RESUMO
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.
Assuntos
Células Artificiais/metabolismo , Grânulos Citoplasmáticos/genética , Proteínas Intrinsicamente Desordenadas/genética , Peptidomiméticos/metabolismo , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Sequência de Aminoácidos , Células Artificiais/citologia , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Elastina/química , Elastina/genética , Elastina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Biológicos , Peptidomiméticos/química , Transição de Fase , Plasmídeos/genética , Plasmídeos/metabolismo , Biossíntese de Proteínas , Engenharia de Proteínas/métodos , RNA/genética , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismoRESUMO
In nature, proteins range from those with highly ordered secondary and tertiary structures to those that completely lack a well-defined three-dimensional structure, termed intrinsically disordered proteins (IDPs). IDPs are generally characterized by one or more segments that have a compositional bias toward small hydrophilic amino acids and proline residues that promote structural disorder and are called intrinsically disordered regions (IDRs). The combination of IDRs with ordered regions and the interactions between the two determine the phase behavior, structure, and function of IDPs. Nature also diversifies the structure of proteins and thereby their functions by hybridization of the proteins with other moieties such as glycans and lipids; for instance, post-translationally glycosylated and lipidated proteins are important cell membrane components. Additionally, diversity in protein structure and function is achieved in nature through cross-linking proteins within themselves or with other domains to create various topologies. For example, an essential characteristic of the extracellular matrix (ECM) is the cross-linking of its network components, including proteins such as collagen and elastin, as well as polysaccharides such as hyaluronic acid (HA). Inspired by nature, synthetic IDP (SynIDP)-based biomaterials can be designed by employing similar strategies with the goal of introducing structural diversity and hence unique physiochemical properties. This Account describes such materials produced over the past decade and following one or more of the following approaches: (1) incorporating highly ordered domains into SynIDPs, (2) conjugating SynIDPs to other moieties through either genetically encoded post-translational modification or chemical conjugation, and (3) engineering the topology of SynIDPs via chemical modification. These approaches introduce modifications to the primary structure of SynIDPs, which are then translated to unique three-dimensional secondary and tertiary structures. Beginning with completely disordered SynIDPs as the point of origin, structure may be introduced into SynIDPs by each of these three unique approaches individually along orthogonal axes or by combinations of the three, enabling bioinspired designs to theoretically span the entire range of three-dimensional structural possibilities. Furthermore, the resultant structures span a wide range of length scales, from nano- to meso- to micro- and even macrostructures. In this Account, emphasis is placed on the physiochemical properties and structural features of the described materials. Conjugates of SynIDPs to synthetic polymers and materials achieved by simple mixing of components are outside the scope of this Account. Related biomedical applications are described briefly. Finally, we note future directions for the design of functional SynIDP-based biomaterials.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Conformação Proteica , Ácido Hialurônico , AminoácidosRESUMO
The formation of biomolecular condensates mediated by a coupling of associative and segregative phase transitions plays a critical role in controlling diverse cellular functions in nature. This has inspired the use of phase transitions to design synthetic systems. While design rules of phase transitions have been established for many synthetic intrinsically disordered proteins, most efforts have focused on investigating their phase behaviors in a test tube. Here, we present a rational engineering approach to program the formation and physical properties of synthetic condensates to achieve intended cellular functions. We demonstrate this approach through targeted plasmid sequestration and transcription regulation in bacteria and modulation of a protein circuit in mammalian cells. Our approach lays the foundation for engineering designer condensates for synthetic biology applications.
Assuntos
Condensados Biomoleculares , Proteínas Intrinsicamente Desordenadas , Animais , Organelas/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , MamíferosRESUMO
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.
