein is applicable to a large subset of recombinant protein and peptide drugs and provides a general methodology for improvement of their pharmacological profiles.