RESUMEN
Multivalent presentation of ligands often enhances receptor activation and downstream signalling. DNA origami offers a precise nanoscale spacing of ligands, a potentially useful feature for therapeutic nanoparticles. Here we use a square-block DNA origami platform to explore the importance of the spacing of CpG oligonucleotides. CpG engages Toll-like receptors and therefore acts to activate dendritic cells. Through in vitro cell culture studies and in vivo tumour treatment models, we demonstrate that square blocks induce Th1 immune polarization when CpG is spaced at 3.5 nm. We observe that this DNA origami vaccine enhances DC activation, antigen cross-presentation, CD8 T-cell activation, Th1-polarized CD4 activation and natural-killer-cell activation. The vaccine also effectively synergizes with anti-PD-L1 for improved cancer immunotherapy in melanoma and lymphoma models and induces long-term T-cell memory. Our results suggest that DNA origami may serve as a platform for controlling adjuvant spacing and co-delivering antigens in vaccines.
Asunto(s)
Vacunas contra el Cáncer , Oligodesoxirribonucleótidos , Animales , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/inmunología , Ratones , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacología , ADN/química , ADN/inmunología , Células Dendríticas/inmunología , Humanos , Ratones Endogámicos C57BL , Islas de CpG , Vacunas de ADN/química , Vacunas de ADN/inmunología , Vacunas de ADN/farmacología , Linfocitos T CD8-positivos/inmunología , Vacunación/métodos , Línea Celular Tumoral , FemeninoRESUMEN
Controlling where and when self-assembly happens is crucial in both biological and synthetic systems as it optimizes the utilization of available resources. We previously reported strictly seed-initiated linear crisscross polymerization with alternating recruitment of single-stranded DNA slats that are aligned in a parallel versus perpendicular orientation with respect to the double-helical axes. However, for some applications, it would be advantageous to produce growth that is faster than what a linear assembly can provide. Here, we implement crisscross polymerization with alternating sets of six parallel slats versus six perpendicular slats and use this framework to explore branching behavior. We present architectures that, respectively, are designed to exhibit primary, secondary, and hyperbranching growth. Thus, amplification via nonlinear crisscross polymerization can provide a route for applications such as low-cost, enzyme-free, and ultrasensitive detection.
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ADN de Cadena Simple , PolimerizacionRESUMEN
Current SARS-CoV-2 vaccines have demonstrated robust induction of neutralizing antibodies and CD4+ T cell activation, however CD8+ responses are variable, and the duration of immunity and protection against variants are limited. Here we repurposed our DNA origami vaccine platform, DoriVac, for targeting infectious viruses, namely SARS-CoV-2, HIV, and Ebola. The DNA origami nanoparticle, conjugated with infectious-disease-specific HR2 peptides, which act as highly conserved antigens, and CpG adjuvant at precise nanoscale spacing, induced neutralizing antibodies, Th1 CD4+ T cells, and CD8+ T cells in naïve mice, with significant improvement over a bolus control. Pre-clinical studies using lymph-node-on-a-chip systems validated that DoriVac, when conjugated with antigenic peptides or proteins, induced promising cellular immune responses in human cells. These results suggest that DoriVac holds potential as a versatile, modular vaccine platform, capable of inducing both humoral and cellular immunities. The programmability of this platform underscores its potential utility in addressing future pandemics.
RESUMEN
The self-assembly of DNA-based monomers into higher-order structures has significant potential for realizing various biomimetic behaviors including algorithmic assembly, ultrasensitive detection, and self-replication. For these behaviors, it is desirable to implement high energetic barriers to undesired spurious nucleation, where such barriers can be bypassed via seed-initiated assembly. Joint-neighbor capture is a mechanism enabling the construction of such barriers while allowing for algorithmic behaviors, such as bit-copying. Cycles of polymerization with division could accordingly be used for implementing exponential growth in self-replicating materials. Previously, we demonstrated crisscross polymerization, a strategy that attains robust seed-dependent self-assembly of single-stranded DNA and DNA-origami monomers via joint-neighbor capture. Here, we expand the crisscross assembly to achieve autonomous, isothermal exponential amplification of ribbons through their concurrent growth and scission via toehold-mediated strand displacement. We demonstrate how this crisscross chain reaction, or 3CR, can be used as a detection strategy through coupling to single- and double-stranded nucleic acid targets and introduce a rule-based stochastic modeling approach for simulating molecular self-assembly behaviors such as crisscross-ribbon scission.