Assuntos
Peptídeos Penetradores de Células , Micelas , Animais , Polipeptídeos Semelhantes à Elastina , Adsorção , Bicamadas Lipídicas/química , Peptídeos/química , Lipossomas Unilamelares/química , Peptídeos Penetradores de Células/química , Mamíferos/metabolismoRESUMO
We report a targeted prodrug delivery platform that can deliver a cytostatic nucleobase analog with high drug loading. We chose fluorouracil (5FU), a drug used to treat various cancers, whose active metabolite 5-fluorodeoxyuridine monophosphate (5-FdUMP) is the antineoplastic agent. We use terminal deoxynucleotidyl transferase (TdT) to polymerize 5-fluorodeoxyuridine triphosphate (5-FdUTP) onto the 3'-end of an aptamer. We find that (i) addition of hydrophobic, unnatural nucleotides at the 3'-end of the 5-FdU polynucleotide by TdT leads to their spontaneous self-assembly into nuclease resistant micelles, (ii) aptamers presented on the micelle corona retain specificity for their cognate receptor on tumor cells, and (iii) the micelles deliver 5FU to tumor cells and exhibit greater cytotoxicity than the free drug. The modular design of our platform, consisting of a targeting moiety, a polynucleotide drug, and a self-assembly domain, can be adapted to encompass a range of polymerizable therapeutic nucleotides and targeting units.
Assuntos
Antineoplásicos , Nanopartículas , Micelas , Polinucleotídeos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Fluoruracila , Nanopartículas/química , Sistemas de Liberação de Medicamentos , Linhagem Celular TumoralRESUMO
Nucleocapsid protein (N-protein) is required for multiple steps in betacoronaviruses replication. SARS-CoV-2-N-protein condenses with specific viral RNAs at particular temperatures making it a powerful model for deciphering RNA sequence specificity in condensates. We identify two separate and distinct double-stranded, RNA motifs (dsRNA stickers) that promote N-protein condensation. These dsRNA stickers are separately recognized by N-protein's two RNA binding domains (RBDs). RBD1 prefers structured RNA with sequences like the transcription-regulatory sequence (TRS). RBD2 prefers long stretches of dsRNA, independent of sequence. Thus, the two N-protein RBDs interact with distinct dsRNA stickers, and these interactions impart specific droplet physical properties that could support varied viral functions. Specifically, we find that addition of dsRNA lowers the condensation temperature dependent on RBD2 interactions and tunes translational repression. In contrast RBD1 sites are sequences critical for sub-genomic (sg) RNA generation and promote gRNA compression. The density of RBD1 binding motifs in proximity to TRS-L/B sequences is associated with levels of sub-genomic RNA generation. The switch to packaging is likely mediated by RBD1 interactions which generate particles that recapitulate the packaging unit of the virion. Thus, SARS-CoV-2 can achieve biochemical complexity, performing multiple functions in the same cytoplasm, with minimal protein components based on utilizing multiple distinct RNA motifs that control N-protein interactions.
Assuntos
Proteínas do Nucleocapsídeo de Coronavírus , RNA de Cadeia Dupla , SARS-CoV-2 , Sítios de Ligação , Proteínas do Nucleocapsídeo de Coronavírus/química , Fosfoproteínas/química , RNA de Cadeia Dupla/genética , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/genética , TemperaturaRESUMO
Complement protein C3dg, a key linkage between innate and adaptive immunity, is capable of stimulating both humoral and cell-mediated immune responses, leading to considerable interest in its use as a molecular adjuvant. However, the potential of C3dg as an adjuvant is limited without ways of controllably assembling multiple copies of it into vaccine platforms. Here, we report a strategy to assemble C3dg into supramolecular nanofibers with excellent compositional control, using ß-tail fusion tags. These assemblies were investigated as therapeutic active immunotherapies, which may offer advantages over existing biologics, particularly toward chronic inflammatory diseases. Supramolecular assemblies based on the Q11 peptide system containing ß-tail-tagged C3dg, B cell epitopes from TNF, and the universal T cell epitope PADRE raised strong antibody responses against both TNF and C3dg, and prophylactic immunization with these materials significantly improved protection in a lethal TNF-mediated inflammation model. Additionally, in a murine model of psoriasis induced by imiquimod, the C3dg-adjuvanted nanofiber vaccine performed as well as anti-TNF monoclonal antibodies. Nanofibers containing only ß-tail-C3dg and lacking the TNF B cell epitope also showed improvements in both models, suggesting that supramolecular C3dg, by itself, played an important therapeutic role. We observed that immunization with ß-tail-C3dg caused the expansion of an autoreactive C3dg-specific T cell population, which may act to dampen the immune response, preventing excessive inflammation. These findings indicate that molecular assemblies displaying C3dg warrant further development as active immunotherapies.
