Name: folding and characterization of a bio-responsive robot from dna origami
Uploaded: 2015-12-03T10:45:00.000+00:00
Duration: 7 min 59 s
Description: Watch this Scientific Journal Video about Folding and Characterization of a Bio-responsive Robot from DNA Origami at JoVE.com

Los autores desean agradecer a S. Douglas para las discusiones y consejos muy valiosos, y todos los miembros del laboratorio Bachelet útil para los debates y el trabajo. Este trabajo es apoyado por becas de la Facultad de Ciencias de la Vida y el Instituto de Nanotecnología y Materiales Avanzados de la Universidad de Bar-Ilan.

1. Preparación de Grapas piscina Mezcla <ol><li> Solicitar liofilizado ADN grapas nanorobot en placas de 96 pocillos que se enumeran en la Tabla 1 (ver Materiales) y normalizar a 10 nmol. Para obtener una descripción detallada del diseño y la arquitectura del nanorobot de ADN ver Ben-Ishay et. Al 14 y Douglas et al. 15). </li><li> Reconstituir cada grapa bien con DNasa / ultrapura RNasa libre a una concentración de 100 mM. Para grapas normalizaron a 10 nmol, reconstituir con 100 l de agua ultrapura. </li><li> Piscina juntos 20 l de cada grapa utilizando una pipeta multicanal y 55 ml cuenca solución estéril. Nota: Dado que la forma nanorobot se compone de 254 capítulos básicos (incluyendo Core, Bordes, manijas y las secuencias de Guías, Tabla 1), la concentración de cada uno de los alimentos básicos en la piscina grapas es 394 nM. Extracción de grapas de la piscina de primera necesidad, o la adición de algunos en diferentes volumes, puede reducir considerablemente el rendimiento, o de otra manera dar lugar a agregados desplegadas. </li></ol> 2. Preparación de la Mezcla de Reacción Fabrication <ol><li> Para una mezcla de reacción estándar de utilizar 40 l de M13mp18 genoma viral circular sola cadena de ADN, correspondientes a 4 pmol de ADN andamio (solución 100 nM de valores, ver Materiales). La concentración final de andamio en la mezcla de fabricación es de 20 nm, un volumen final de 200 l. <ol><li> Ajuste la cantidad de ADN de andamio de acuerdo a las necesidades específicas; sin embargo, mantener la concentración final de ADN de andamio en la mezcla de reacción a una constante 20 nM. </li></ol></li><li> Añadir grapas para llegar a un andamio a la proporción de grapas de 1 a 10, respectivamente. Para una nM concentración de ADN andamio 20, cada uno de concentración final los 254 grapas &#39;es 200 nM. Para un volumen final de reacción l 200 añadir 102 l de la mezcla de alimentos básicos de la piscina (sección 1.2). <ol><li> Añadir secuencias específicas Gate separado a the mezcla de reacción de plegado en este momento. Estos oligonucleótidos se piden por separado y requieren purificación por HPLC. Asegúrese de que los oligos Gate están presentes en un uno y diez minutos andamio para Puerta relación de secuencia, es decir, 200 nM de cada oligo para una nM concentración andamio 20. Para un volumen de reacción de plegado 200 l, añadir 0,4 l para cada uno de los oligo de cuatro Gate en un stock de concentración 100 mM. </li></ol></li><li> Añadir 10x tampón TAE de stock para llegar a una concentración final de 1x TAE (40 mM Tris-Acetato, EDTA 1 mM). Para un volumen de reacción de plegado 200 l, añadir 20 l de 10x TAE. </li><li> Añadir 1 M de MgCl 2 a una concentración final de 10 mM. Para un