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Técnicas Biosensibles , ADN de Cadena Simple , ADN/química , Polimerizacion , Técnicas de Amplificación de Ácido NucleicoRESUMEN
DNA origami is a popular nanofabrication strategy that employs self-assembly of a long single scaffold strand, typically less than 10 kilobases in length, with hundreds of shorter staple strands into a desired shape. In particular, origami arranged as a single-layer rectangle has proven popular as flat pegboards that can display functionalities at staple-strand breakpoints, off the sides of the constituent double helices, with a â¼5.3 nm rhombic-lattice spacing. For applications that demand tighter spacing, functionalities can be displayed instead on the termini of helices of multilayer DNA origami. However, pegboards with the greatest addressable surface area are often found to be the most versatile. Given the practical limitations of the length of the scaffold that can be easily realized, designs that minimize the length of each helix would have advantages for maximizing the number of helices and therefore the number of addressable pixels on each terminal surface. Here we present an architecture for multilayer DNA origami displaying flush terminal interfaces from over 200 helices that each are only 5.3 turns in length. We characterize an example using cryo-EM imaging paired with single-particle analysis for further analysis of the global structure.
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ADN , Nanoestructuras , Conformación de Ácido Nucleico , ADN/química , Nanoestructuras/química , Nanotecnología/métodosRESUMEN
The ability to accurately map the 3D geometry of single-molecule complexes in trace samples is a challenging goal that would lead to new insights into molecular mechanics and provide an approach for single-molecule structural proteomics. To enable this, we have developed a high-resolution force spectroscopy method capable of measuring multiple distances between labeled sites in natively folded protein complexes. Our approach combines reconfigurable nanoscale devices, we call DNA nanoswitch calipers, with a force-based barcoding system to distinguish each measurement location. We demonstrate our approach by reconstructing the tetrahedral geometry of biotin-binding sites in natively folded streptavidin, with 1.5-2.5 Å agreement with previously reported structures.
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Biotina , Nanotecnología , Estreptavidina/química , Biotina/química , Nanotecnología/métodos , Sitios de Unión , ADNRESUMEN
The ability to accurately map the 3D geometry of single-molecule complexes in trace samples would lead to new insights into molecular mechanics and provide an approach for single-molecule structural proteomics. To enable this, we have developed a high-resolution force-spectroscopy method capable of measuring multiple distances between labeled sites in natively folded protein complexes. Our approach combines reconfigurable nanoscale devices we call DNA Nanoswitch Calipers, which we have previously introduced, with a force-based barcoding system to distinguish each measurement location. We demonstrate our approach by reconstructing the tetrahedral geometry of biotin-binding sites in natively folded streptavidin, with 1.5-2.5 Å agreement to previously reported structures.
RESUMEN
Living systems achieve robust self-assembly across a wide range of length scales. In the synthetic realm, nanofabrication strategies such as DNA origami have enabled robust self-assembly of submicron-scale shapes from a multitude of single-stranded components. To achieve greater complexity, subsequent hierarchical joining of origami can be pursued. However, erroneous and missing linkages restrict the number of unique origami that can be practically combined into a single design. Here we extend crisscross polymerization, a strategy previously demonstrated with single-stranded components, to DNA-origami 'slats' for fabrication of custom multi-micron shapes with user-defined nanoscale surface patterning. Using a library of ~2,000 strands that are combinatorially arranged to create unique DNA-origami slats, we realize finite structures composed of >1,000 uniquely addressable slats, with a mass exceeding 5 GDa, lateral dimensions of roughly 2 µm and a multitude of periodic structures. Robust production of target crisscross structures is enabled through strict control over initiation, rapid growth and minimal premature termination, and highly orthogonal binding specificities. Thus crisscross growth provides a route for prototyping and scalable production of structures integrating thousands of unique components (that is, origami slats) that each is sophisticated and molecularly precise.