Assuntos
Complemento C3d/imunologia , Nanofibras/química , Psoríase/prevenção & controle , Vacinas/imunologia , Adjuvantes Imunológicos/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Células Cultivadas , Epitopos/química , Epitopos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/imunologia , Vacinas/químicaRESUMO
Antigen tests to detect SARS-CoV-2 have emerged as a promising rapid diagnostic method for COVID-19, but they are unable to differentiate between variants of concern (VOCs). Here, we report a rapid point-of-care test (POC-T), termed CoVariant-SPOT, that uses a set of antibodies that are either tolerant or intolerant to spike protein mutations to identify the likely SARS-CoV-2 strain concurrent with COVID-19 diagnosis using antibodies targeting the nucleocapsid protein. All reagents are incorporated into a portable, multiplexed, and sensitive diagnostic platform built upon a nonfouling polymer brush. To validate CoVariant-SPOT, we tested recombinant SARS-CoV-2 proteins, inactivated viruses, and nasopharyngeal swab samples from COVID-19 positive and negative individuals and showed that CoVariant-SPOT can readily distinguish between two VOCs: Delta and Omicron. We believe that CoVariant-SPOT can serve as a valuable adjunct to next-generation sequencing to rapidly identify variants using a scalable and deployable POC-T, thereby enhancing community surveillance efforts worldwide and informing treatment selection.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Teste para COVID-19 , AnticorposRESUMO
The ongoing Coronavirus disease 2019 (COVID-19) pandemic illustrates the need for sensitive and reliable tools to diagnose and monitor diseases. Traditional diagnostic approaches rely on centralized laboratory tests that result in long wait times to results and reduce the number of tests that can be given. Point-of-care tests (POCTs) are a group of technologies that miniaturize clinical assays into portable form factors that can be run both in clinical areas --in place of traditional tests-- and outside of traditional clinical settings --to enable new testing paradigms. Hallmark examples of POCTs are the pregnancy test lateral flow assay and the blood glucose meter. Other uses for POCTs include diagnostic assays for diseases like COVID-19, HIV, and malaria but despite some successes, there are still unsolved challenges for fully translating these lower cost and more versatile solutions. To overcome these challenges, researchers have exploited innovations in colloid and interface science to develop various designs of POCTs for clinical applications. Herein, we provide a review of recent advancements in lateral flow assays, other paper based POCTs, protein microarray assays, microbead flow assays, and nucleic acid amplification assays. Features that are desirable to integrate into future POCTs, including simplified sample collection, end-to-end connectivity, and machine learning, are also discussed in this review.
RESUMO
The development of platinum(Pt)-drugs for cancer therapy has stalled, as no new Pt-drugs have been approved in over a decade. Packaging small molecule drugs into nanoparticles is a way to enhance their therapeutic efficacy. To date, there has been no direct comparison of relative merits of the choice of Pt oxidation state in the same nanoparticle system that would allow its optimal design. To address this lacuna, we designed a recombinant asymmetric triblock polypeptide (ATBP) that self-assembles into rod-shaped micelles and chelates Pt(II) or enables covalent conjugation of Pt(IV) with similar morphology and stability. Both ATBP-Pt(II) and ATBP-Pt(IV) nanoparticles enhanced the half-life of Pt by â¼45-fold, but ATBP-Pt(IV) had superior tumor regression efficacy compared to ATBP-Pt(II) and cisplatin. These results suggest loading Pt(IV) into genetically engineered nanoparticles may yield a new generation of more effective platinum-drug nanoformulations.
Assuntos
Antineoplásicos , Nanopartículas , Neoplasias , Pró-Fármacos , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cisplatino/química , Cisplatino/uso terapêutico , Camundongos , Nanopartículas/química , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Peptídeos/uso terapêutico , Platina/química , Pró-Fármacos/químicaRESUMO
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.