volumen de reacción plegable 200 l, añadir 2 l de 1 M de MgCl2. </li><li> Añadir 36 l de DNasa / RNasa libre de agua ultrapura a un alcanzar un volumen final de 200 l. </li><li> Muestras Vortex y alícuota de 100 l en viales de PCR. Nota: Consulte la especificación termociclador con respecto a los volúmenes de reacción máximos que se utilizarán.Reducir el volumen para cumplir con estos límites no reducirá los rendimientos obtenidos. Los volúmenes de reacción por encima del máximo especificado pongan en peligro los rendimientos. </li></ol> 3. Temperatura de recocido Rampa de Reacción Fabrication <ol><li> Termociclador Programa como sigue: <ol><li> Rampa 85 ° C a 60 ° C a una velocidad de 5 min / ° C. </li><li> Rampa de 60 ° C a 4 ° C a una velocidad de 75 min / ° C. </li><li> Mantenga a 4 ° C indefinidamente. </li></ol></li><li> Después de la fabricación ha terminado almacenar las muestras a -20 ° C. </li></ol> 4. La eliminación de exceso de grapas <ol><li> Añadir 100 l de mezcla de reacción de plegado a un filtro de centrífuga de 0,5 ml con un MWCO de 100 kDa. Guardar una muestra de 10 l de nanorobots pre-purificación para su posterior análisis. </li><li> Centrifugar durante 10 min a 9600 x g. </li><li> Añadir 400 l de tampón de plegamiento (1X TAE, 10 mM de MgCl 2). </li><li> Repita los pasos 4,2 y 4,3 veces más. </li><li>Centrifugar durante 5 min a 9600 x g. </li><li> Recuperar el concentrado colocando el filtro al revés en un tubo de microcentrífuga limpio y centrifugado durante 1 min a 9600 x g. Volúmenes finales pueden variar dependiendo del volumen inicial de mezcla de reacción de fabricación que se puso en el filtro. Típicamente se obtiene un volumen final de 25-60 l de muestra nanorobot concentrado. </li><li> Medir la concentración de ADN en las muestras a través de espectrofotómetro a 260 nm. Utilice el peso molecular de 5,3 mg / pmol en el cálculo de las concentraciones molares de muestras nanorobot. Nota: El coeficiente de extinción molar para dsDNA, 50 g / OD 260, es suficiente para la mayoría de las aplicaciones. 48 mg / OD 260 tiene en cuenta la timina poli ssDNA se extiende a las grapas bordes. </li></ol> 5. agarosa electroforesis en gel de Análisis de plegado Nanorobots <ol><li> Preparación de TBE 0,5x (45 mM Tris-borato, EDTA 1 mM), 2% en gel de agarosa suplementado con 10 mM de MgCl 2 </sub&gt; (adaptado de Ernesto Castro et al 21.): <ol><li> Preparar tampón TBE 0,5x diluyendo 6,25 ml de 10x TBE reserva de estabilización en 118,75 ml de ddH 2 O. </li><li> Disolver 2,5 g de agarosa en 125 ml de tampón TBE 0,5x. </li><li> Hervir en el microondas hasta que la agarosa se disuelva completamente. </li><li> Añadir 1,25 ml de 1 M de MgCl 2 a la concentración final de 10 mM. </li><li> Añadir 7 l de 10 mg / ml de bromuro de etidio. </li><li> Espere a que la solución a ligeramente fresco y llenar la bandeja de gel antes de que se solidifique el gel de agarosa. Instale peine deseado inmediatamente. </li><li> Preparar 1 L TBE 0,5x tampón mediante la adición de 50 ml de 10x tampón TBE Stock y 10 ml de 1 M de MgCl 2 a 940 ml de ddH 2 O. </li><li> Una vez gel es complemento sólido tampón al dispositivo de electroforesis y el dispositivo puesto en un baño de hielo. </li></ol></li><li> Cargar 1 g de ADN total de la nanorobots pre-depuración (paso 3.2), y la purificación posterior (paso 4.7), junto