RESUMEN
Due to their capacity to conduct complex organic transformations, enzymes find extensive use in medical and industrial settings. Unfortunately, enzymes are limited by their poor stability when exposed to harsh non-native conditions. While a host of methods have been developed to stabilize enzymes in non-native conditions, recent research into the synthesis of polymer-enzyme biohybrids using reversible deactivation radical polymerization approaches has demonstrated the potential of increased enzymatic activity in both native and non-native environments. In this manuscript, we utilize the enzyme lipase, as a model system, to explore the impact that modulation of grafted polymer molecular weight has on enzyme activity in both aqueous and organic media. We studied the properties of these hybrids using both solution-phase enzyme activity methods and coarse-grain modeling to assess the impact of polymer grafting density and grafted polymer molecular weight on enzyme activity to gain a deeper insight into this understudied property of the biohybrid system.
Asunto(s)
Biocatálisis , Lipasa/química , Lipasa/metabolismo , Multimerización de Proteína , Simulación de Dinámica Molecular , Estructura Cuaternaria de Proteína , Agua/químicaRESUMEN
The targeted delivery of polymers to neurons is a challenging yet important goal for polymer based drug delivery. We prepared a fentanyl based atom transfer radical polymerization (ATRP) initiator to target the Mu opioid receptor (MOR) for neuronal targeting. We incorporated our recently discovered rigid acrylate linking group into the initiator to retain a high degree of binding to the MOR and grafted random or block copolymers of poly(oligo(ethylene oxide) methacrylate)-block-(glycidyl methacrylate). Trifluoroethanol promoted amine ring opening of the glycidyl methacrylate was used for post-polymerization modification of the fentanyl initiated polymers to attach a near-infrared fluorescent dye (ADS790WS) or to build a targeted siRNA delivery system via modification with secondary amines. We examined the biocompatibility, cellular internalization, and siRNA binding properties of our polymer library in a green fluorescent protein expressing SY SH5Y neuroblastoma cell-line.
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Sistemas de Liberación de Medicamentos/métodos , Polímeros/farmacocinética , ARN Interferente Pequeño/farmacocinética , Línea Celular Tumoral , Fentanilo , Colorantes Fluorescentes , Humanos , Neuroblastoma/diagnóstico por imagen , Neuronas/metabolismo , Polimerizacion , Polímeros/química , ARN Interferente Pequeño/metabolismo , Receptores Opioides muRESUMEN
The synthesis of a sulfoxide-based water-soluble polymer, poly(2-(methylsulfinyl)ethyl acrylate) (polyMSEA), a polymeric analogue of DMSO, by atom transfer radical polymerization (ATRP) is reported. Well-defined linear polymers were synthesized using relatively low amounts of copper catalyst (1000 or 100 ppm). Two types of star polymers were synthesized by either an "arm-first" approach or a "core-first" approach using a biodegradable ß-cyclodextrin core. The glass transition temperatures of both the linear polymer (16 °C) and star polymer (32 °C) were determined by differential scanning calorimetry (DSC). The lower critical solution temperature (LCST) of poly(MSEA) was estimated to be ca. 140 °C by extrapolating the LCST of a series of copolymers with NIPAM. Cytotoxicity tests revealed that both the linear and star polymers have low toxicity, even at concentrations up to 3 mg/mL.
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Materiales Biocompatibles/administración & dosificación , Dimetilsulfóxido/química , Polímeros/administración & dosificación , Materiales Biocompatibles/química , Rastreo Diferencial de Calorimetría , Catálisis , Supervivencia Celular/efectos de los fármacos , Células HEK293 , Humanos , Polimerizacion , Polímeros/química , Temperatura de Transición , Agua/químicaRESUMEN
The MspJI modification-dependent restriction endonuclease recognizes 5-methylcytosine or 5-hydroxymethylcytosine in the context of CNN(G/A) and cleaves both strands at fixed distances (N(12)/N(16)) away from the modified cytosine at the 3'-side. We determined the crystal structure of MspJI of Mycobacterium sp. JLS at 2.05-Å resolution. Each protein monomer harbors two domains: an N-terminal DNA-binding domain and a C-terminal endonuclease. The N-terminal domain is structurally similar to that of the eukaryotic SET and RING-associated domain, which is known to bind to a hemi-methylated CpG dinucleotide. Four protein monomers are found in the crystallographic asymmetric unit. Analytical gel-filtration and ultracentrifugation measurements confirm that the protein exists as a tetramer in solution. Two monomers form a back-to-back dimer mediated by their C-terminal endonuclease domains. Two back-to-back dimers interact to generate a tetramer with two double-stranded DNA cleavage modules. Each cleavage module contains two active sites facing each other, enabling double-strand DNA cuts. Biochemical, mutagenesis and structural characterization suggest three different monomers of the tetramer may be involved respectively in binding the modified cytosine, making the first proximal N(12) cleavage in the same strand and then the second distal N(16) cleavage in the opposite strand. Both cleavage events require binding of at least a second recognition site either in cis or in trans.