Assuntos
Biopolímeros/química , Sistemas de Liberação de Medicamentos , Elastina/fisiologia , Peptídeos/química , Engenharia de Proteínas/métodos , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Portadores de Fármacos , Escherichia coli , Ácidos Graxos/química , Humanos , Hidrogéis , Peso Molecular , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Polímeros , Seda , TemperaturaRESUMO
Small-scale production of biologics has great potential for enhancing the accessibility of biomanufacturing. By exploiting cell-material feedback, we have designed a concise platform to achieve versatile production, analysis and purification of diverse proteins and protein complexes. The core of our technology is a microbial swarmbot, which consists of a stimulus-sensitive polymeric microcapsule encapsulating engineered bacteria. By sensing the confinement, the bacteria undergo programmed partial lysis at a high local density. Conversely, the encapsulating material shrinks responding to the changing chemical environment caused by cell growth, squeezing out the protein products released by bacterial lysis. This platform is then integrated with downstream modules to enable quantification of enzymatic kinetics, purification of diverse proteins, quantitative control of protein interactions and assembly of functional protein complexes and multienzyme metabolic pathways. Our work demonstrates the use of the cell-material feedback to engineer a modular and flexible platform with sophisticated yet well-defined programmed functions.
Assuntos
Proteínas de Bactérias/metabolismo , Bioengenharia , Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Reatores Biológicos , Regulação da Expressão Gênica , Engenharia Genética , PlasmídeosRESUMO
Valency is a fundamental principle to control macromolecular interactions and is used to target specific cell types by multivalent ligand-receptor interactions using self-assembled nanoparticle carriers. At the concentrations encountered in solid tumors upon systemic administration, these nanoparticles are, however, likely to show critical micelle concentration (CMC)-dependent disassembly and thus loss of function. To overcome this limitation, core-crosslinkable micelles of genetically encoded resilin-/elastin-like diblock polypeptides were recombinantly synthesized. The amphiphilic constructs were covalently photo-crosslinked through the genetically encoded unnatural amino acid para-azidophenylalanine in their hydrophobic block and they carried different anticancer ligands on their hydrophilic block: the wild-type tenth human fibronectin type III domain, a GRGDSPAS peptide-both targeting αvß3 integrin-and an engineered variant of the third fibronectin type III domain of tenascin C that is a death receptor 5 agonist. Although uncrosslinked micelles lost most of their targeting ability below their CMC, the crosslinked analogues remained active at concentrations up to 1000-fold lower than the CMC, with binding affinities that are comparable to antibodies.
Assuntos
Elastina , Neoplasias , Elastina/genética , Humanos , Proteínas de Insetos , Micelas , Neoplasias/tratamento farmacológico , Neoplasias/genética , Peptídeos/genéticaRESUMO
Diblock copolymers are valued for their ability to form thin films with nanoscale features that typically reflect those of their microphase-separated structures in concentrated solution. Here, we show that such self-assembled structures can be easily formed with diblock copolymers composed of thermally responsive polypeptides, such as resilin-like polypeptides (RLP) and elastin-like polypeptides (ELP), by exploiting the inverse temperature transition behavior of ELPs in aqueous media. Specifically, we examine the self-assembly of a series of RLP-b-ELP diblock copolypeptides in concentrated aqueous solution (30 and 50 wt %) by small-angle X-ray scattering (SAXS). By systematically varying RLP block length and temperature (10-45 °C), we observed microphase separation into hexagonally packed cylinders and lamellae. By analyzing the observed order-order transitions (OOT) and order-disorder transitions (ODT), we determined that self-assembly in this system is primarily driven by polymer-solvent interactions. While these thermally responsive polymers showed clear ODTs and OOTs at certain temperatures, temperature only had a weak effect on the spacing of the resulting nanostructures. In contrast, we found that nanostructure spacing was far more sensitive to RLP block length. Finally, we used atomic force microscopy (AFM) to demonstrate that spin casting RLP-b-ELP diblock copolypeptides also produce nanostructured thin films with spacings that correlate with those in concentrated solution.
Assuntos
Elastina , Proteínas de Insetos , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
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.
