RESUMO
Synthetic high-density lipoprotein (HDL) mimics have emerged as promising therapeutic agents. However, approaches to date have been unable to reproduce key features of spherical HDLs, which are the most abundant human HDL species. Here, we report the synthesis and characterization of spherical HDL mimics using lipid-conjugated organic core scaffolds. The core design motif constrains and orients phospholipid geometry to facilitate the assembly of soft-core nanoparticles that are approximately 10 nm in diameter and resemble human HDLs in their size, shape, surface chemistry, composition, and protein secondary structure. These particles execute salient HDL functions, including efflux of cholesterol from macrophages, cholesterol delivery to hepatocytes, support lecithin:cholesterol acyltransferase activity, and suppress inflammation. These results represent a significant step toward a genuine functional mimic of human HDLs.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Materiais Biomiméticos/química , Portadores de Fármacos/química , Nanopartículas Metálicas/química , Fosfatidiletanolaminas/química , Materiais Biomiméticos/síntese química , Colesterol/metabolismo , DNA/química , Portadores de Fármacos/síntese química , Ouro/química , Células Hep G2 , Humanos , Inflamação/tratamento farmacológico , Lipoproteínas HDL/química , Lipossomos/química , Monócitos/metabolismo , Subunidade p50 de NF-kappa B/metabolismoRESUMO
Complementary tetrahedral small molecule-DNA hybrid (SMDH) building blocks have been combined to form nucleic acid-based polymeric nanoparticles without the need for an underlying template or scaffold. The sizes of these particles can be tailored in a facile fashion by adjusting assembly conditions such as SMDH concentration, assembly time, and NaCl concentration. Notably, these novel particles can be stabilized and transformed into functionalized spherical nucleic acid (SNA) structures through the incorporation of capping DNA strands conjugated with functional groups. These results demonstrate a systematic, efficient strategy for the construction and surface functionalization of well-defined, size-tunable nucleic acid particles from readily accessible molecular building blocks. Furthermore, because these nucleic acid-based polymeric nanoparticles exhibited enhanced cellular internalization and resistance to DNase I compared to free synthetic nucleic acids, they should have a plethora of applications in diagnostics and therapeutics.
Assuntos
DNA/química , Nanopartículas/química , Sequência de Bases , Linhagem Celular , Permeabilidade da Membrana Celular , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Humanos , Modelos Moleculares , Nanopartículas/metabolismo , Nanopartículas/ultraestrutura , Nanotecnologia , Conformação de Ácido NucleicoRESUMO
Two complementary small-molecule-DNA hybrid (SMDH) building blocks have been combined to form well-defined supramolecular cage dimers at DNA concentrations as high as 102 µM. This was made possible by combining a flexible small-molecule core and three DNA arms of moderate lengths (<20 base pairs). These results were successfully modeled by coarse-grained molecular dynamics simulations, which also revealed that the formation of ill-defined networks in the case of longer DNA arms can be significantly biased by the presence of deep kinetic traps. Notably, melting point studies revealed that cooperative melting behavior can be used as a means to distinguish the relative propensities for dimer versus network formation from complementary flexible three-DNA-arm SMDH (fSMDH3) components: sharp, enhanced melting transitions were observed for assemblies that result mostly in cage dimers, while no cooperative melting behavior was observed for assemblies that form ill-defined networks.
Assuntos
DNA/química , Dimerização , Simulação de Dinâmica Molecular , Eletroforese em Gel de Poliacrilamida NativaRESUMO
An acid-degradable polymer-caged lipoplex (PCL) platform consisting of a cationic lipoplex core and a biocompatible, pH-responsive polymer shell has been developed for the effective delivery of small interfering RNA (siRNA) through a combination of facile loading, rapid acid-triggered release, cellular internalization, and effective endosomal escape. In vitro testing of this degradable PCL delivery platform reveals â¼45- and â¼2.5-fold enhancement of enhanced green fluorescent protein knockdown in cancer cells in comparison to either free siRNA or siRNA-loaded non-acid-degradable lipoplex formulations, respectively.
