RESUMO
Peptide surfactants (PEPS) are studied to capture and retain rare earth elements (REEs) at air-water interfaces to enable REE separations. Peptide sequences, designed to selectively bind REEs, depend crucially on the position of ligands within their binding loop domain. These ligands form a coordination sphere that wraps and retains the cation. We study variants of lanthanide binding tags (LBTs) designed to complex strongly with Tb3+. The peptide LBT5- (with net charge -5) is known to bind Tb3+ and adsorb with more REE cations than peptide molecules, suggesting that undesired non-specific coulombic interactions occur. Rheological characterization of interfaces of LBT5- and Tb3+ solutions reveal the formation of an interfacial gel. To probe whether this gelation reflects chelation among intact adsorbed LBT5-:Tb3+ complexes or destruction of the binding loop, we study a variant, LBT3-, designed to form net neutral LBT3-:Tb3+ complexes. Solutions of LBT3- and Tb3+ form purely viscous layers in the presence of excess Tb3+, indicating that each peptide binds a single REE in an intact coordination sphere. We introduce the variant RR-LBT3- with net charge -3 and anionic ligands outside of the coordination sphere. We find that such exposed ligands promote interfacial gelation. Thus, a nuanced requirement for interfacial selectivity of PEPS is proposed: that anionic ligands outside of the coordination sphere must be avoided to prevent the non-selective recruitment of REE cations. This view is supported by simulation, including interfacial molecular dynamics simulations, and interfacial metadynamics simulations of the free energy landscape of the binding loop conformational space.
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Noble gases have well-established biological effects, yet their molecular mechanisms remain poorly understood. Here, we investigated, both experimentally and computationally, the molecular modes of xenon (Xe) action in bacteriophage T4 lysozyme (T4L). By combining indirect gassing methods with a colorimetric lysozyme activity assay, a reversible, Xe-specific (20 ± 3)% inhibition effect was observed. Accelerated molecular dynamic simulations revealed that Xe exerts allosteric inhibition on the protein by expanding a C-terminal hydrophobic cavity. Xe-induced cavity expansion results in global conformational changes, with long-range transduction distorting the active site where peptidoglycan binds. Interestingly, the peptide substrate binding site that enables lysozyme specificity does not change conformation. Two T4L mutants designed to reshape the C-terminal Xe cavity established a correlation between cavity expansion and enzyme inhibition. This work also highlights the use of Xe flooding simulations to identify new cryptic binding pockets. These results enrich our understanding of Xe-protein interactions at the molecular level and inspire further biochemical investigations with noble gases.
Assuntos
Muramidase , Xenônio , Xenônio/química , Xenônio/metabolismo , Muramidase/química , Gases Nobres/química , Gases Nobres/metabolismo , Sítios de Ligação , ProteínasRESUMO
Many RNA delivery strategies require efficient endosomal uptake and release. To monitor this process, we developed a 2'-OMe RNA-based ratiometric pH probe with a pH-invariant 3'-Cy5 and 5'-FAM whose pH sensitivity is enhanced by proximal guanines. The probe, in duplex with a DNA complement, exhibits a 48.9-fold FAM fluorescence enhancement going from pH 4.5 to pH 8.0 and reports on both endosomal entrapment and release when delivered to HeLa cells. In complex with an antisense RNA complement, the probe constitutes an siRNA mimic capable of protein knockdown in HEK293T cells. This illustrates a general approach for measuring the localization and pH microenvironment of any oligonucleotide.
