RESUMO
Template-directed macromolecule synthesis is a hallmark of living systems. Inspired by this natural process, several fundamentally novel mechanisms for template-directed assembly of nucleic acid analogues have been developed. Although these approaches have broad significance, including potential applications in biotechnology and implications for the origins of life, there are unresolved challenges in how to characterize in detail the complex assembly equilibria associated with dynamic templated reactions. Here we describe mechanistic studies of template-directed dynamic assembly for thioester peptide nucleic acid (tPNA), an informational polymer that responds to selection pressures under enzyme-free conditions. To overcome some of the inherent challenges of mechanistic studies of dynamic oligomers, we designed, synthesized, and implemented tPNA-DNA conjugates. The DNA primer region affords a high level of control over the location and register of the tPNA backbone in relation to the template strand. We characterized the degree and kinetics of dynamic nucleobase mismatch correction at defined backbone positions. Furthermore, we report the fidelity of dynamic assembly in tPNA as a function of position along the peptide backbone. Finally, we present theoretical studies that explore the level of fidelity that can be expected for an oligomer having a given hybridization affinity in dynamic templated reactions and provide guidance for the future development of sequence self-editing polymers and materials. As our results demonstrate, the use of molecular conjugates of constitutionally static and dynamic polymers establishes a new methodology for expediting the characterization of the complex chemical equilibria that underlie the assembly of dynamic informational polymers.
Assuntos
Ácidos Nucleicos Peptídicos/química , Moldes Genéticos , Cromatografia Líquida de Alta Pressão , DNA/química , Cinética , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/síntese química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Although the functional specificity and catalytic versatility of enzymes have been exploited in numerous settings, controlling the spatial and temporal activity of enzymes remains challenging. Here we describe an approach for programming the function of streptokinase (SK), a protein that is clinically used as a blood "clot buster" therapeutic. We show that the fibrinolytic activity resulting from the binding of SK to the plasma proenzyme plasminogen (Pg) can be effectively regulated (turned "OFF" and "ON") by installing an intrasteric regulatory feature using a DNA-linked protease inhibitor modification. We describe the design rationale, synthetic approach, and functional characterization of two generations of intrasterically regulated SK-Pg constructs and demonstrate dose-dependent and sequence-specific temporal control in fibrinolytic activity in response to short predesignated DNA inputs. The studies described establish the feasibility of a new enzyme-programming approach and serves as a step toward advancing a new generation of programmable enzyme therapeutics.
Assuntos
DNA/farmacologia , Desenho de Fármacos , Ativadores de Plasminogênio/farmacologia , Inibidores de Proteases/farmacologia , Estreptoquinase/antagonistas & inibidores , DNA/química , Humanos , Ativadores de Plasminogênio/síntese química , Ativadores de Plasminogênio/química , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Estreptoquinase/metabolismoRESUMO
We describe an isothermal, enzyme-free method to detect single nucleotide differences between oligonucleotides of close homology. The approach exploits kinetic differences in toe-hold-mediated, nucleic acid strand-displacement reactions to detect single nucleotide polymorphisms (SNPs) with essentially "digital" precision. The theoretical underpinning, experimental analyses, predictability, and accuracy of this new method are reported. We demonstrate detection of biologically relevant SNPs and single nucleotide differences in the let-7 family of microRNAs. The method is adaptable to microarray formats, as demonstrated with on-chip detection of SNP variants involved in susceptibility to the therapeutic agents abacavir, Herceptin, and simvastatin.
Assuntos
Pareamento Incorreto de Bases/genética , Técnicas Biossensoriais , Técnicas de Amplificação de Ácido Nucleico , Nucleotídeos/análise , Cinética , Nucleotídeos/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
A thermodynamic approach to peptide macrocyclization inspired by the cyclization of non-ribosomal peptide aldehydes is presented. The method provides access to structurally diverse macrocycles by exploiting the reactivity of transient macrocyclic peptide imines toward inter- and intramolecular nucleophiles. Reactions are performed in aqueous media, in the absence of side chain protecting groups, and are tolerant of all proteinogenic functional groups. Macrocyclic products bearing non-native and rigidifying structural motifs, isotopic labels, and a variety of bioorthogonal handles are prepared, along with analogues of four distinct natural products. Structural interrogation of the linear and macrocyclic peptides using variable-temperature NMR and circular dichroism suggests that preorganization of linear substrates is not a prerequisite for macrocyclization.