Assuntos
Proteínas Intrinsicamente Desordenadas , Aminoácidos , Organelas , Polímeros , ÁguaRESUMO
The detection of biomarkers in blood often requires extensive and time-consuming sample preparation to remove blood cells and concentrate the biomarker(s) of interest. We demonstrate proof-of-concept for a chip-based, acoustofluidic method that enables the rapid capture and isolation of a model protein biomarker (i.e., streptavidin) from blood for off-chip quantification. Our approach makes use of two key components - namely, soluble, thermally responsive polypeptides fused to ligands for the homogeneous capture of biomarkers from whole blood and silicone microparticles functionalized with similar, tethered, thermally responsive polypeptides. When the two components are mixed together and subjected to a mild thermal trigger, the thermally responsive moieties undergo a phase transition, causing the untethered (soluble) polypeptides to co-aggregate with the particle-bound polypeptides. The mixture is then diluted with warm buffer and injected into a microfluidic channel supporting a bulk acoustic standing wave. The biomarker-bearing particles migrate to the pressure antinodes, whereas blood cells migrate to the pressure node, leading to rapid separation with efficiencies exceeding 90% in a single pass. The biomarker-bearing particles can then be analyzed via flow cytometry, with a limit of detection of 0.75 nM for streptavidin spiked in blood plasma. Finally, by cooling the solution below the solubility temperature of the polypeptides, greater than 75% of the streptavidin is released from the microparticles, offering a unique approach for downstream analysis (e.g., sequencing or structural analysis). Overall, this methodology has promise for the detection, enrichment and analysis of some biomarkers from blood and other complex biological samples.
Assuntos
Acústica , Análise Química do Sangue , Microfluídica , Citometria de Fluxo , Som , EstreptavidinaRESUMO
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.
Assuntos
Bioimpressão , Ouro , Imunoensaio , Prata , Humanos , Nanotecnologia , Testes Imediatos , PolímerosRESUMO
Small-molecule therapeutics demonstrate suboptimal pharmacokinetics and bioavailability due to their hydrophobicity and size. One way to overcome these limitations-and improve their efficacy-is to use "stealth" macromolecular carriers that evade uptake by the reticuloendothelial system. Although unstructured polypeptides are of increasing interest as macromolecular drug carriers, current recombinant polypeptides in the clinical pipeline typically lack stealth properties. We address this challenge by developing new unstructured polypeptides, called zwitterionic polypeptides (ZIPPs), that exhibit "stealth" behavior in vivo. We show that conjugating paclitaxel to a ZIPP imparts amphiphilicity to the polypeptide chain that is sufficient to drive its self-assembly into micelles. This in turn increases the half-life of paclitaxel by 17-fold compared to free paclitaxel, and by 1.6-fold compared to the nonstealth control, i.e., ELP-paclitaxel. Treatment of mice bearing highly aggressive prostate or colon cancer with a single dose of ZIPP-paclitaxel nanoparticles leads to near-complete eradication of the tumor, and these nanoparticles have a wider therapeutic window than Abraxane, an FDA-approved taxane nanoformulation.
Assuntos
Paclitaxel Ligado a Albumina/uso terapêutico , Antineoplásicos/uso terapêutico , Nanoconjugados/uso terapêutico , Neoplasias/tratamento farmacológico , Paclitaxel/uso terapêutico , Peptídeos/uso terapêutico , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Nus , Nanoconjugados/análise , Paclitaxel/farmacocinética , Peptídeos/farmacocinética , Resultado do TratamentoRESUMO
Phase separation is thought to underlie spatial and temporal organization that is required for controlling biochemical reactions in cells. Multivalence of interaction motifs, also known as stickers, is a defining feature of proteins that drive phase separation. Intrinsically disordered proteins with stickers uniformly distributed along the linear sequence can serve as scaffold molecules that drive phase separation. The sequence-intrinsic contributions of disordered proteins to phase separation can be discerned by computing or measuring sequence-specific phase diagrams. These help to delineate the combinations of protein concentration and a suitable control parameter, such as temperature, that support phase separation. Here, we present an approach that combines detailed simulations with a numerical adaptation of an analytical Gaussian cluster theory to enable the calculation of sequence-specific phase diagrams. Our approach leverages the known equivalence between the driving forces for single-chain collapse in dilute solutions and the driving forces for phase separation in concentrated solutions. We demonstrate the application of the theory-aided computations through calculation of phase diagrams for a set of archetypal intrinsically disordered low-complexity domains. We also leverage theories to compute sequence-specific percolation lines and thereby provide a thermodynamic framework for hardening transitions that have been observed for many biomolecular condensates.
Assuntos
Proteínas Intrinsicamente Desordenadas , Transição de Fase , Temperatura , TermodinâmicaRESUMO
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.