Assuntos
Endossomos , Corantes Fluorescentes , Humanos , Células HeLa , RNA Interferente Pequeno/metabolismo , Células HEK293 , Concentração de Íons de Hidrogênio , Endossomos/metabolismo , Corantes Fluorescentes/metabolismoRESUMO
Protein interiors contain void space that can bind small gas molecules. Determination of gas pathways and kinetics in proteins has been an intriguing and challenging task. Here, we combined computational methods and the hyperpolarized xenon-129 chemical exchange saturation transfer (hyper-CEST) NMR technique to investigate xenon (Xe) exchange kinetics in maltose-binding protein (MBP). A salt bridge â¼9 Å from the Xe-binding site formed upon maltose binding and slowed the Xe exchange rate, leading to a hyper-CEST 129Xe signal from maltose-bound MBP. Xe dissociation occurred faster than dissociation of the salt bridge, as shown by 13C NMR spectroscopy and variable-B1 hyper-CEST experiments. "Xe flooding" molecular dynamics simulations identified a surface hydrophobic site, V23, that has good Xe binding affinity. Mutations at this site confirmed its role as a secondary exchange pathway in modulating Xe diffusion. This shows the possibility for site-specifically controlling xenon protein-solvent exchange. Analysis of the available MBP structures suggests a biological role of MBP's large hydrophobic cavity to accommodate structural changes associated with ligand binding and protein-protein interactions.
Assuntos
Xenônio , Proteínas Ligantes de MaltoseRESUMO
The affinity of small molecules for biomolecular cavities is tuned through a combination of primary and secondary interactions. It has been challenging to mimic these features in organic synthetic host molecules, however, where the cavities tend to be highly symmetric and nonpolar, and less amenable to chemical manipulation. Here, a host molecule composed of a TREN ligand and cyclotriveratrylene moiety was investigated. Size-matched polar guests were encapsulated within the cavity via triple protonation of the TREN moiety with various sulfonic acids. X-ray crystallography confirmed guest encapsulation and identified three methanesulfonates, p-toluenesulfonates, or 2-naphthalenesulfonates hydrogen-bonded with H3TREN at the periphery of the cavity. These structurally diverse counteranions were shown by 1H NMR spectroscopy to differentially regulate guest access at the three portals, and to undergo competitive displacement in solution. This work reveals "counteranion tuning" to be a simple and powerful strategy for modulating host-guest affinity, as applied here in a TREN-hemicryptophane.
Assuntos
Hidrogênio , Cristalografia por Raios X , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos MolecularesRESUMO
Supercharged proteins exhibit high solubility and other desirable properties, but no engineered superpositively charged enzymes have previously been made. Superpositively charged variants of proteins such as green fluorescent protein have been efficiently encapsulated within Archaeoglobus fulgidus thermophilic ferritin (AfFtn). Encapsulation by supramolecular ferritin can yield systems with a variety of sequestered cargo. To advance applications in enzymology and green chemistry, we sought a general method for supercharging an enzyme that retains activity and is compatible with AfFtn encapsulation. The zinc metalloenzyme human carbonic anhydrase II (hCAII) is an attractive encapsulation target based on its hydrolytic activity and physiologic conversion of carbon dioxide to bicarbonate. A computationally designed variant of hCAII contains positively charged residues substituted at 19 sites on the protein's surface, resulting in a shift of the putative net charge from -1 to +21. This designed hCAII(+21) exhibits encapsulation within AfFtn without the need for fusion partners or additional reagents. The hCAII(+21) variant retains esterase activity comparable to the wild type and spontaneously templates the assembly of AfFtn 24mers around itself. The AfFtn-hCAII(+21) host-guest complex exhibits both greater activity and thermal stability when compared to hCAII(+21). Upon immobilization on a solid support, AfFtn-hCAII(+21) retains enzymatic activity and exhibits an enhancement of activity at elevated temperatures.