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Enzimas de Restricción del ADN/química , División del ADN , Enzimas de Restricción del ADN/metabolismo , Proteínas de Unión al ADN/química , Dimerización , Modelos Moleculares , Mycobacterium/enzimología , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
MspJI is a novel modification-dependent restriction endonuclease that cleaves at a fixed distance away from the modification site. Here, we present the biochemical characterization of several MspJI homologs, including FspEI, LpnPI, AspBHI, RlaI, and SgrTI. All of the enzymes specifically recognize cytosine C5 modification (methylation or hydroxymethylation) in DNA and cleave at a constant distance (N(12)/N(16)) away from the modified cytosine. Each displays its own sequence context preference, favoring different nucleotides flanking the modified cytosine. By cleaving on both sides of fully modified CpG sites, they allow the extraction of 32-base long fragments around the modified sites from the genomic DNA. These enzymes provide powerful tools for direct interrogation of the epigenome. For example, we show that RlaI, an enzyme that prefers (m)CWG but not (m)CpG sites, generates digestion patterns that differ between plant and mammalian genomic DNA, highlighting the difference between their epigenomic patterns. In addition, we demonstrate that deep sequencing of the digested DNA fragments generated from these enzymes provides a feasible method to map the modified sites in the genome. Altogether, the MspJI family of enzymes represent appealing tools of choice for method development in DNA epigenetic studies.
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Enzimas de Restricción del ADN , Epigénesis Genética , Epigenómica/métodos , Técnicas Genéticas , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Mapeo Cromosómico/métodos , Biología Computacional , ADN/química , ADN/genética , ADN/aislamiento & purificación , Metilación de ADN , Enzimas de Restricción del ADN/genética , Enzimas de Restricción del ADN/metabolismo , Biblioteca de Genes , Células HeLa , Humanos , Células Jurkat , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
PvuRts1I is a modification-dependent restriction endonuclease that recognizes 5-hydroxymethylcytosine (5hmC) as well as 5-glucosylhydroxymethylcytosine (5ghmC) in double-stranded DNA. Using PvuRts1I as the founding member, we define a family of homologous proteins with similar DNA modification-dependent recognition properties. At the sequence level, these proteins share a few uniquely conserved features. We show that these enzymes introduce a double-stranded cleavage at the 3'-side away from the recognized modified cytosine. The distances between the cleavage sites and the modified cytosine are fixed within a narrow range, with the majority being 11-13 nt away in the top strand and 9-10 nt away in the bottom strand. The recognition sites of these enzymes generally require two cytosines on opposite strand around the cleavage sites, i.e. 5'-CN(11-13)↓N(9-10)G-3'/3'-GN(9-10)↓N(11-13)C-5', with at least one cytosine being modified for efficient cleavage. As one potential application for these enzymes is to provide useful tools for selectively mapping 5hmC sites, we have compared the relative selectivity of a few PvuRts1I family members towards different forms of modified cytosines. Our results show that the inherently different relative selectivity towards modified cytosines can have practical implications for their application. By using AbaSDFI, a PvuRts1I homolog with the highest relative selectivity towards 5ghmC, to analyze rat brain DNA, we show it is feasible to map genomic 5hmC sites close to base resolution. Our study offers unique tools for determining more accurate hydroxymethylomes in mammalian cells.