RESUMO
Inhibition of histone deacetylase (HDAC) enzymes has emerged as a target for development of cancer chemotherapy. Four compounds have gained approval for clinical use by the Food and Drug Administration in the US, and several are currently in clinical trials. However, none of these compounds possesses particularly good isozyme selectivity, which would be a highly desirable feature in a tool compound. Whether selective inhibition of individual HDAC isozymes will provide improved drug candidates remains to be seen. Nevertheless, it has been speculated that using macrocyclic compounds to target HDAC enzymes might hold an advantage over the use of traditional hydroxamic-acid-containing inhibitors, which rely on chelation to the conserved active-site zinc ion. Here we review the literature on macrocyclic HDAC inhibitors obtained from natural sources and on structure-activity relationship studies inspired by these molecules, as well as on efforts aimed at fully synthetic macrocyclic HDAC inhibitors.
Assuntos
Produtos Biológicos/química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Compostos Macrocíclicos/química , Sítios de Ligação , Produtos Biológicos/síntese química , Produtos Biológicos/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/química , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/metabolismo , Simulação de Acoplamento Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Relação Estrutura-AtividadeRESUMO
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that is caused by inactivating mutations in the Survival of motor neuron 1 (SMN1) gene, resulting in decreased SMN protein expression. Humans possess a paralog gene, SMN2, which contains a splicing defect in exon 7 leading to diminished expression of full-length, fully functional SMN protein. Increasing SMN2 expression has been a focus of therapeutic development for SMA. Multiple studies have reported the efficacy of histone deacetylase inhibitors (HDACi) in this regard. However, clinical trials involving HDACi have been unsatisfactory, possibly because previous efforts to identify HDACi to treat SMA have employed non-neuronal cells as the screening platform. To address this issue, we generated an SMA-patient specific, induced pluripotent stem cell (iPSC) derived neuronal cell line that contains homogenous Tuj1+neurons. We screened a small library of cyclic tetrapeptide HDACi using this SMA neuronal platform and discovered compounds that elevate SMN2 expression by an impressive twofold or higher. These candidates are also capable of forming gems intranuclearly in SMA neurons, demonstrating biological activity. Our study identifies new potential HDACi therapeutics for SMA screened using a disease-relevant SMA neuronal cellular model.
Assuntos
Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Atrofia Muscular Espinal/tratamento farmacológico , Neurônios/efeitos dos fármacos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Atrofia Muscular Espinal/genética , Neurogênese , Neurônios/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Regulação para Cima/efeitos dos fármacosRESUMO
We have observed that molecular constructs based on multiple apoA-I mimetic peptides attached to a branched scaffold display promising anti-atherosclerosis functions in vitro. Building on these promising results, we now describe chronic in vivo studies to assess anti-atherosclerotic efficacy of HDL-like nanoparticles assembled from a trimeric construct, administered over 10 weeks either ip or orally to LDL receptor-null mice. When dosed ip, the trimer-based nanolipids markedly reduced plasma LDL-cholesterol levels by 40%, unlike many other apoA-I mimetic peptides, and were substantially atheroprotective. Surprisingly, these nanoparticles were also effective when administered orally at a dose of 75 mg/kg, despite the peptide construct being composed of l-amino acids and being undetectable in the plasma. The orally administered nanoparticles reduced whole aorta lesion areas by 55% and aortic sinus lesion volumes by 71%. Reductions in plasma cholesterol were due to the loss of non-HDL lipoproteins, while plasma HDL-cholesterol levels were increased. At a 10-fold lower oral dose, the nanoparticles were marginally effective in reducing atherosclerotic lesions. Intriguingly, analogous results were obtained with nanolipids of the corresponding monomeric peptide. These nanolipid formulations provide an avenue for developing orally efficacious therapeutic agents to manage atherosclerosis.
