RESUMO
The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Proteínas Serina-Treonina Quinases , Camundongos , Humanos , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citocinas , Inflamação/tratamento farmacológico , Isoformas de Proteínas , Anti-Inflamatórios/farmacologia , Imunidade Inata , Fatores de TranscriçãoRESUMO
Controlling excitonic energy transfer at the molecular level is a key requirement for transitioning nanophotonics research to viable devices with the main inspiration coming from biological light-harvesting antennas that collect and direct light energy with near-unity efficiency using Förster resonance energy transfer (FRET). Among putative FRET processes, point-to-plane FRET between donors and acceptors arrayed in two-dimensional sheets is predicted to be particularly efficient with a theoretical 1/r4 energy transfer distance (r) dependency versus the 1/r6 dependency seen for a single donor-acceptor interaction. However, quantitative validation has been confounded by a lack of robust experimental approaches that can rigidly place dyes in the required nanoscale arrangements. To create such assemblies, we utilize a DNA brick scaffold, referred to as a DNA block, which incorporates up to five two-dimensional planes with each displaying from 1 to 12 copies of five different donor, acceptor, or intermediary relay dyes. Nanostructure characterization along with steady-state and time-resolved spectroscopic data were combined with molecular dynamics modeling and detailed numerical simulations to compare the energy transfer efficiencies observed in the experimental DNA block assemblies to theoretical expectations. Overall, we demonstrate clear signatures of sheet regime FRET, and from this we provide a better understanding of what is needed to realize the benefits of such energy transfer in artificial dye networks along with FRET-based sensing and imaging.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Nanoestruturas , Corantes , DNA , Análise EspectralRESUMO
Because of its attractive cost and yield, hierarchical assembly, in which constituent structures of lower hierarchy share a majority of components, is an appealing approach to scale up DNA self-assembly. A few strategies have already been investigated to combine preformed DNA nanostructures. In this study, we present a new hierarchical assembly method based on four-way toehold-mediated strand displacement to facilitate the combination of preformed DNA structural units. Employing such a method, we have constructed a series of higher-order structures composed of 5, 7, 9, 11, 13, and 15 preformed units respectively.
RESUMO
Nucleic acids (DNA and RNA) are widely used to construct nanometre-scale structures with ever increasing complexity, with possible application in fields such as structural biology, biophysics, synthetic biology and photonics. The nanostructures are formed through one-pot self-assembly, with early kilodalton-scale examples containing typically tens of unique DNA strands. The introduction of DNA origami, which uses many staple strands to fold one long scaffold strand into a desired structure, has provided access to megadalton-scale nanostructures that contain hundreds of unique DNA strands. Even larger DNA origami structures are possible, but manufacturing and manipulating an increasingly long scaffold strand remains a challenge. An alternative and more readily scalable approach involves the assembly of DNA bricks, which each consist of four short binding domains arranged so that the bricks can interlock. This approach does not require a scaffold; instead, the short DNA brick strands self-assemble according to specific inter-brick interactions. First-generation bricks used to create three-dimensional structures are 32 nucleotides long, consisting of four eight-nucleotide binding domains. Protocols have been designed to direct the assembly of hundreds of distinct bricks into well formed structures, but attempts to create larger structures have encountered practical challenges and had limited success. Here we show that DNA bricks with longer, 13-nucleotide binding domains make it possible to self-assemble 0.1-1-gigadalton, three-dimensional nanostructures from tens of thousands of unique components, including a 0.5-gigadalton cuboid containing about 30,000 unique bricks and a 1-gigadalton rotationally symmetric tetramer. We also assembled a cuboid that contains around 10,000 bricks and about 20,000 uniquely addressable, 13-base-pair 'voxels' that serves as a molecular canvas for three-dimensional sculpting. Complex, user-prescribed, three-dimensional cavities can be produced within this molecular canvas, enabling the creation of shapes such as letters, a helicoid and a teddy bear. We anticipate that with further optimization of structure design, strand synthesis and assembly procedure even larger structures could be accessible, which could be useful for applications such as positioning functional components.