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Citosina/análogos & derivados , Enzimas de Restricción del ADN/metabolismo , 5-Metilcitosina/análogos & derivados , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Secuencia Conservada , Citosina/análisis , Citosina/metabolismo , División del ADN , Enzimas de Restricción del ADN/química , Enzimas de Restricción del ADN/clasificación , Genómica , Datos de Secuencia Molecular , Ratas , Especificidad por SustratoRESUMEN
Mrr superfamily of homologous genes in microbial genomes restricts modified DNA in vivo. However, their biochemical properties in vitro have remained obscure. Here, we report the experimental characterization of MspJI, a remote homolog of Escherichia coli's Mrr and show it is a DNA modification-dependent restriction endonuclease. Our results suggest MspJI recognizes (m)CNNR (R = G/A) sites and cleaves DNA at fixed distances (N(12)/N(16)) away from the modified cytosine at the 3' side (or N(9)/N(13) from R). Besides 5-methylcytosine, MspJI also recognizes 5-hydroxymethylcytosine but is blocked by 5-glucosylhydroxymethylcytosine. Several other close homologs of MspJI show similar modification-dependent endonuclease activity and display substrate preferences different from MspJI. A unique feature of these modification-dependent enzymes is that they are able to extract small DNA fragments containing modified sites on genomic DNA, for example â¼32 bp around symmetrically methylated CG sites and â¼31 bp around methylated CNG sites. The digested fragments can be directly selected for high-throughput sequencing to map the location of the modification on the genomic DNA. The MspJI enzyme family, with their different recognition specificities and cleavage properties, provides a basis on which many future methods can build to decode the epigenomes of different organisms.
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Enzimas de Restricción del ADN/metabolismo , Secuencia de Aminoácidos , ADN/química , ADN/metabolismo , Enzimas de Restricción del ADN/química , Enzimas de Restricción del ADN/clasificación , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/clasificación , Genoma , Células HeLa , Humanos , Datos de Secuencia Molecular , Mycobacterium/enzimología , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Modified DNA bases are widespread in biology. 5-Methylcytosine (mC) is a predominant epigenetic marker in higher eukaryotes involved in gene regulation, development, aging, cancer, and disease. Recently, 5-hydroxymethylcytosine (hmC) was identified in mammalian brain tissue and stem cells. However, most of the currently available assays cannot distinguish mC from hmC in DNA fragments. We investigate here the physical properties of DNA with modified cytosines, in efforts to develop a physical tool that distinguishes mC from hmC in DNA fragments. Molecular dynamics simulations reveal that polar cytosine modifications affect internal base pair dynamics, while experimental evidence suggest a correlation between the modified cytosine's polarity, DNA flexibility, and duplex stability. On the basis of these physical differences, solid-state nanopores can rapidly discriminate among DNA fragments with mC or hmC modification by sampling a few hundred molecules in the solution. Further, the relative proportion of hmC in the sample can be determined from the electronic signature of the intact DNA fragment.
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5-Metilcitosina/química , Citosina/análogos & derivados , ADN/química , Animales , Citosina/química , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
The ability to manipulate the electrophysiology of electrically active cells and tissues has enabled a deeper understanding of healthy and diseased tissue states. This has primarily been achieved via input/output (I/O) bioelectronics that interface engineered materials with biological entities. Stable long-term application of conventional I/O bioelectronics advances as materials and processing techniques develop. Recent advancements have facilitated the development of graphene-based I/O bioelectronics with a wide variety of functional characteristics. Engineering the structural, physical, and chemical properties of graphene nanostructures and integration with modern microelectronics have enabled breakthrough high-density electrophysiological investigations. Here, we review recent advancements in 2D and 3D graphene-based I/O bioelectronics and highlight electrophysiological studies facilitated by these emerging platforms. Challenges and present potential breakthroughs that can be addressed via graphene bioelectronics are discussed. We emphasize the need for a multidisciplinary approach across materials science, micro-fabrication, and bioengineering to develop the next generation of I/O bioelectronics.
RESUMEN
Objective.Three-dimensional (3D) neuronal spheroid culture serves as a powerful model system for the investigation of neurological disorders and drug discovery. The success of such a model system requires techniques that enable high-resolution functional readout across the entire spheroid. Conventional microelectrode arrays and implantable neural probes cannot monitor the electrophysiology (ephys) activity across the entire native 3D geometry of the cellular construct.Approach.Here, we demonstrate a 3D self-rolled biosensor array (3D-SR-BA) integrated with a 3D cortical spheroid culture for simultaneousin vitroephys recording, functional Ca2+imaging, while monitoring the effect of drugs. We have also developed a signal processing pipeline to detect neural firings with high spatiotemporal resolution from the ephys recordings based on established spike sorting methods.Main results.The 3D-SR-BAs cortical spheroid interface provides a stable, high sensitivity recording of neural action potentials (<50µV peak-to-peak amplitude). The 3D-SR-BA is demonstrated as a potential drug screening platform through the investigation of the neural response to the excitatory neurotransmitter glutamate. Upon addition of glutamate, the neural firing rates increased notably corresponding well with the functional Ca2+imaging.Significance.Our entire system, including the 3D-SR-BA integrated with neuronal spheroid culture, enables simultaneous ephys recording and functional Ca2+imaging with high spatiotemporal resolution in conjunction with chemical stimulation. We demonstrate a powerful toolset for future studies of tissue development, disease progression, and drug testing and screening, especially when combined with native spheroid cultures directly extracted from humans.