Assuntos
Preparações de Ação Retardada/química , RNA Interferente Pequeno/administração & dosagem , Linhagem Celular Tumoral , Preparações de Ação Retardada/metabolismo , Endossomos/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Concentração de Íons de Hidrogênio , Polímeros/química , Polímeros/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
All eukaryotic cells require a minimal iron threshold to sustain anabolic metabolism. However, the mechanisms by which cells sense iron to regulate anabolic processes are unclear. Here we report a previously undescribed eukaryotic pathway for iron sensing in which molecular iron is required to sustain active histone demethylation and maintain the expression of critical components of the pro-anabolic mTORC1 pathway. Specifically, we identify the iron-binding histone-demethylase KDM3B as an intrinsic iron sensor that regulates mTORC1 activity by demethylating H3K9me2 at enhancers of a high-affinity leucine transporter, LAT3, and RPTOR. By directly suppressing leucine availability and RAPTOR levels, iron deficiency supersedes other nutrient inputs into mTORC1. This process occurs in vivo and is not an indirect effect by canonical iron-utilizing pathways. Because ancestral eukaryotes share homologues of KDMs and mTORC1 core components, this pathway probably pre-dated the emergence of the other kingdom-specific nutrient sensors for mTORC1.
Assuntos
Histonas , Transdução de Sinais , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Leucina/metabolismo , Histonas/genética , Histonas/metabolismo , Ferro/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , DesmetilaçãoRESUMO
As a platform for "turn-on" DNA sensing, the level of oxidation of graphene oxide strongly affects its fluorescence quenching ability and binding interactions to single-stranded oligodeoxyribonucleotides (ssODNs), leading to a broad range of sensitivity. Fine-tuning the level of oxidation of graphene oxide yields a DNA-detection platform that is highly sensitive in serum and biological media.
Assuntos
Técnicas Biossensoriais/métodos , DNA/análise , Fluorescência , Grafite/química , Espectroscopia de Ressonância Magnética , Microscopia de Força Atômica , Nanopartículas/química , Nanopartículas/ultraestrutura , Oligodesoxirribonucleotídeos/química , Compostos de Quinolínio/químicaRESUMO
A pillared-paddlewheel type metal-organic framework material featuring bodipy- and porphyrin-based struts, and capable of harvesting light across the entire visible spectrum, has been synthesized. Efficient-essentially quantitative-strut-to-strut energy transfer (antenna behavior) was observed for the well-organized donor-acceptor assembly consituting the ordered MOF structure.
Assuntos
Compostos de Boro/química , Porfirinas/química , Transferência de Energia , Luz , Modelos MolecularesRESUMO
Highly stable and stimuli/pH-responsive ultrasmall polymer-grafted nanobins (usPGNs) have been developed by grafting a small amount (10 mol %) of short (4.3 kDa) cholesterol-terminated poly(acrylic acid) (Chol-PAA) into an ultrasmall unilamellar vesicle (uSUV). The usPGNs are stable against fusion and aggregation over several weeks, exhibiting over 10-fold enhanced cargo retention in biologically relevant media at pH 7.4 in comparison with the parent uSUV template. Coarse-grained molecular dynamics (CGMD) simulations confirm that the presence of the cholesterol moiety can greatly stabilize the lipid bilayer. They also show extended PAA chain conformations that can be interpreted as causing repulsion between colloidal particles, thus stabilizing them against fusion. Notably, CGMD predicted a clustering of the Chol-PAA chains on the lipid bilayer under acidic conditions due to intra- and interchain hydrogen bonding, leading to the destabilization of local membrane areas. This explains the experimental observation that usPGNs can be triggered to release a significant amount of cargo upon acidification to pH 5. These developments put the lipid-bilayer-embedded Chol-PAA in stark contrast with traditional poly(acrylic acid) systems where the molar mass (Mn) of the polymer chains must exceed 16.5 kDa to achieve stimuli-responsive changes in conformation. They also distinguish the small usPGNs from the much-larger polymer-caged nanobin platform where the Chol-PAA chains must be covalently cross-linked to engender stimuli-responsive behaviors.