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Nanoestructuras , Nanotecnología , Nanotecnología/métodos , Nanoestructuras/química , Conformación de Ácido Nucleico , ADN/químicaRESUMEN
Nanoscale stepper motors such as kinesin and dynein play a key role in numerous natural processes such as mitotic spindle formation during cell division or intracellular organelle transport. Their high efficacy in terms of operational speed and processivity has inspired the investigation of biomimetic technologies based on the use of programmable molecules. In particular, several designs of molecular walkers have been explored using DNA nanotechnology. Here, we study the actuation of a DNA-origami walker on a DNA-origami track based on three principles: 1) octapedal instead of bipedal walking for greater redundancy; 2) three pairs of orthogonal sequences, each of which fuels one repeatable stepping phase for cyclically driven motion with controlled directionality based on strain-based step selection; 3) designed size of only 3.5 nm per step on an origami track. All three principles are innovative in the sense that earlier demonstrations of steppers relied on a maximum of four legs on at least four orthogonal sequences to drive cyclic stepping, and took steps much larger than 3.4 nm in size. Using gel electrophoresis and negative-stain electron microscopy, we demonstrate cyclic actuation of DNA-origami structures through states defined by three sets of specific sequences of anchor points. However, this mechanism was not able to provide the intended control over directionality of movement. DNA-origami-based stepper motors will offer a future platform for investigating how increasing numbers of legs can be exploited to achieve robust stepping with relatively small step sizes.
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Nanoestructuras , Nanotecnología , Nanotecnología/métodos , ADN/química , Dineínas/química , Cinesinas/química , Nanoestructuras/química , Conformación de Ácido NucleicoRESUMEN
Decoding the identity of biomolecules from trace samples is a longstanding goal in the field of biotechnology. Advances in DNA analysis have substantially affected clinical practice and basic research, but corresponding developments for proteins face challenges due to their relative complexity and our inability to amplify them. Despite progress in methods such as mass spectrometry and mass cytometry, single-molecule protein identification remains a highly challenging objective. Towards this end, we combine DNA nanotechnology with single-molecule force spectroscopy to create a mechanically reconfigurable DNA nanoswitch caliper capable of measuring multiple coordinates on single biomolecules with atomic resolution. Using optical tweezers, we demonstrate absolute distance measurements with ångström-level precision for both DNA and peptides, and using multiplexed magnetic tweezers, we demonstrate quantification of relative abundance in mixed samples. Measuring distances between DNA-labelled residues, we perform single-molecule fingerprinting of synthetic and natural peptides, and show discrimination, within a heterogeneous population, between different posttranslational modifications. DNA nanoswitch calipers are a powerful and accessible tool for characterizing distances within nanoscale complexes that will enable new applications in fields such as single-molecule proteomics.
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ADN/química , Nanotecnología , Imagen Individual de Molécula , Secuencia de Aminoácidos , Calibración , Péptidos/química , Procesamiento Proteico-Postraduccional , Reproducibilidad de los Resultados , Análisis EspectralRESUMEN
Natural biomolecular assemblies such as actin filaments or microtubules can exhibit all-or-nothing polymerization in a kinetically controlled fashion. The kinetic barrier to spontaneous nucleation arises in part from positive cooperativity deriving from joint-neighbor capture, where stable capture of incoming monomers requires straddling multiple subunits on a filament end. For programmable DNA self-assembly, it is likewise desirable to suppress spontaneous nucleation to enable powerful capabilities such as all-or-nothing assembly of nanostructures larger than a single DNA origami, ultrasensitive detection, and more robust algorithmic assembly. However, existing DNA assemblies use monomers with low coordination numbers that present an effective kinetic barrier only for slow, near-reversible growth conditions. Here we introduce crisscross polymerization of elongated slat monomers that engage beyond nearest neighbors which sustains the kinetic barrier under conditions that promote fast, irreversible growth. By implementing crisscross slats as single-stranded DNA, we attain strictly seed-initiated nucleation of crisscross ribbons with distinct widths and twists.