a un andamioMuestra de ADN y un marcador de ADN 1 kb, sobre el gel. Un ejemplo se da en la Figura 2. Volúmenes reales de muestras dependen de las concentraciones obtenidas después de la purificación. Cada muestras se carga con tampón de carga en una proporción de 1: volumen final 6. </li><li> Ajuste la fuente de poder de 80 V y ejecutar gel durante 3 horas. Ejecutar el gel en un baño lleno de hielo y agua. Nanorobots se desarrollarán y aparecer como una mancha si el gel de agarosa se calienta durante la electroforesis. Durante la electroforesis agregue hielo adicional para mantener el dispositivo de calefacción. </li><li> Ver gel sobre una mesa de UV (Figura 2). </li></ol> 6. tinción negativa de nanorobot con uranilo-formiato <ol><li> Preparación de solución madre-uranilo formiato 2% (adaptado de Castro et al 23.): <ol><li> Pesar 100 mg de polvo de formiato de uranilo en un tubo de 15 ml. </li><li> Hervir DNasa / RNasa agua ultrapura gratuita por 3 minutos para des-oxigenado. </li><li> Añadir 5 ml de agua caliente desoxigenada (~ 60 ° C)en el tubo de 15 ml que contiene el polvo de uranilo-formiato (del paso anterior). Cierre bien la tapa, envoltura en papel de aluminio y agitar rigurosamente durante 10 minutos (tubo sujetar a un vórtice). Solución debería aparecer turbia con un color amarillo. </li><li> Filtrar la solución a través de una jeringa de 0,2 micras fi ltro. Solución debe quedado claro. </li><li> Alícuota de 200 l de la solución en tubos de microcentrífuga. </li><li> Centrifugar a máxima velocidad durante 5 minutos en una centrífuga de mesa a las muestras de la piscina en la parte inferior del tubo. </li></ol></li><li> La carga de la muestra a la red y tinción negativa: <ol><li> Descongelar una alícuota de 200 l de solución de uranilo-formiato 2% (preparado en la sección 6.1). Añadir 10 l de solución 0,5 M de NaOH y agitar rigurosamente durante 3 min. Centrifugar a máxima velocidad durante 5 minutos usando una centrífuga de mesa. </li><li> Mientras tanto, la rejilla de descarga luminiscente usando aire de la habitación a 0,2 mbar y 25 mA durante 30 segundos. </li><li> Añadir 15 l de 2 nM muestra nanorobot después de la purificación (SECTIen 4.7) en parte superior de la rejilla (celebrada con pinzas) y dejar absorber durante 1 min. Utilice papel de filtro para drenar el exceso de líquido mediante la colocación de la rejilla en la parte superior del papel de filtro. No toque la superficie de la rejilla. </li><li> Añadir 10 l de la uranilo-formiato 2% (del paso 6.2.1) en la parte superior de la rejilla (celebrada con pinzas). </li><li> Utilizar rápidamente papel de filtro para drenar el exceso de líquido mediante la colocación de la rejilla en la parte superior del papel de filtro. No toque la superficie de la rejilla. </li><li> Repita los pasos 6.2.4 y 6.2.5. </li><li> Añadir 10 l de la uranilo-formiato 2% (del paso 6.2.1) en la parte superior de la rejilla (celebrada con pinzas). Deje solución de tinción absorber durante 30 s. </li><li> Utilice papel de filtro para drenar el exceso de líquido mediante la colocación de la rejilla en la parte superior del papel de filtro. No toque la superficie de la rejilla. </li><li> Deje que la parrilla se seque por completo durante al menos 30 min, boca arriba sobre un papel de filtro, antes de la inyección en el TEM. </li></ol></li></ol>