RESUMO
High throughput analysis of DNA in low concentration and small volume is an important issue and a continuing challenge in the field of DNA microarray and sensor. Recently, we have demonstrated that the DNA microarray on nano-scale controlled surface provides ample space for hybridization resulting in the best discrimination efficiency for SNP analysis. Here, we report the utility of the nano-scale controlled surface in conjunction with a multiply tagged protein. Application of streptavidin-fluorophore conjugates in combination with the highly controlled surface that suppresses non-specific binding of DNA allows highly sensitive detection of DNA while maintaining superior SNP discrimination efficiency comparable to our earlier results. The sensitivity of DNA microarray on the mesospaced surface is two orders of magnitude higher than that of the generic surface when a streptavidin-fluorophore conjugate was employed, and the detection limit on the former surface was found to be 50 fM of 15-mer target DNA. Various streptavidin-fluorophore conjugates including streptavidin-Cy3, streptavidin-Cy5, streptavidin-Alexa Flour 555 and streptavidin-phycoerythrin were examined.
Assuntos
Corantes Fluorescentes , Nanotecnologia , Análise de Sequência com Séries de Oligonucleotídeos , Estreptavidina , Antracenos , Sensibilidade e EspecificidadeRESUMO
We have developed new surface to ensure a proper spacing between immobilized biomolecules. While DNA microarray on this surface provided each probe DNA with ample space for hybridization with incoming target DNAs, the microarray showed enhanced discrimination efficiency for various types of single nucleotide polymorphism. The high discrimination efficiency holds for all tested cases (100:<1 for internal mismatched cases; 100:<28 for terminal mismatched ones). In addition, by investigating influence of hybridization temperature and washing condition on the fluorescence intensity and the discrimination efficiency with and without controlled mesospacing, it was observed that the nanoscale-controlled surface showed good discrimination efficiency in a wide range of temperature (37-50 degrees C), and hybridization behavior on the surface was in agreement with the solution one. Intriguingly, it was found that washing process after the hybridization was critical for the high discrimination efficiency. For the particular case, washing process was so efficient that only 30 s washing was sufficient to reach the optimal discrimination ratio.
Assuntos
Nanotecnologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polimorfismo de Nucleotídeo Único , Antracenos/química , Pareamento Incorreto de Bases , Sondas de Oligonucleotídeos/química , Propriedades de Superfície , TemperaturaRESUMO
Selectivity and sensitivity in the detection of single nucleotide polymorphisms (SNPs) are among most important attributes to determine the performance of DNA microarrays. We previously reported the generation of a novel mesospaced surface prepared by applying dendron molecules on the solid surface. DNA microarrays that were fabricated on the dendron-modified surface exhibited outstanding performance for the detection of single nucleotide variation in the synthetic oligonucleotide DNA. DNA microarrays on the dendron-modified surface were subjected to the detection of single nucleotide variations in the exons 5-8 of the p53 gene in genomic DNAs from cancer cell lines. DNA microarrays on the dendron-modified surface clearly discriminated single nucleotide variations in hotspot codons with high selectivity and sensitivity. The ratio between the fluorescence intensity of perfectly matched duplexes and that of single nucleotide mismatched duplexes was >5-100 without sacrificing signal intensity. Our results showed that the outstanding performance of DNA microarrays fabricated on the dendron-modified surface is strongly related to novel properties of the dendron molecule, which has the conical structure allowing mesospacing between the capture probes. Our microarrays on the dendron-modified surface can reduce the steric hindrance not only between the solid surface and target DNA, but also among immobilized capture probes enabling the hybridization process on the surface to be very effective. Our DNA microarrays on the dendron-modified surface could be applied to various analyses that require accurate detection of SNPs.