Assuntos
Algoritmos , DNA/química , DNA/síntese química , Nanoestruturas/química , Nanotecnologia , Conformação de Ácido Nucleico , Animais , Tomografia com Microscopia Eletrônica , Imageamento Tridimensional , Nucleotídeos/química , Rotação , Análise de Sequência de DNA , UrsidaeRESUMO
Fluorescence microscopy allows specific target detection down to the level of single molecules and has become an enabling tool in biological research. To transduce the biological information to an imageable signal, we have developed a variety of fluorescent probes, such as organic dyes or fluorescent proteins with different colors. Despite their success, a limitation on constructing small fluorescent probes is the lack of a general framework to achieve precise and programmable control of critical optical properties, such as color and brightness. To address this challenge, we introduce metafluorophores, which are constructed as DNA nanostructure-based fluorescent probes with digitally tunable optical properties. Each metafluorophore is composed of multiple organic fluorophores, organized in a spatially controlled fashion in a compact sub-100-nm architecture using a DNA nanostructure scaffold. Using DNA origami with a size of 90 × 60 nm2, substantially smaller than the optical diffraction limit, we constructed small fluorescent probes with digitally tunable brightness, color, and photostability and demonstrated a palette of 124 virtual colors. Using these probes as fluorescent barcodes, we implemented an assay for multiplexed quantification of nucleic acids. Additionally, we demonstrated the triggered in situ self-assembly of fluorescent DNA nanostructures with prescribed brightness upon initial hybridization to a nucleic acid target.
Assuntos
DNA/química , Corantes Fluorescentes/química , Nanoestruturas/química , Fenômenos Biofísicos , Microscopia de Fluorescência , Nanotecnologia , Conformação de Ácido Nucleico , Ácidos Nucleicos/químicaRESUMO
Collections of DNA sequences can be rationally designed to self-assemble into predictable three-dimensional structures. The geometric and functional diversity of DNA nanostructures created to date has been enhanced by improvements in DNA synthesis and computational design. However, existing methods for structure characterization typically image the final product or laboriously determine the presence of individual, labelled strands using gel electrophoresis. Here we introduce a new method of structure characterization that uses barcode extension and next-generation DNA sequencing to quantitatively measure the incorporation of every strand into a DNA nanostructure. By quantifying the relative abundances of distinct DNA species in product and monomer bands, we can study the influence of geometry and sequence on assembly. We have tested our method using 2D and 3D DNA brick and DNA origami structures. Our method is general and should be extensible to a wide variety of DNA nanostructures.
Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA/ultraestrutura , Nanoestruturas/ultraestrutura , Nanotecnologia/métodos , Sequência de Bases , DNA/química , Eletroforese em Gel de Ágar , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Nanoestruturas/química , Conformação de Ácido Nucleico , Análise de Sequência de DNARESUMO
DNA nanostructures are a useful technology for precisely organizing and manipulating nanomaterials. The DNA bricks method is a modular and versatile platform for applications requiring discrete or periodic structures with complex three-dimensional features. Here, we describe how structures are designed from the fundamental strand architecture through assembly and characterization of the formed structures.
Assuntos
DNA/química , DNA/genética , Nanoestruturas/química , Nanotecnologia/métodos , Conformação de Ácido NucleicoRESUMO
A small and compact DNA cube with zeptoliter volume is constructed by means of a generalized DNA brick concept using short synthetic oligonucleotides with varying lengths. By mimicking design principles from the DNA origami technique, the DNA cube offers higher stability and assembly yields compared to other approaches. Its potential application as nanoscale fluorescent probe is demonstrated using super-resolution imaging.