Assuntos
Apolipoproteína A-I , Lipoproteínas HDL/química , Lipoproteínas HDL/farmacologia , Nanopartículas/química , Receptores de LDL/metabolismo , Animais , Feminino , Lipoproteínas HDL/metabolismo , Camundongos , Camundongos Knockout , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Receptores de LDL/genéticaRESUMO
The lipid bilayer membranes are Nature's dynamic structural motifs that individualize cells and keep ions, proteins, biopolymers and metabolites confined in the appropriate location. The compartmentalization and isolation of these molecules from the external media facilitate the sophisticated functions and connections between the different biological processes accomplished by living organisms. However, cells require assistance from minimal energy shortcuts for the transport of molecules across membranes so that they can interact with the exterior and regulate their internal environments. Ion channels and pores stand out from all other possible transport mechanisms due to their high selectivity and efficiency in discriminating and transporting ions or molecules across membrane barriers. Nevertheless, the complexity of these smart "membrane holes" has driven researchers to develop simpler artificial structures with comparable performance to the natural systems. As a broad range of supramolecular interactions have emerged as efficient tools for the rational design and preparation of stable 3D superstructures, these results have stimulated the creativity of chemists to design synthetic mimics of natural active macromolecules and even to develop artificial structures with functions and properties. In this Account, we highlight results from our laboratories on the construction of artificial ion channel models that exploit the self-assembly of conformationally flat cyclic peptides (CPs) into supramolecular nanotubes. Because of the straightforward synthesis of the cyclic peptide monomers and the complete control over the internal diameter and external surface properties of the resulting hollow tubular suprastructure, CPs are the optimal candidates for the fabrication of ion channels. The ion channel activity and selective transport of small molecules by these structures are examples of the great potential that cyclic peptide nanotubes show for the construction of functional artificial transmembrane transporters. Our experience to date suggests that the next steps for achieving conceptual devices with better performance and selectivity will derive from the topological control over cyclic peptide assembly and the functionalization of the lumen.
Assuntos
Canais Iônicos/química , Modelos Biológicos , Nanotubos de Peptídeos/química , Peptídeos Cíclicos/químicaRESUMO
We describe an approach for engineering peptide-lipid nanoparticles that function similarly to high-density lipoprotein (HDL). Branched, multivalent constructs, bearing multiple 23- or 16-amino-acid peptides, were designed, synthesized, and combined with phospholipids to produce nanometer-scale discoidal HDL-like particles. A variety of biophysical techniques were employed to characterize the constructs, including size exclusion chromatography, analytical ultracentrifuge sedimentation, circular dichroism, transmission electron microscopy, and fluorescence spectroscopy. The nanoparticles functioned in vitro (human and mouse plasma) and in vivo (mice) to rapidly remodel large native HDLs into small lipid-poor HDL particles, which are key acceptors of cholesterol in reverse cholesterol transport. Fluorescent labeling studies showed that the constituents of the nanoparticles readily distributed into native HDLs, such that the peptide constructs coexisted with apolipoprotein A-I (apoA-I), the main structural protein in HDLs. Importantly, nanolipid particles containing multivalent peptides promoted efficient cellular cholesterol efflux and were functionally superior to those derived from monomeric apoA-I mimetic peptides. The multivalent peptide-lipid nanoparticles were also remarkably stable toward enzymatic digestion in vitro and displayed long half-lives and desirable pharmacokinetic profiles in mice, providing a real practical advantage over previously studied linear or tandem helical peptides. Encouragingly, a two-week exploratory efficacy study in a widely used animal model for atherosclerosis research (LDLr-null mice) using nanoparticles constructed from a trimeric peptide demonstrated an exceptional 50% reduction in the plasma total cholesterol levels compared to the control group. Altogether, the studies reported here point to an attractive avenue for designing synthetic, HDL-like nanoparticles, with potential for treating atherosclerosis.