Assuntos
Proteínas Arqueais/química , Anidrase Carbônica II/química , Enzimas Imobilizadas/química , Ferritinas/química , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Archaeoglobus fulgidus/enzimologia , Anidrase Carbônica II/genética , Anidrase Carbônica II/isolamento & purificação , Anidrase Carbônica II/metabolismo , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/isolamento & purificação , Enzimas Imobilizadas/metabolismo , Ferritinas/genética , Ferritinas/isolamento & purificação , Ferritinas/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Cryptophane host molecules provide ultrasensitive contrast agents for 129Xe NMR/MRI. To investigate key features of cryptophane-Xe sensing behavior, we designed a novel water-soluble cryptophane with a pendant hydrophobic adamantyl moiety, which has good affinity for a model receptor, beta-cyclodextrin (ß-CD). Adamantyl-functionalized cryptophane-A (AFCA) was synthesized and characterized for Xe affinity, 129Xe NMR signal, and aggregation state at varying AFCA and ß-CD concentrations. The Xe-AFCA association constant was determined by fluorescence quenching, KA = 114,000 ± 5000 M-1 at 293 K, which is the highest reported affinity for a cryptophane host in phosphate-buffered saline (pH 7.2). No hyperpolarized (hp) 129Xe NMR peak corresponding to AFCA-bound Xe was directly observed at high (100 µM) AFCA concentration, where small cryptophane aggregates were observed, and was only detected at low (15 µM) AFCA concentration, where the sensor remained fully monomeric in solution. Additionally, we observed no change in the chemical shift of AFCA-encapsulated 129Xe after ß-CD binding to the adamantyl moiety and a concomitant lack of change in the size distribution of the complex, suggesting that a change in the aggregation state is necessary to elicit a 129Xe NMR chemical shift in cryptophane-based sensing. These results aid in further elucidating the recently discovered aggregation phenomenon, highlight limitations of cryptophane-based Xe sensing, and offer insights into the design of monomeric, high-affinity Xe sensors.
Assuntos
Compostos Policíclicos/química , Xenônio/química , beta-Ciclodextrinas/química , Técnicas Biossensoriais , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Compostos Policíclicos/síntese química , Isótopos de XenônioRESUMO
Conditionally activated ("caged") oligonucleotides provide useful spatiotemporal control for studying dynamic biological processes, e.g., regulating in vivo gene expression or probing specific oligonucleotide targets. This review summarizes recent advances in caging strategies, which involve different stimuli in the activation step. Oligo cyclization is a particularly attractive caging strategy, which simplifies the probe design and affords oligo stabilization. Our laboratory developed an efficient synthesis for circular caged oligos, and a circular caged antisense DNA oligo was successfully applied in gene regulation. A second technology is Transcriptome In Vivo Analysis (TIVA), where caged oligos enable mRNA isolation from single cells in living tissue. We highlight our development of TIVA probes with improved caging stability. Finally, we illustrate the first protease-activated oligo probe, which was designed for caspase-3. This expands the toolkit for investigating the transcriptome under a specific physiologic condition (e.g., apoptosis), particularly in specimens where light activation is impractical.
Assuntos
Regulação da Expressão Gênica/genética , Oligonucleotídeos Antissenso/química , Oligonucleotídeos/química , Animais , Ciclização/genética , Ativação Enzimática/genética , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Humanos , Luz , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/genética , RNA Mensageiro/genética , Biologia Sintética/métodosRESUMO
Dysregulation of cellular ribose uptake can be indicative of metabolic abnormalities or tumorigenesis. However, analytical methods are currently limited for quantifying ribose concentration in complex biological samples. Here, we utilize the highly specific recognition of ribose by ribose-binding protein (RBP) to develop a single-protein ribose sensor detectable via a sensitive NMR technique known as hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST). We demonstrate that RBP, with a tunable ribose-binding site and further engineered to bind xenon, enables the quantitation of ribose over a wide concentration range (nM to mM). Ribose binding induces the RBP "closed" conformation, which slows Xe exchange to a rate detectable by hyper-CEST. Such detection is remarkably specific for ribose, with the minimal background signal from endogenous sugars of similar size and structure, for example, glucose or ribose-6-phosphate. Ribose concentration was measured for mammalian cell lysate and serum, which led to estimates of low-mM ribose in a HeLa cell line. This highlights the potential for using genetically encoded periplasmic binding proteins such as RBP to measure metabolites in different biological fluids, tissues, and physiologic states.
Assuntos
Proteínas de Escherichia coli/química , Proteínas Periplásmicas de Ligação/química , Ribose/análise , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Periplásmicas de Ligação/isolamento & purificação , Proteínas Periplásmicas de Ligação/metabolismo , Ribose/metabolismo , Isótopos de XenônioRESUMO
Light-activated ("caged") oligonucleotides provide a strategy for modulating the activity of antisense oligos, siRNA, miRNA, aptamers, DNAzymes, and mRNA-capturing probes with high spatiotemporal resolution. However, the near-UV and visible wavelengths that promote these bond-breaking reactions poorly penetrate living tissue, which limits some biological applications. To address this issue, we describe the first example of a protease-activated oligonucleotide probe, capable of reporting on caspase-3 during cellular apoptosis. The 2'-F RNA-peptide substrate-peptide nucleic acid (PNA) hairpin structure was generated in 30% yield in a single bioconjugation step.