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Técnicas Biosensibles , Esferoides Celulares , Humanos , Microelectrodos , Sistema Nervioso , NeuronasRESUMEN
Multidrug (Mdr) transporters are membrane proteins that actively export structurally dissimilar drugs from the cell, thereby rendering the cell resistant to toxic compounds. Similar to substrate-specific transporters, Mdr transporters also undergo substrate-induced conformational changes. However, the mechanism by which a variety of dissimilar substrates are able to induce similar transport-compatible conformational responses in a single transporter remains unclear. To address this major aspect of Mdr transport, we studied the conformational behavior of the Escherichia coli Mdr transporter MdfA. Our results show that indeed, different substrates induce similar conformational changes in the transporter. Intriguingly, in addition, we observed that compounds other than substrates are able to confer similar conformational changes when covalently attached at the putative Mdr recognition pocket of MdfA. Taken together, the results suggest that the Mdr-binding pocket of MdfA is conformationally sensitive. We speculate that the same conformational switch that usually drives active transport is triggered promiscuously by merely occupying the Mdr-binding site.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas de Transporte de Membrana/metabolismo , Sitios de Unión , Transporte Biológico , Membrana Celular/metabolismo , Detergentes/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Endopeptidasa K/química , Maleimidas/farmacología , Proteínas de Transporte de Membrana/química , Modelos Químicos , Conformación Molecular , Polietilenglicoles/química , Conformación Proteica , Especificidad por SustratoRESUMEN
PURPOSE: To establish a consistent and affordable, high quality porcine anterior segment perfusion and transduction model that allows direct visualization of the trabecular meshwork. METHODS: Porcine anterior segments were cultured within 2 hours of death by removing lens and uvea and securing in a specially designed petri dish with a thin bottom to allow direct visualization of the trabecular meshwork with minimal distortion. Twenty-two control eyes (CO) with a constant flow rate were compared to eight gravity perfused eyes (COgr, 15 mm Hg). We established gene delivery to the TM using eGFP expressing feline immunodeficiency virus (FIV) vector GINSIN at 108 transducing units (TU) per eye (GINSIN_8, n = 8) and 107 TU (GINSIN_7, n = 8). Expression was assessed for 14 days before histology was obtained. RESULTS: Pig eyes were a reliable source for consistent and high quality anterior segment cultures with a low failure rate of 12%. Control eyes had an intraocular pressure (IOP) of 15.8 ± 1.9 mm Hg at fixed pump perfusion with 3 µL/min compared to gravity perfused COgr with imputed 3.7 ± 1.6 µL/min. Vector GINSIN_8 eyes experienced a transient posttransduction IOP increase of 44% that resolved at 48 hours; this was not observed in GINSIN_7 eyes. Expression was higher in GINSIN_8 than in GINSIN_7 eyes. Trabecular meshwork architecture was well preserved. CONCLUSIONS: Compared with previously used human donor eyes, this inexpensive porcine anterior segment perfusion model is of sufficient, repeatable high quality to develop strategies of TM bioengineering. Trabecular meshwork could be observed directly. Despite significant anatomic differences, effects of transduction replicate the main aspects of previously explored human, feline and rodent models.
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Segmento Anterior del Ojo/metabolismo , Humor Acuoso/metabolismo , Glaucoma/terapia , Presión Intraocular , Perfusión/métodos , Malla Trabecular/patología , Animales , Segmento Anterior del Ojo/patología , Gatos , Células Cultivadas , Modelos Animales de Enfermedad , Glaucoma/metabolismo , Glaucoma/patología , Humanos , PorcinosRESUMEN
The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3' to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5' to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.
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Enzimas de Restricción del ADN/química , Enzimas de Restricción del ADN/genética , Mutagénesis , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Dominio Catalítico , ADN/química , Activación Enzimática , Variación Genética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Alineación de Secuencia , Especificidad por SustratoRESUMEN
MdfA is a prototypic secondary multidrug transporter from Escherichia coli, which recognizes and exports a broad spectrum of structurally and electrically dissimilar toxic compounds. Here we review recent studies of MdfA, which, on the one hand, provide advanced understanding of certain aspects of secondary multidrug transport, and, on the other, address major mechanistic questions, some of which remain to be elucidated. Using biochemical, genetic, and physiological approaches, we have revealed several surprisingly promiscuous properties of MdfA including its multidrug recognition capacity, proton recognition determinants, aspects of energy utilization, and physiological role.