Assuntos
Lipoproteínas HDL/química , Nanopartículas/química , Peptídeos/química , Animais , Aterosclerose/tratamento farmacológico , Modelos Animais de Doenças , Humanos , Lipoproteínas HDL/sangue , Lipoproteínas HDL/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Modelos Moleculares , Estrutura Molecular , Nanopartículas/uso terapêutico , Peptídeos/sangue , Peptídeos/uso terapêutico , Receptores de LDL/deficiênciaRESUMO
An amphipathic alpha-helical peptide (C5A) derived from the membrane anchor domain of the hepatitis C virus (HCV) NS5A protein is virocidal for HCV at submicromolar concentrations in vitro. C5A prevents de novo HCV infection and suppresses ongoing infection by inactivating both extra- and intracellular infectious particles, and it is nontoxic in vitro and in vivo at doses at least 100-fold higher than required for antiviral activity. Mutational analysis indicates that C5A's amphipathic alpha-helical structure is necessary but not sufficient for its virocidal activity, which depends on its amino acid composition but not its primary sequence or chirality. In addition to HCV, C5A inhibits infection by selected flaviviruses, paramyxoviruses, and HIV. These results suggest a model in which C5A destabilizes viral membranes based on their lipid composition, offering a unique therapeutic approach to HCV and other viral infections.
Assuntos
Antivirais/farmacologia , Hepacivirus/metabolismo , Hepatite C/genética , Hepatite C/prevenção & controle , Peptídeos/farmacologia , Replicação Viral/efeitos dos fármacos , Sequência de Aminoácidos , Linhagem Celular , Dicroísmo Circular , Testes Imunológicos de Citotoxicidade , Hepacivirus/genética , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Peptídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Relação Estrutura-Atividade , Sais de Tetrazólio , TiazóisRESUMO
The gut microbiome is a malleable microbial community that can remodel in response to various factors, including diet, and contribute to the development of several chronic diseases, including atherosclerosis. We devised an in vitro screening protocol of the mouse gut microbiome to discover molecules that can selectively modify bacterial growth. This approach was used to identify cyclic D,L-α-peptides that remodeled the Western diet (WD) gut microbiome toward the low-fat-diet microbiome state. Daily oral administration of the peptides in WD-fed LDLr-/- mice reduced plasma total cholesterol levels and atherosclerotic plaques. Depletion of the microbiome with antibiotics abrogated these effects. Peptide treatment reprogrammed the microbiome transcriptome, suppressed the production of pro-inflammatory cytokines (including interleukin-6, tumor necrosis factor-α and interleukin-1ß), rebalanced levels of short-chain fatty acids and bile acids, improved gut barrier integrity and increased intestinal T regulatory cells. Directed chemical manipulation provides an additional tool for deciphering the chemical biology of the gut microbiome and might advance microbiome-targeted therapeutics.
Assuntos
Aterosclerose/microbiologia , Microbioma Gastrointestinal , Animais , Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Aterosclerose/sangue , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Biomarcadores/metabolismo , Colesterol/sangue , Dieta Ocidental , Comportamento Alimentar , Feminino , Microbioma Gastrointestinal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Fatores Imunológicos/farmacologia , Camundongos Endogâmicos C57BL , Modelos Biológicos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Receptores de LDL/metabolismo , Proteínas de Junções Íntimas/metabolismo , Transcrição GênicaRESUMO
Defined broadly, molecular translators are constructs that can convert any designated molecular input into a unique output molecule. In particular, the development of universal nucleic acid translators would be of significant practical value in view of the expanding biomedical importance of gene diagnostics. Currently, diagnostic assays for nucleic acids must be individually developed and optimized for each new sequence because inputs for one assay are sequence-specific and are therefore incompatible with any other assay designed for the detection of a different nucleic acid. However, if a desired nucleic acid sequence could be translated in vitro into a predetermined nucleic acid output for which there is already a known diagnostic assay, then that single assay could be easily adapted to detect nearly any strand. Here we investigate PCR-independent isothermal molecular translation strategies that function without the need for post-translation purification and can be implemented with commercially available components. Translation yields up to 96% are obtained in 5 min at room temperature with minimal background reaction (<1%) and with discrimination of single nucleotide polymorphisms in the input sequence. Furthermore, we apply these translators to adapt a high-gain HIV diagnostic system for high-throughput detection of hepatitis C, avian influenza (H5N1), and smallpox without making changes to the underlying assay. Finally, we show the feasibility of translating small-molecule interactions into nucleic acid outputs by demonstrating the utility of a DNA aptamer for translating adenosine into a readily detectable output DNA sequence. Additionally, equilibrium expressions are described in order to facilitate rational engineering of aptameric translators for label-free detection of any molecule that an aptamer can recognize.