Assuntos
Apoptose , Caspases/metabolismo , Sondas de Oligonucleotídeos/metabolismo , Sequência de Bases , Caspase 3/metabolismo , Ativação Enzimática , Células HeLa , Humanos , Sondas de Oligonucleotídeos/química , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/metabolismoRESUMO
We investigate the magnetic resonance properties and exchange kinetics of guest molecules in a series of hetero-bimetallic capsules, [ConFe4-nL6]4- (n = 1-3), where L2- = 4,4'-bis[(2-pyridinylmethylene)amino]-[1,1'-biphenyl]-2,2'-disulfonate. H bond networks between capsule sulfonates and guanidinium cations promote the crystallization of [ConFe4-nL6]4-. The following four isostructural crystals are reported: two guest-free forms, (C(NH2)3)4[Co1.8Fe2.2L6]·69H2O (1) and (C(NH2)3)4[Co2.7Fe1.3L6]·73H2O (2), and two Xe- and CFCl3-encapsulated forms, (C(NH2)3)4[(Xe)0.8Co1.8Fe2.2L6]·69H2O (3) and (C(NH2)3)4[(CFCl3)Co2.0Fe2.0L6]·73H2O (4), respectively. Structural analyses reveal that Xe induces negligible structural changes in 3, while the angles between neighboring phenyl groups expand by ca. 3° to accommodate the much larger guest, CFCl3, in 4. These guest-encapsulated [ConFe4-nL6]4- molecules reveal 129Xe and 19F chemical shift changes of ca. -22 and -10 ppm at 298 K, respectively, per substitution of low-spin FeII by high-spin CoII. Likewise, the temperature dependence of the 129Xe and 19F NMR resonances increases by 0.1 and 0.06 ppm/K, respectively, with each additional paramagnetic CoII center. The optimal temperature for hyperpolarized (hp) 129Xe chemical exchange saturation transfer (hyper-CEST) with [ConFe4-nL6]4- capsules was found to be inversely proportional to the number of CoII centers, n, which is consistent with the Xe chemical exchange accelerating as the portals expand. The systematic study was facilitated by the tunability of the [M4L6]4- capsules, further highlighting these metal-organic systems for developing responsive sensors with highly shifted 129Xe resonances.
RESUMO
We investigated Xe binding in a previously reported paramagnetic metal-organic tetrahedral capsule, [Co4L6]4-, where L2- = 4,4'-bis[(2-pyridinylmethylene)amino][1,1'-biphenyl]-2,2'-disulfonate. The Xe-inclusion complex, [XeCo4L6]4-, was confirmed by 1H NMR spectroscopy to be the dominant species in aqueous solution saturated with Xe gas. The measured Xe dissociation rate in [XeCo4L6]4-, koff = 4.45(5) × 102 s-1, was at least 40 times greater than that in the analogous [XeFe4L6]4- complex, highlighting the capability of metal-ligand interactions to tune the capsule size and guest permeability. The rapid exchange of 129Xe nuclei in [XeCo4L6]4- produced significant hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST) NMR signal at 298 K, detected at a concentration of [XeCo4L6]4- as low as 100 pM, with presaturation at -89 ppm, which was referenced to solvated 129Xe in H2O. The saturation offset was highly temperature-dependent with a slope of -0.41(3) ppm/K, which is attributed to hyperfine interactions between the encapsulated 129Xe nucleus and electron spins on the four CoII centers. As such, [XeCo4L6]4- represents the first example of a paramagnetic hyper-CEST (paraHYPERCEST) sensor. Remarkably, the hyper-CEST 129Xe NMR resonance for [XeCo4L6]4- (δ = -89 ppm) was shifted 105 ppm upfield from the diamagnetic analogue [XeFe4L6]4- (δ = +16 ppm). The Xe inclusion complex was further characterized in the crystal structure of (C(NH2)3)4[Xe0.7Co4L6]·75 H2O (1). Hydrogen bonding between capsule-linker sulfonate groups and exogenous guanidinium cations, (C(NH2)3)+, stabilized capsule-capsule interactions in the solid state and also assisted in trapping a Xe atom (â¼42 Å3) in the large (135 Å3) cavity of 1. Magnetic susceptibility measurements confirmed the presence of four noninteracting, magnetically anisotropic high-spin CoII centers in 1. Furthermore, [Co4L6]4- was found to be stable toward aggregation and oxidation, and the CEST performance of [XeCo4L6]4- was unaffected by biological macromolecules in H2O. These results recommend metal-organic capsules for fundamental investigations of Xe host-guest chemistry as well as applications with highly sensitive 129Xe-based sensors.