Assuntos
Antracenos/química , Genes p53 , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polimorfismo de Nucleotídeo Único , Linhagem Celular Tumoral , Sondas de DNA/química , Fluorescência , Heterozigoto , HumanosRESUMO
Since we observed that dendron-assembled surface provided high single nucleotide polymorphism discrimination efficiency for DNA microarrays, and that the binding yield for streptavidin increased when biotin was immobilized on top of it, the nanoscale-controlled surface is examined for surface plasmon field-enhanced fluorescence spectroscopy (or SPFS). Firstly, a silica film was coated onto a gold substrate using the sol-gel technique, followed by the covalent immobilization of a layer of second-generation dendrons with a DNA catcher strand at their apex. The thickness of the inorganic interlayer (d=33 nm) was effectively suppressing fluorescence quenching. Thus, the kinetics and affinity characteristics of DNA hybridization could be investigated very sensitively by SPFS. The kinetic rate constants found for DNA hybridization on the dendron-modified surface were larger than those reported for a streptavidin-modified surface by one order of magnitude, except for dissociation rate constant for a single mismatched case. In addition, we observed that the DNA on the cone-shaped linker maintained its capability to capture DNA target strands even after extended storage at ambient conditions.
Assuntos
DNA/química , Dendrímeros/química , Dióxido de Silício/química , Espectrometria de Fluorescência/métodos , Ressonância de Plasmônio de Superfície/métodos , Géis , Cinética , Hibridização de Ácido NucleicoRESUMO
The nanoassembly behavior of trivalent small molecule-DNA hybrids (SMDH3s) was investigated as a function of core geometry and supramolecular flexibility through a synergistic experimental-modeling study. While complementary SMDH3s possessing a highly flexible tetrahedral trivalent core primarily assemble into nanoscale caged dimers, the nanoassemblies of SMDH3 comonomers with rigid pyramidal and trigonal cores yield fewer caged dimers and more large-oligomer networks. Specifically, the rigid pyramidal SMDH3 comonomers tend to form smaller nanosized aggregates (dimers, tetramers, and hexamers) upon assembly, attributable to the small (<109°) branch-core-branch angle of the pyramidal core. In contrast, the more-rigid trigonal planar SMDH3 comonomers have a larger (â¼120°) branch-core-branch angle, which spaces their DNA arms farther apart, facilitating the formation of larger nanoassemblies (≥nonamers). The population distributions of these nanoassemblies were successfully captured by coarse-grained molecular dynamics (CGMD) simulations over a broad range of DNA concentrations. CGMD simulations can also forecast the effect of incorporating Tn spacer units between the hydridizing DNA arms and the rigid organic cores to increase the overall flexibility of the SMDH3 comonomers. Such "decoupling" of the DNA arms from the organic core was found to result in preferential formation of nanoscale dimers up to an optimal spacer length, beyond which network formation takes over due to entropic factors. This excellent agreement between the simulation and experimental results confirms the versatility of the CGMD model as a useful and reliable tool for elucidating the nanoassembly of SMDH-based building blocks.
Assuntos
DNA/química , Simulação de Dinâmica Molecular , Nanoestruturas , PolímerosRESUMO
Microarrays of biomolecules are emerging as powerful tools for genomics, proteomics, and clinical assays, since they make it possible to screen biologically important binding events in a parallel and high throughput fashion. Because the microarrays are fabricated on a solid support, coating of the surface and immobilization strategy of the biomolecules are major issues for successful microarray fabrication. This review deals with both DNA microarrays and protein microarrays, and focuses on the various modification approaches for the two-dimensional surface materials and three-dimensional ones. In addition, the immobilization strategies including adsorption, covalent attachment, physical entrapment, and affinity attachment of the biomolecules are summarized, and advantage and limitation of representative efforts are discussed.
Assuntos
Manufaturas , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise Serial de Proteínas/instrumentação , Propriedades de SuperfícieRESUMO
A PCN theranostic platform comprises a doxorubicin (DXR)-loaded liposomal core and an acid-sensitive polymer shell that is functionalized with Herceptin and GdIII-based MRI contrast agents. In vitro testing reveals a 14-fold increase in DXR-based cytotoxicity versus a non-targeted analogue and an 120-fold improvement in cellular GdIII-uptake in comparison with clinically approved DOTA-GdIII, leading to significant T1 MRI contrast enhancement.