Assuntos
Hepatite C/diagnóstico , Influenza Aviária/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Varíola/diagnóstico , Adenosina/genética , Animais , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Aves , DNA/análise , DNA/genética , Hepatite C/genética , Humanos , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/genética , Polimorfismo de Nucleotídeo Único , RNA Viral/análise , RNA Viral/genética , Varíola/genéticaRESUMO
Histone deacetylases (HDACs) are a family of enzymes found in bacteria, fungi, plants, and animals that profoundly affect cellular function by catalyzing the removal of acetyl groups from -N-acetylated lysine residues of various protein substrates including histones, transcription factors, alpha-tubulin, and nuclear importers. Although the precise roles of HDAC isoforms in cellular function are not yet completely understood, inhibition of HDAC activity has emerged as a promising approach for reversing the aberrant epigenetic states associated with cancer and other chronic diseases. Potent new isoform-selective HDAC inhibitors would therefore help expand our understanding of the HDAC enzymes and represent attractive lead compounds for drug design, especially if combined with high-resolution structural analyses of such inhibitors to shed light on the three-dimensional pharmacophoric features necessary for the future design of more potent and selective compounds. Here we present structural and functional analyses of a series of beta-amino-acid-containing HDAC inhibitors inspired by cyclic tetrapeptide natural products. To survey a diverse ensemble of pharmacophoric configurations, we systematically varied the position of the beta-amino acid, amino acid chirality, functionalization of the Zn(2+)-coordinating amino acid side chain, and alkylation of the backbone amide nitrogen atoms around the macrocycle. In many cases, the compounds were a single conformation in solution and exhibited potent activities against a number of HDAC isoforms as well as effective antiproliferative and cytotoxic activities against human tumor cells. High-resolution NMR solution structures were determined for a selection of the inhibitors, providing a useful means of correlating detailed structural information with potency. The structure-based approach described here is expected to furnish valuable insights toward the future design of more selective HDAC inhibitors.
Assuntos
Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Inibidores de Histona Desacetilases/síntese química , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Isoenzimas , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/síntese química , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
We report the design, synthesis, membrane activity, biophysical characterization, and in vitro antibacterial activities of cationic cyclic D,L-alpha-glycopeptides bearing d-glucosamine (GlcNH(2)), D-galactose (Gal), or D-mannose (Man) glycosyl side chains.
Assuntos
Antibacterianos/química , Glicopeptídeos/química , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Animais , Antibacterianos/síntese química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Eritrócitos/efeitos dos fármacos , Glicopeptídeos/síntese química , Glicopeptídeos/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Células NIH 3T3 , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , EstereoisomerismoRESUMO
We describe the preparation and characterization of polythioesters composed of alternating alpha-amino acid and alpha-thioglycolic acid residues that undergo dynamic constitutional exchange under mild conditions. The polymers are assembled via reversible ring-opening polymerizations of 1,4-thiazine-2,5-diones and related monomers in solution-phase conditions that do not require the use of transition metal catalysts. Because 1,4-thiazine-2,5-diones can be derived in part from alpha-amino acids, a variety of side chain functionalized monomers in optically pure forms could readily be accessed. In addition, the resulting polythioesters have the potential for intra- and inter-chain hydrogen bonding, which is known to impart materials properties to other previously studied polyamides. The studies reported here could be useful in advancing a new class of biodegradable polymers and furthermore suggest that dynamic constitutional exchange could be exploited to modify many known synthetic and natural polythioesters.
Assuntos
Ésteres/química , Polímeros/química , Tiazinas/química , Polímeros/síntese químicaRESUMO
Fooling enzymes with mock amides: Analogues of apicidin, a cyclic-tetrapeptide inhibitor of histone deacetylase (HDAC), were designed with a 1,4- or 1,5-disubstituted 1,2,3-triazole in place of a backbone amide bond to fix the bond in question in either a trans-like or a cis-like configuration. Thus, the binding affinity of distinct peptide conformations (see picture) could be probed. One analogue proved in some cases to be superior to apicidin as an HDAC inhibitor.