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Genetically encoded (GE) contrast agents detectable by magnetic resonance imaging (MRI) enable non-invasive visualization of gene expression and cell proliferation at virtually unlimited penetration depths. Using hyperpolarized 129 Xe in combination with chemical exchange saturation transfer, an MR contrast approach known as hyper-CEST, enables ultrasensitive protein detection and biomolecular imaging. GE MRI contrast agents developed to date include nanoscale proteinaceous gas vesicles as well as the monomeric bacterial proteins TEM-1 ß-lactamase (bla) and maltose binding protein (MBP). To improve understanding of hyper-CEST NMR with proteins, structural and computational studies were performed to further characterize the Xe-bla interaction. X-ray crystallography validated the location of a high-occupancy Xe binding site predicted by MD simulations, and mutagenesis experiments confirmed this Xe site as the origin of the observed CEST contrast. Structural studies and MD simulations with representative bla mutants offered additional insight regarding the relationship between local protein structure and CEST contrast.
Assuntos
Isótopos de Xenônio/química , beta-Lactamases/química , Sítio Alostérico , Sítios de Ligação , Meios de Contraste/química , Cristalografia por Raios X , Limite de Detecção , Proteínas Ligantes de Maltose/química , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
We report the development of a fast and accurate fluorescence-based assay for amidine linked to cellulose membranes and Sepharose gel. The assay is founded on the glyoxal reaction, which involves reaction of an amidine group with glyoxal and an aromatic aldehyde, leading to the formation of a fluorophore that can be analyzed and quantified by fluorescence spectroscopy and imaging. While the assay has been reported previously for aromatic amidine estimation in solution phase, here we describe its adaptation and application to amidine linked to diverse forms of solid matrices, particularly benzamidine Sepharose and benzamidine-linked cellulose membranes. These functionalized porous matrices find important application in purification of serine proteases. The efficacy of a protein separation device is determined by, among other factors, the ligand (amidine) density. Hence, a sensitive and reproducible method for amidine quantitation in solid phase is needed. The glyoxal reaction was carried out on microbead-sized Sepharose gel and cellulose membranes. Calibration curves were developed for each phase, which established linearity in the range of 0-0.45 µmol per mL amidine for free amidine in solution, 0-0.45 µmol amidine per mL Sepharose gel, and 0-0.48 µmol per mL cellulose membrane. The assay showed high accuracy (~ 3.4% error), precision (RSD < 2%), and reproducibility. Finally, we show how this fluorescent labeling (glyoxal) method can provide a tool for imaging membranes and ligand distribution through confocal laser scanning microscopy. Graphical abstract.