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Hemos descrito la fabricación, la purificación, y la visualización del nanorobot ADN. Después de la fabricación del chasis hexagonal del dispositivo, la función de la nanorobot está programado con la simple introducción de la carga específica y hebras de detección para el robot que encontrar fácilmente su posición designada debido a la complementariedad de enlaces de hidrógeno con los sitios de conexión disponibles sola hebra 14 , 15,22. El protocolo de fa...

Los usos de los ácidos nucleicos de la nanotecnología son asombrosos. La maleabilidad de la base de emparejamiento Watson-Crick, así como la facilidad y bajo costo relativo de la síntesis a gran escala de oligonucleótidos medida 2 ha generado una explosión de aplicaciones 3 y la investigación en el campo de la nanotecnología de ADN. Nanotecnología de ADN estructural, basado en el 4,5 unión Seeman inmóvil como un bloque de construcción fundamental hace uso de ADN como una unidad elemental auto-montaje para la construcción de formas arbitrarias 6-8. El reciente desarrollo de la DNA andamiaje origami 9 técnica permite la construcción de complejos 2D / 3D nano-arquitecturas de 10-12 con una precisión sub-nanométrica y es una vía eficaz para la construcción de nuevos objetos funcionales con el aumento de la complejidad y la diversidad asombrosa. El proceso de construcción se basa en un ADN monocatenario largo andamio, por lo general deriva de una genom virale, que se puede plegar a través de la hibridación de cientos de oligos cortos cadena de ADN individuales denomina grapas. La alta resolución estructural obtenida por esta técnica es el resultado directo de las dimensiones naturales de la doble hélice del ADN, mientras que la reproducibilidad de la fabricación es el resultado de la adaptación de las secuencias cortas de grapas de una sola hebra para facilitar la complementariedad máxima de enlaces de hidrógeno alcanzable. Con el uso de una temperatura de recocido rampa lenta la más baja energía diseñado, termodinámicamente preferido nanoestructura se alcanza en altos rendimientos y fidelidad. La fácil aplicación de las reglas de diseño de unión en un código informático permitió el desarrollo de herramientas CAD, como caDNAno 13, que muy a simplificar la tarea de diseñar estructuras grandes y complejas que contienen cientos de uniones conectadas. Anteriormente hemos descrito el diseño de un nanorobot de ADN con la ayuda de la herramienta de 14,15 caDNAno. Aquí representan la fabricación yvisualización, a través de microscopía electrónica de transmisión (TEM), de la nanorobot, un nanodispositivo hexagonal hueca 3D, con unas dimensiones de 35 x 35 x 50 nm 3, diseñado para sufrir un cambio conformacional importante en respuesta a un estímulo predeterminados y presente de carga específica, tales como proteínas o ácidos nucleicos oligos, secuestrado en el interior. Mientras que 12 estaciones de carga están disponibles dentro del chasis hueco, el número real de carga con destino diferente con el tamaño de la carga. Moléculas de carga van desde pequeñas moléculas de ADN a las enzimas, anticuerpos y 5-10 nanopartículas de oro nm. Cargocan o bien ser uniforme o heterogénea, de manera que cada nanorobot contiene una mezcla de diferentes moléculas. Sensing se logra a través de dos puertas de bloqueo doble diseño helicoidal para detectar proteínas, ácidos nucleicos u otros productos químicos, basado ya sea en aptasensor 16,17 o cadena de ADN desplazamiento 18 tecnologías. Los recientes desarrollos en protocolos de selección de aptámeros 19-21 permiten el diseño de nanorobots respondera una gama cada vez mayor de las moléculas y los tipos de células. Un trabajo anterior mostró una nanorobot que lleva un anticuerpo específico, que tras la unión a su antígeno puede transmitir o bien un inhibidor o una señal prolífico en el interior de tipos específicos de células en una población de células mixtas 15. Una característica interesante de estos nanodispositivos es su capacidad para realizar aún más tareas complejas y el control de la lógica con la introducción de diferentes subtipos nanorobot en una sola población. Recientemente hemos demostrado subtipos específicos de nanorobots que realizan, ya sea como reguladores positivos o negativos, controlando una población efectora que contiene una molécula de carga activa 22. El protocolo que se presenta aquí describe la fabricación, purificación y obtención de imágenes de un nanorobot cerrada con secuencias de sensores aptámero que se unen selectivamente a PDGF para facilitar la apertura de la nanorobot 15,22. El proceso de fabricación descrito es similar a la nproceso de fabricación anorobot representado inicialmente por Douglas et al. 15 con los cambios destinados a reducir la duración total del proceso, al tiempo que aumenta las tasas de rendimiento y purificación.