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ADN/química , Polimerizacion , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , ADN de Cadena Simple , Cinética , Microtúbulos/metabolismoRESUMEN
DNA origami, in which a long scaffold strand is assembled with a many short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. However, currently the design and optimization of custom 3D DNA-origami shapes is a barrier to rapid application to new areas. Here we introduce a modular barrel architecture, and demonstrate hierarchical assembly of a 100 megadalton DNA-origami barrel of ~90 nm diameter and ~250 nm height, that provides a rhombic-lattice canvas of a thousand pixels each, with pitch of ~8 nm, on its inner and outer surfaces. Complex patterns rendered on these surfaces were resolved using up to twelve rounds of Exchange-PAINT super-resolution microscopy. We envision these structures as versatile nanoscale pegboards for applications requiring complex 3D arrangements of matter, which will serve to promote rapid uptake of this technology in diverse fields beyond specialist groups working in DNA nanotechnology.
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ADN/química , Imagenología Tridimensional , Conformación de Ácido Nucleico , Dimerización , Modelos MolecularesRESUMEN
Selective isolation of DNA is crucial for applications in biology, bionanotechnology, clinical diagnostics and forensics. We herein report a smart methanol-responsive polymer (MeRPy) that can be programmed to bind and separate single- as well as double-stranded DNA targets. Captured targets are quickly isolated and released back into solution by denaturation (sequence-agnostic) or toehold-mediated strand displacement (sequence-selective). The latter mode allows 99.8% efficient removal of unwanted sequences and 79% recovery of highly pure target sequences. We applied MeRPy for the depletion of insulin, glucagon, and transthyretin cDNA from clinical next-generation sequencing (NGS) libraries. This step improved the data quality for low-abundance transcripts in expression profiles of pancreatic tissues. Its low cost, scalability, high stability and ease of use make MeRPy suitable for diverse applications in research and clinical laboratories, including enhancement of NGS libraries, extraction of DNA from biological samples, preparative-scale DNA isolations, and sorting of DNA-labeled non-nucleic acid targets.
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ADN/aislamiento & purificación , Polímeros de Estímulo Receptivo , Secuencia de Bases/genética , ADN/genética , ADN/metabolismo , ADN Complementario/genética , ADN de Cadena Simple/genética , Electroforesis en Gel de Poliacrilamida , Fraccionamiento de Campo-Flujo , Glucagón/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Insulina/genética , Metanol , Páncreas/metabolismo , Prealbúmina/genética , TranscriptomaRESUMEN
Phospho-lipid bilayer nanodiscs have gathered much scientific interest as a stable and tunable membrane mimetic for the study of membrane proteins. Until recently the size of the nanodiscs that could be produced was limited to ~ 16 nm. Recent advances in nanodisc engineering such as covalently circularized nanodiscs (cND) and DNA corralled nanodiscs (DCND) have opened up the possibility of engineering nanodiscs of size up to 90 nm. This enables widening the application of nanodiscs from single membrane proteins to investigating large protein complexes and biological processes such as virus-membrane fusion and synaptic vesicle fusion. Another aspect of exploiting the large available surface area of these novel nanodiscs could be to engineer more realistic membrane mimetic systems with features such as membrane asymmetry and curvature. In this review, we discuss the recent technical developments in nanodisc technology leading to construction of large nanodiscs and examine some of the implicit applications.
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Precise fabrication of semiconducting carbon nanotubes (CNTs) into densely aligned evenly spaced arrays is required for ultrascaled technology nodes. We report the precise scaling of inter-CNT pitch using a supramolecular assembly method called spatially hindered integration of nanotube electronics. Specifically, by using DNA brick crystal-based nanotrenches to align DNA-wrapped CNTs through DNA hybridization, we constructed parallel CNT arrays with a uniform pitch as small as 10.4 nanometers, at an angular deviation <2° and an assembly yield >95%.