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Cryptophane-based biosensors are promising agents for the ultrasensitive detection of biomedically relevant targets via 129Xe NMR. Dynamic light scattering revealed that cryptophanes form water-soluble aggregates tens to hundreds of nanometers in size. Acridine orange fluorescence quenching assays allowed quantitation of the aggregation state, with critical concentrations ranging from 200 nM to 600 nM, depending on the cryptophane species in solution. The addition of excess carbonic anhydrase (CA) protein target to a benzenesulfonamide-functionalized cryptophane biosensor (C8B) led to C8B disaggregation and produced the expected 1:1 C8B-CA complex. C8B showed higher affinity at 298 K for the cytoplasmic isozyme CAII than the extracellular CAXII isozyme, which is a biomarker of cancer. Using hyper-CEST NMR, we explored the role of stoichiometry in detecting these two isozymes. Under CA-saturating conditions, we observed that isozyme CAII produces a larger 129Xe NMR chemical shift change (δ = 5.9 ppm, relative to free biosensor) than CAXII (δ = 2.7 ppm), which indicates the strong potential for isozyme-specific detection. However, stoichiometry-dependent chemical shift data indicated that biosensor disaggregation contributes to the observed 129Xe NMR chemical shift change that is normally assigned to biosensor-target binding. Finally, we determined that monomeric cryptophane solutions improve hyper-CEST saturation contrast, which enables ultrasensitive detection of biosensor-protein complexes. These insights into cryptophane-solution behavior support further development of xenon biosensors, but will require reinterpretation of the data previously obtained for many water-soluble cryptophanes.
Assuntos
Técnicas Biossensoriais , Anidrases Carbônicas/análise , Técnicas Eletroquímicas , Nanoestruturas/química , Ressonância Magnética Nuclear Biomolecular , Compostos Policíclicos/química , Técnicas Biossensoriais/instrumentação , Anidrases Carbônicas/isolamento & purificação , Anidrases Carbônicas/metabolismo , Técnicas Eletroquímicas/instrumentação , Fluorescência , Humanos , Solubilidade , Isótopos de XenônioRESUMO
Light-activated ("caged") antisense oligonucleotides are powerful molecules for regulating gene expression at submicron spatial resolution through the focal modulation of endogenous cellular processes. Cyclized caged oligos are particularly promising structures because of their inherent stability and similarity to naturally occurring circular DNA and RNA molecules. Here, we introduce an efficient route for cyclizing an antisense oligodeoxynucleotide incorporating a photocleavable linker. Oligo cyclization was achieved for several sequences in nearly quantitative yields through intramolecular copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Caging stability and light activation were characterized by FRET efficiency, denaturing gel assay, and melting temperature measurements. Finally, a cyclized caged oligo was designed to target gfap, and it gave a tenfold reduction in glial fibrillary acidic protein upon photoactivation in astrocytes.
Assuntos
Química Click/métodos , Oligonucleotídeos Antissenso/síntese química , Optogenética/métodos , Alcinos/síntese química , Alcinos/química , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Azidas/síntese química , Azidas/química , Sequência de Bases , Carbocianinas/síntese química , Carbocianinas/química , Catálise , Cobre/química , Ciclização , Reação de Cicloadição/métodos , Expressão Gênica/efeitos da radiação , Proteína Glial Fibrilar Ácida/genética , Humanos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genéticaRESUMO
Genetic incorporation of biologically orthogonal functional groups into macromolecules has the potential to yield efficient, controlled, reproducible, site-specific conjugation of affinity ligands, contrast agents, or therapeutic cargoes. Here, we applied this approach to ferritin, a ubiquitous iron-storage protein that self-assembles into multimeric nanocages with remarkable stability, size uniformity (12 nm), and endogenous capacity for loading and transport of a variety of inorganic and organic cargoes. The unnatural amino acid, 4-azidophenylalanine (4-AzF), was incorporated at different sites in the human ferritin light chain (hFTL) to allow site-specific conjugation of alkyne-containing small molecules or affinity ligands to the exterior surface of the nanocage. The optimal positioning of the 4-AzF residue was evaluated by screening a library of variants for the efficiency of copper-free click conjugation. One of the engineered ferritins, hFTL-5X, was found to accommodate â¼14 small-molecule fluorophores (AlexaFluor 488) and 3-4 IgG molecules per nanocage. Intravascular injection in mice of radiolabeled hFTL-5X carrying antibody to cell adhesion molecule ICAM-1, but not control IgG, enabled specific targeting to the lung due to high basal expression of ICAM-1 (43.3 ± 6.99 vs 3.48 ± 0.14%ID/g for Ab vs IgG). Treatment of mice with endotoxin known to stimulate inflammatory ICAM-1 overexpression resulted in 2-fold enhancement of pulmonary targeting (84.4 ± 12.89 vs 43.3 ± 6.99%ID/g). Likewise, injection of fluorescent, ICAM-targeted hFTL-5X nanocages revealed the effect of endotoxin by enhancement of near-infrared signal, indicating potential utility of this approach for both vascular targeting and imaging.