<table><tbody><tr><td>DNase/RNase free distilled water</td><td>Gibco</td><td>10977</td><td></td></tr><tr><td>M13mp18 ssDNA scaffold</td><td>NEB</td><td>N4040S</td><td></td></tr><tr><td>10x TAE</td><td>Gibco</td><td>15558-042</td><td></td></tr><tr><td>1 M MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Ambion</td><td>AM9530G</td><td></td></tr><tr><td>Amicon Ultra 0.5 ml centrifugal filter 100K MWCO</td><td>Amicon</td><td>UFC510024</td><td></td></tr><tr><td>Agarose</td><td>Promega</td><td>V3125</td><td></td></tr><tr><td>TBE buffer</td><td>Promega</td><td>V4251</td><td></td></tr><tr><td>Ethidium bromide 10&amp;nbsp;mg/ml solution</td><td>Sigma Aldrich</td><td>E1510</td><td></td></tr><tr><td>1 kb DNA marker</td><td>NEB</td><td>N3232S</td><td></td></tr><tr><td>Loading Dye</td><td>NEB</td><td>B7021S</td><td></td></tr><tr><td>uranyl formate</td><td>polysciences</td><td>24762</td><td></td></tr><tr><td>carbon-coated TEM grids&amp;nbsp;</td><td>Science services</td><td>EFCF400-Cu-50</td><td></td></tr><tr><td>Thermal Cycler c1000 Touch</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Glow Discharge K100X</td><td>Emitech</td><td></td><td></td></tr><tr><td>UV table Gel Doc EZ Imager</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>NanoDrop 2000c</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>TEM FEI-G12</td><td>Tecnai</td><td></td><td></td></tr></tbody></table>

folding and characterization of a bio-responsive robot from dna origami

El nanorobot de ADN es un dispositivo nanométrico hexagonal hueco, diseñado para abrirse en respuesta a estímulos específicos y presente de carga secuestradas en el interior. Ambos estímulos y carga se pueden adaptar según las necesidades específicas. Aquí se describe el protocolo de fabricación nanorobot de ADN, con el uso de la técnica de origami de ADN. El procedimiento se inicia mediante la mezcla de alimentos básicos de ADN de una sola hebra cortos en una mezcla de valores que se añade a una larga y circular, andamio ADN de una sola hebra en presencia de un tampón de plegado. Un ciclador térmico estándar de está programado para disminuir gradualmente la temperatura de reacción de mezcla para facilitar el recocido-grapas a andamio, que es la fuerza de guía detrás el plegado de la nanorobot. Una vez que la reacción de plegado 60 hr es completa, el exceso de grapas se descartan usando un filtro de centrífuga, seguido por visualización a través de electroforesis en gel de agarosa (AGE). Por último, la fabricación con éxito de la nanorobot es verificado por microscopía electrónica de transmisión (TEM),con el uso de uranilo-formiato como tinción negativa.

Los resultados representativos se muestran en la Figura 2A. Todos los carriles contienen 1 g de ADN total, medida a través de espectrofotómetro (OD 260). En comparación con el andamio de ADN de una sola hebra circular (carril 2), nanorobots se ven obstaculizados en el gel debido a su peso molecular más alto, el resultado de grapas hibridación con el ADN de andamio (Carril 3. Red flecha). La banda de bajo peso molecular en el carril 3 representa el exceso de alimentos bá...

Watch this Scientific Journal Video about Folding and Characterization of a Bio-responsive Robot from DNA Origami at JoVE.com

Plegado y Caracterización de un Robot Bio-Responsivo de DNA Origami | Química | Video

Folding and Characterization of a Bio-responsive Robot from DNA Origami

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

Plegable y caracterización de un Robot Bio-sensible de ADN Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

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DNA Structure and Function

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14.5K vistas.  Bar-Ilan University. DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM). 