RESUMEN
DNA nanostructures (DNs) have garnered a large amount of interest as a potential therapeutic modality. However, DNs are prone to nuclease-mediated degradation and are unstable in low Mg2+ conditions; this greatly limits their utility in physiological settings. Previously, PEGylated oligolysines were found to protect DNs against low-salt denaturation and to increase nuclease resistance by up to â¼400-fold. Here we demonstrate that glutaraldehyde cross-linking of PEGylated oligolysine-coated DNs extends survival by up to another â¼250-fold to >48 h during incubation with 2600 times the physiological concentration of DNase I. DNA origami with cross-linked oligolysine coats are non-toxic and are internalized into cells more readily than non-cross-linked origami. Our strategy provides an off-the-shelf and generalizable method for protecting DNs in vivo.
Asunto(s)
Reactivos de Enlaces Cruzados/metabolismo , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Glutaral/metabolismo , Polilisina/metabolismo , Supervivencia Celular/efectos de los fármacos , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/toxicidad , ADN/química , ADN/toxicidad , Glutaral/química , Glutaral/toxicidad , Células HEK293 , Humanos , Hidrólisis , Nanoestructuras/química , Nanoestructuras/toxicidad , Conformación de Ácido Nucleico , Polietilenglicoles/química , Polietilenglicoles/metabolismo , Polietilenglicoles/toxicidad , Polilisina/química , Polilisina/toxicidadRESUMEN
Cells often spatially organize biomolecules to regulate biological interactions. Synthetic mimicry of complex spatial organization may provide a route to similar levels of control for artificial systems. As a proof-of-principle, we constructed an RNA-extruding nanofactory using a DNA-origami barrel with an outer diameter of 60 nm as a chassis for integrated rolling-circle transcription and processing of RNA through spatial organization of DNA templates, RNA polymerases, and RNA endonucleases. The incorporation efficiency of molecular components was quantified to be roughly 50% on designed sites within the DNA-origami chassis. Each integrated nanofactory with RNA-producing units, composed of DNA templates and RNA polymerases, produced 100 copies of target RNA in 30 min on average. Further integration of RNA endonucleases that cleave rolling-circle transcripts from concatemers into monomers resulted in 30% processing efficiency. Disabling spatial organization of molecular components on DNA origami resulted in suppression of RNA production as well as processing.
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ARN Polimerasas Dirigidas por ADN/metabolismo , ADN/metabolismo , Endorribonucleasas/metabolismo , Nanotecnología , ARN/biosíntesis , ADN/química , Tamaño de la Partícula , ARN/química , Propiedades de SuperficieRESUMEN
There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in â¼40-50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 µl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.
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ADN de Cadena Simple/síntesis química , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Proteína 9 Asociada a CRISPR/metabolismo , Reparación del ADN/efectos de los fármacos , ADN de Cadena Simple/química , Marcación de Gen/métodos , Células HEK293 , Humanos , Metanol/química , Metanol/farmacología , Mutagénesis Sitio-Dirigida/métodos , Polímeros/química , Factores de TiempoRESUMEN
DNA-based devices often operate through a series of toehold-mediated strand-displacement reactions. To achieve cycling, fluidic mixing can be used to introduce 'recovery' strands to reset the system. However, such mixing can be cumbersome, non-robust, and wasteful of materials. Here we demonstrate mixing-free thermal cycling of DNA devices that operate through associative strand-displacement cascades. These cascades are favored at low temperatures due to the primacy of a net increase in base pairing, whereas rebinding of 'recovery' strands is favored at higher temperatures due to the primacy of a net release of strands. The temperature responses of the devices could be modulated by adjustment of design parameters such as the net increase of base pairs and the concentrations of strands. Degradation of function was not observable even after 500 thermal cycles. We experimentally demonstrated simple digital-logic circuits that evaluate at 35°C and reset after transient heating to 65°C. Thus associative strand displacement enables robust thermal cycling of DNA-based devices in a closed system.