RESUMO
Aqueous dispersions of graphene oxide are inherently unstable in the presence of electrolytes, which screen the electrostatic surface charge on these nanosheets and induce irreversible aggregation. Two complementary strategies, utilizing either electrostatic or steric stabilization, have been developed to enhance the stability of graphene oxide in electrolyte solutions, allowing it to stay dispersed in cell culture media and serum. The electrostatic stabilization approach entails further oxidation of graphene oxide to low C/O ratio (~1.1) and increases ionic tolerance of these nanosheets. The steric stabilization technique employs an amphiphilic block copolymer that serves as a noncovalently bound surfactant to minimize the aggregate-inducing nanosheet-nanosheet interactions. Both strategies can stabilize graphene oxide nanosheets with large dimensions (>300 nm) in biological media, allowing for an enhancement of >250% in the bioconjugation efficiency of streptavidin in comparison to untreated nanosheets. Notably, both strategies allow the stabilized nanosheets to be readily taken up by cells, demonstrating their excellent performance as potential drug-delivery vehicles.
Assuntos
Materiais Biocompatíveis/química , Portadores de Fármacos/química , Grafite/química , Nanocápsulas/química , Nanocápsulas/ultraestrutura , Óxidos/química , Difusão , Estabilidade de Medicamentos , Eletrólitos/química , Células HeLa , Humanos , Teste de Materiais , SoluçõesRESUMO
Enzymatic on-chip DNA polymerization can be utilized to elongate surface-bound primers with DNA polymerase and to enhance the signal in the detection of target DNAs on the solid support. In order to investigate the steric effect of the enzymatic reaction on the solid support, we compared the efficiency of on-chip DNA polymerization on a high-density surface with that on a spacing-controlled surface. The spacing-controlled, 9-acid dendron-coated surface exhibited approximately 8-fold higher efficiency of on-chip DNA polymerization compared with the high-density surface. The increase in fluorescence intensity during the on-chip DNA polymerization could be fit to an exponential equation, and the saturation level of the 9-acid dendron slide was 7 times higher than that of the high-density slide. The on-chip DNA polymerization was employed to measure the transcription level of nine genes related to epithelial-to-mesenchymal transition in hepatocellular carcinoma cells. Compared to the high-density surface, the dendron-coated surface exhibited a lower detection limit in the on-chip DNA polymerization and higher correlation with transcription levels as determined by quantitative real-time PCR. Our results suggest that control of the lateral spacing of DNA strands on the solid support should significantly enhance the accessibility of DNA polymerase and the efficiency of the on-chip DNA polymerization.
Assuntos
Materiais Revestidos Biocompatíveis/química , DNA Polimerase Dirigida por DNA/química , DNA/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polímeros/química , Teste de MateriaisRESUMO
Single-molecule force measurement methods have attracted increasing interest over recent years for the development of novel approaches for biomolecular screening. However, many of these developments are currently hindered by the available biomolecule surface attachment methods, in that it is still not trivial to create surfaces and devices with highly defined surface functionality and/or uniformity. Here we offer a new approach to address such issues based on the formation of dendron arrays. Through the measurement of forces between dendron surfaces functionalized with complementary DNA oligonucleotides, we observed several unique properties of the surfaces modified via this approach. The capability to record attractive or "jump-in" forces associated with molecular binding events is one of them. Additionally, these events occur in greater than 80% of measurements, and the forces are dependent on the number of complementary DNA bases of the associating strands while being insensitive to the measurement rate. Combined with a narrow distribution of both attractive forces and unbinding forces we suggest such functionalized surfaces offer a significant advance for fast and accurate force-based studies of oligonucleotide hybridization.
Assuntos
Antracenos/química , Técnicas Biossensoriais/métodos , DNA/análise , Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Pareamento de Bases , Sequência de Bases , DNA/química , DNA Complementar/química , Microscopia de Força Atômica , Conformação de Ácido NucleicoRESUMO
We have prepared solid substrates modified with a cone-shaped dendron that generates mesospacing (3.2 nm on average) on the surface. This nanoscale-controlled surface provided an ideal DNA microarray in which each probe DNA strand was given ample space for the incoming target DNA, resulting in selectivity as high as that in solution (100: < 1). In addition, high hybridization yield confirms that DNA probes on the mesospaced surface are sterically unhindered for the hybridization.