Video: Folding and Characterization of a Bio-responsive Robot from DNA Origami

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

Origami Inspired autoensamblaje de partículas modeladas y Reconfigurable

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Investigación

Química

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

Autoensamblaje de formas bidimensionales complejas de una sola hebra de ADN Azulejos

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

Preparación de Mica y Silicon Sustratos para el Análisis de Origami ADN y Experimentación

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

Diseño de un Robot Bio-respuesta a partir del ADN Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

Bioingeniería

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Producción de Conjuntos de Motores de Dynein y Kinesin en Nanoestructuras de Origami de ADN para Observación de MoléculaS Simples

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Bioquímica

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Here, a protocol for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using natural cationic polysaccharide chitosan and synthetic linear polyethyleneimine (LPEI) coatings is presented.

Terapia génica inspirado POLICATIÓN recubrimiento para protección de nanoestructuras de Origami de ADN

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in ...

Assembly of Gold Nanorods into Chiral Plasmonic Metamolecules Using DNA Origami Templates

The inherent addressability of DNA origami structures makes them ideal templates for the arrangement of metal nanoparticles into complex plasmonic nanostructures. The high spatial precision of a DNA origami-templated assembly allows controlling the coupling between plasmonic resonances of individual particles and enables tailoring optical properties of the constructed nanostructures. Recently, chiral plasmonic systems attracted a lot of attention due to the strong correlation between the spatial configuration of plasmonic assemblies and their optical responses (e.g., circular dichroism [CD]). In this protocol, we describe the whole workflow for the generation of DNA origami-based chiral assemblies of gold nanorods (AuNRs). The protocol includes a detailed description of the design principles and experimental procedures for the fabrication of DNA origami templates, the synthesis of AuNRs, and the assembly of origami-AuNR structures. In addition, the characterization of structures using transmission electron microscopy (TEM) and CD spectroscopy is included. The described protocol is not limited to chiral configurations and can be adapted for the construction of various plasmonic architectures.

We describe the detailed protocol for the DNA origami-based assembly of gold nanorods into chiral plasmonic metamolecules with strong chiroptical responses. The protocol is not limited to chiral configurations and can be easily adapted for the fabrication of various plasmonic architectures.

Asamblea de nanobarras de oro en Metamolecules plasmónica quirales mediante plantillas de Origami de ADN

The inherent addressability of DNA origami structures makes them ideal templates for the arrangement of metal nanoparticles into complex plasmonic ...

DNA Origami-Mediated Substrate Nanopatterning of Inorganic Structures for Sensing Applications

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA nanofabrication technique is called DNA origami, and it allows high-throughput synthesis of accurate and highly versatile structures with nanometer-level precision. Here, it is shown how the spatial information of DNA origami can be transferred to metallic nanostructures by combining the bottom-up DNA origami with the conventionally used top-down lithography approaches. This allows fabrication of billions of tiny nanostructures in one step onto selected substrates. The method is demonstrated using bowtie DNA origami to create metallic bowtie-shaped antenna structures on silicon nitride or sapphire substrates. The method relies on the selective growth of a silicon oxide layer on top of the origami deposition substrate, thus resulting in a patterning mask for following lithographic steps. These nanostructure-equipped surfaces can be further used as molecular sensors (e.g., surface-enhanced Raman spectroscopy (SERS)) and in various other optical applications at the visible wavelength range owing to the small feature sizes (sub-10 nm). The technique can be extended to other materials through methodological modifications; therefore, the resulting optically active surfaces may find use in development of metamaterials and metasurfaces.

Here, we describe a protocol to create discrete and accurate inorganic nanostructures on substrates using DNA origami shapes as guiding templates. The method is demonstrated by creating plasmonic gold bowtie-shaped antennas on a transparent substrate (sapphire).

Nanopatrón de nanopatrón de nanopatrón de sustrato mediado por origami de ADN para aplicaciones de cizallamiento

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Automatizado de montaje robotizada manipulación líquido de dispositivos modulares de la DNA

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

Diseño y síntesis de un ADN Reconfigurable acordeón Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...