Assuntos
Azidas/química , Ferritinas/química , Corantes Fluorescentes/química , Imunoconjugados/química , Molécula 1 de Adesão Intercelular/análise , Imagem Óptica/métodos , Fenilalanina/análogos & derivados , Alcinos/síntese química , Alcinos/química , Animais , Azidas/síntese química , Química Click/métodos , Ferritinas/síntese química , Corantes Fluorescentes/síntese química , Humanos , Inflamação/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Camundongos , Nanoestruturas/química , Fenilalanina/síntese química , Fenilalanina/químicaRESUMO
Incorporation of photoresponsive molecules within soft materials can provide spatiotemporal control over bulk properties and address challenges in targeted delivery and mechanical variability. However, the kinetics of in situ photochemical reactions are often slow and typically employ ultraviolet wavelengths. Here, we present a novel photoactive crosslinker Ru(bipyridine)2 (3-pyridinaldehyde)2 (RuAldehyde), which was reacted with hydrazide-functionalized hyaluronic acid to form hydrogels capable of encapsulating protein cargo. Visible light irradiation (400-500â nm) initiated rapid ligand exchange on the ruthenium center, which degraded the hydrogel within seconds to minutes, depending on gel thickness. An exemplar enzyme cargo, TEM1 ß-lactamase, was loaded into and photoreleased from the Ru-hydrogel. To expand their applications, Ru-hydrogels were also processed into microgels using a microfluidic platform.
RESUMO
Protein cage self-assembly enables encapsulation and sequestration of small molecules, macromolecules, and nanomaterials for many applications in bionanotechnology. Notably, wild-type thermophilic ferritin from Archaeoglobus fulgidus (AfFtn) exists as a stable dimer of four-helix bundle proteins at a low ionic strength, and the protein forms a hollow assembly of 24 protomers at a high ionic strength (â¼800 mM NaCl). This assembly process can also be initiated by highly charged gold nanoparticles (AuNPs) in solution, leading to encapsulation. These data suggest that salt solutions or charged AuNPs can shield unfavorable electrostatic interactions at AfFtn dimer-dimer interfaces, but specific "hot-spot" residues controlling assembly have not been identified. To investigate this further, we computationally designed three AfFtn mutants (E65R, D138K, and A127R) that introduce a single positive charge at sites along the dimer-dimer interface. These proteins exhibited different assembly kinetics and thermodynamics, which were ranked in order of increasing 24mer propensity: A127R < wild type < D138K ⪠E65R. E65R assembled into the 24mer across a wide range of ionic strengths (0-800 mM NaCl), and the dissociation temperature for the 24mer was 98 °C. X-ray crystal structure analysis of the E65R mutant identified a more compact, closed-pore cage geometry. A127R and D138K mutants exhibited wild-type ability to encapsulate and stabilize 5 nm AuNPs, whereas E65R did not encapsulate AuNPs at the same high yields. This work illustrates designed protein cages with distinct assembly and encapsulation properties.
Assuntos
Proteínas Arqueais/química , Archaeoglobus fulgidus/química , Ferritinas/química , Proteínas Arqueais/genética , Archaeoglobus fulgidus/genética , Cristalografia por Raios X , Composição de Medicamentos , Ferritinas/genética , Ouro/química , Nanopartículas Metálicas/química , Modelos Moleculares , Mutação Puntual , Multimerização Proteica , Eletricidade Estática , TermodinâmicaRESUMO
Transcriptome profiling of single cells resident in their natural microenvironment depends upon RNA capture methods that are both noninvasive and spatially precise. We engineered a transcriptome in vivo analysis (TIVA) tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA tag in combination with RNA sequencing (RNA-seq), we analyzed transcriptome variance among single neurons in culture and in mouse and human tissue in vivo. Our data showed that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology is, to our knowledge, the first noninvasive approach for capturing mRNA from live single cells in their natural microenvironment.