Assuntos
DNA/química , DNA/ultraestrutura , Corantes Fluorescentes/síntese química , Nanopartículas/química , Nanopartículas/ultraestrutura , Tamanho da PartículaRESUMO
The ability to assemble functional materials with precise spatial arrangements is important for applications ranging from protein crystallography to photovoltaics. Here, we describe a general framework for constructing two-dimensional crystals with prescribed depths and sophisticated three-dimensional features. The crystals are self-assembled from single-stranded DNA components called DNA bricks. We demonstrate the experimental construction of DNA brick crystals that can grow to micrometre size in their lateral dimensions with precisely controlled depths up to 80 nm. They can be designed to pack DNA helices at angles parallel or perpendicular to the plane of the crystal and to display user-specified sophisticated three-dimensional nanoscale features, such as continuous or discontinuous cavities and channels.
Assuntos
DNA/química , Cristalização , DNA/ultraestrutura , Ouro/química , Nanopartículas Metálicas/química , Microscopia Eletrônica de Varredura , Conformação de Ácido NucleicoRESUMO
Nucleic acids have been used to create diverse synthetic structural and dynamic systems. Toehold-mediated strand displacement has enabled the construction of sophisticated circuits, motors, and molecular computers. Yet it remains challenging to demonstrate complex structural reconfiguration in which a structure changes from a starting shape to another arbitrarily prescribed shape. To address this challenge, we have developed a general structural-reconfiguration method that utilizes the modularly interconnected architecture of single-stranded DNA tile and brick structures. The removal of one component strand reveals a newly exposed toehold on a neighboring strand, thus enabling us to remove regions of connected component strands without the need to modify the strands with predesigned external toeholds. By using this method, we reconfigured a two-dimensional rectangular DNA canvas into diverse prescribed shapes. We also used this method to reconfigure a three-dimensional DNA cuboid.
Assuntos
DNA/química , Nanoestruturas , Conformação de Ácido Nucleico , Microscopia de Força AtômicaRESUMO
We describe a simple and robust method to construct complex three-dimensional (3D) structures by using short synthetic DNA strands that we call "DNA bricks." In one-step annealing reactions, bricks with hundreds of distinct sequences self-assemble into prescribed 3D shapes. Each 32-nucleotide brick is a modular component; it binds to four local neighbors and can be removed or added independently. Each 8-base pair interaction between bricks defines a voxel with dimensions of 2.5 by 2.5 by 2.7 nanometers, and a master brick collection defines a "molecular canvas" with dimensions of 10 by 10 by 10 voxels. By selecting subsets of bricks from this canvas, we constructed a panel of 102 distinct shapes exhibiting sophisticated surface features, as well as intricate interior cavities and tunnels.
Assuntos
Pareamento de Bases , DNA de Cadeia Simples/química , Modelos Químicos , Nanoestruturas/química , Nanotecnologia/métodos , Nucleotídeos/química , DNA de Cadeia Simples/síntese químicaRESUMO
"Humanized" mice offer a window into aspects of human physiology that are otherwise inaccessible. The best available methods for liver humanization rely on cell transplantation into immunodeficient mice with liver injury but these methods have not gained widespread use due to the duration and variability of hepatocyte repopulation. In light of the significant progress that has been achieved in clinical cell transplantation through tissue engineering, we sought to develop a humanized mouse model based on the facile and ectopic implantation of a tissue-engineered human liver. These human ectopic artificial livers (HEALs) stabilize the function of cryopreserved primary human hepatocytes through juxtacrine and paracrine signals in polymeric scaffolds. In contrast to current methods, HEALs can be efficiently established in immunocompetent mice with normal liver function. Mice transplanted with HEALs exhibit humanized liver functions persistent for weeks, including synthesis of human proteins, human drug metabolism, drug-drug interaction, and drug-induced liver injury. Here, mice with HEALs are used to predict the disproportionate metabolism and toxicity of "major" human metabolites using multiple routes of administration and monitoring. These advances may enable manufacturing of reproducible in vivo models for diverse drug development and research applications.