Assuntos
Inibidores Enzimáticos/química , Inibidores de Histona Desacetilases , Peptídeos Cíclicos/química , Triazóis/química , Histona Desacetilases/metabolismo , Conformação Proteica , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A would-be amide: A 1,4-disubstituted 1,2,3-triazole was used as a surrogate for a trans amide bond to create a library of 16 diastereomeric pseudotetrapeptides as beta-turn mimetics. High-resolution structural analysis indicated that these scaffolds adopt distinct, rigid, conformationally homogeneous beta-turn-like structures (see example), some of which bind somatostatin receptor subtypes selectively, and some of which show broad-spectrum activity.
Assuntos
Desenho de Fármacos , Compostos Heterocíclicos/química , Oligopeptídeos/química , Receptores de Somatostatina/química , Somatostatina/análogos & derivados , Cristalografia por Raios X , Compostos Heterocíclicos/síntese química , Humanos , Conformação Molecular , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Receptores de Somatostatina/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The ability to monitor DNA polymerase activity with single-nucleotide resolution has been the cornerstone of a number of advanced single-molecule DNA sequencing concepts. Toward this goal, we report the first observation of the base-by-base DNA polymerase activity with single-base resolution at the single-molecule level. We describe the design and characterization of a supramolecular nanopore device capable of detecting up to nine consecutive DNA polymerase-catalyzed single-nucleotide primer extensions with high sensitivity and spatial resolution (Assuntos
DNA de Cadeia Simples/biossíntese
, DNA Polimerase Dirigida por DNA/química
, Nanotecnologia/instrumentação
, Nanotecnologia/métodos
, Nucleotídeos/química
, Toxinas Bacterianas/química
, Catálise
, Primers do DNA/química
, DNA de Cadeia Simples/química
, Proteínas Hemolisinas/química
, Polietilenoglicóis/química
RESUMO
Biosensors fabricated on the nanoscale offer exciting new avenues in the quest for better understanding and characterization of biological systems. Porous silicon is an ideal nanostructured material for the construction of optical transducer matrices because it is easily functionalized with biomolecular probes and displays strong optical interferences. Here, we show that certain transition metal complexes, including nickel(II)cyclam, are able to induce corrosion in porous silicon films rapidly, generating a strong optical interferometric signal that originates from the porous layer. We subsequently exploit this effect to design a transducer for ligand-receptor recognition. With a nickel(II)cyclam derivative as a catalytic label, DNA- and avidin-binding events are detected by time-lapse interferometric reflectance spectroscopy in a fast, simple, and inexpensive fashion.
Assuntos
Óptica e Fotônica , Silício/química , Silício/metabolismo , Avidina/metabolismo , Sequência de Bases , Biotina/metabolismo , Catálise , Corrosão , Sondas de DNA/genética , Sondas de DNA/metabolismo , Compostos Heterocíclicos/química , Interferometria , Ligantes , Níquel/química , Oxirredução , Porosidade , Ligação Proteica , Coloração e Rotulagem , Propriedades de Superfície , Fatores de Tempo , TransdutoresRESUMO
One of the fundamental goals of molecular computing is to reproduce the tenets of digital logic, such as component modularity and hierarchical circuit design. An important step toward this goal is the creation of molecular logic gates that can be rationally wired into multi-level circuits. Here we report the design and functional characterization of a complete set of modular DNA-based Boolean logic gates (AND, OR, and AND-NOT) and further demonstrate their wiring into a three-level circuit that exhibits Boolean XOR (exclusive OR) function. The approach is based on solid-supported DNA logic gates that are designed to operate with single-stranded DNA inputs and outputs. Since the solution-phase serves as the communication medium between gates, circuit wiring can be achieved by designating the DNA output of one gate as the input to another. Solid-supported logic gates provide enhanced gate modularity versus solution-phase systems by significantly simplifying the task of choosing appropriate DNA input and output sequences used in the construction of multi-level circuits. The molecular logic gates and circuits reported here were characterized by coupling DNA outputs to a single-input REPORT gate and monitoring the resulting fluorescent output signals.