Stability of DNA origami nanoarrays in cell lysate

ChemInform, 2014

DNA's remarkable molecular recognition properties, flexibility and structural features make it one of the most promising scaffolds to design a variety of nanostructures. During the past decades, two major methods have been developed for the construction of DNA nanomaterials in a programmable way, both generating nanostructures in one, two and three dimensions: the tile-based assembly process, which provides a useful tool to construct large and simple structures, and the DNA origami method, suitable for the production of smaller, more sophisticated and well defined structures. Proteins, nanoparticles and other functional elements have been specifically positioned into designed patterns on these structures. They can also act as templates to study chemical reactions, help in the structural determination of proteins and be used as platform for genomic and drug delivery applications. In this review we examine recent progresses towards the potential use of DNA nanostructures for molecular and cellular biology.

ChemBioChem, 2014

DNA's remarkable molecular recognition properties, flexibility and structural features make it one of the most promising scaffolds to design a variety of nanostructures. During the past decades, two major methods have been developed for the construction of DNA nanomaterials in a programmable way, both generating nanostructures in one, two and three dimensions: the tile-based assembly process, which provides a useful tool to construct large and simple structures, and the DNA origami method, suitable for the production of smaller, more sophisticated and well defined structures. Proteins, nanoparticles and other functional elements have been specifically positioned into designed patterns on these structures. They can also act as templates to study chemical reactions, help in the structural determination of proteins and be used as platform for genomic and drug delivery applications. In this review we examine recent progresses towards the potential use of DNA nanostructures for molecular and cellular biology.

ACS Nano, 2018

Here we present a strategy for the fabrication of biomimetic nanoarrays, based on the use of DNA origami, that permit the multivalent investigation of ligand-receptor molecule interactions in cancer cell spreading, with nanoscale spatial resolution and single-molecule control. We employed DNA-origami to control the nanoscale spatial organization of integrin-and epidermal growth factor (EGF)-binding ligands that modulate epidermal cancer cell behaviour. By organizing these multivalent DNA nanostructures in nanoarray configurations on nanopatterned surfaces, we demonstrated the cooperative behaviour of integrin-and EGF-ligands in the spreading of human cutaneous melanoma cells: this cooperation was shown to depend on both the number and ratio of the selective ligands employed. Notably, the multivalent biochips we have developed allowed for this cooperative effect to be demonstrated with single-molecule control and nanoscale spatial resolution. By and large, the platform presented here is of general applicability for the study, with molecular control, of different multivalent interactions governing biological processes, from the function of cell-surface receptors, to protein-ligand binding and pathogen inhibition.

Journal of The American Chemical Society, 2009

The ability to organize materials is a core goal of bionanotechnology. Biomedically relevant examples include the organization of cells into predictable architectures on surfaces 1,2 and the delivery of diverse molecules to cells. 3 Cell surface engineering 4 seeks to localize nanoscale materials such as proteins, 5 carbon nanotubes, 6 synthetic bioactive polymers, 7 vault nanoparticles, 8 and polyelectrolyte multilayer patches 9 onto cellular membranes. Cells have been assembled into microtissues using DNA-mediated interactions. 10 DNA scaffolds, which are nanoarrays built from repeating DNA motifs, have been used for multicomponent interactions 11 to position small peptides, 12 streptavidin, 13,14 antibodies, 15,16 and inorganic materials 17,18 on the array surface into controlled networks and to detect proteins, 19,20 DNA, 21 and RNA. We demonstrate how selfassembled DNA arrays can be directed to the surface of cells, first through biotin-streptavidin interactions and second through specific antibody-cell surface interactions. The versatile cargo-carrying ability of arrays for directing cell-surface interactions, cell-cell bridging, and positioning multiple cells onto a DNA fabric is explored.

Nanoscale, 2014

Nanoparticle arrays self-assembled in the absence of site-bridging, steric hindrance, and electrostatic repulsion.

DNA origami refers to the technique of assembling single-stranded DNA template molecules into target two- and three-dimensional shapes at the nanoscale. This is accomplished by annealing templates with hundreds of DNA strands and then binding them through the specific base-pairing of complementary bases. The inherent properties of these DNA molecules—molecular recognition, self-assembly, programmability, and structural predictability—has given rise to intriguing applications from drug delivery systems to uses in circuitry in plasmonic devices. The first book to examine this important subfield, DNA Origami brings together leading experts from all fields to explain the current state and future directions of this cutting-edge avenue of study. The book begins by providing a detailed examination of structural design and assembly systems and their applications. As DNA origami technology is growing in popularity in the disciplines of chemistry, materials science, physics, biophysics, biology, and medicine, interdisciplinary studies are classified and discussed in detail. In particular, the book focuses on DNA origami used for creating new functional materials (combining chemistry and materials science; DNA origami for single-molecule analysis and measurements (as applied in physics and biophysics); and DNA origami for biological detection, diagnosis and therapeutics (medical and biological applications). DNA Origami readers will also find: A complete guide for newcomers that brings together fundamental and developmental aspects of DNA origami technology Contributions by a leading team of experts that bring expert views from different angles of the structural developments and applications of DNA origami An emerging and impactful research topic that will be of interest in numerous multidisciplinary areas A helpful list of references provided at the end of each chapter to give avenues for further study Given the wide scope found in this groundbreaking work, DNA Origami is a perfect resource for nanotechnologists, biologists, biophysicists, chemists, materials scientists, medical scientists, and pharmaceutical researchers.

Angewandte Chemie International Edition, 2010

Molecules

DNA origami nanocarriers have emerged as a promising tool for many biomedical applications, such as biosensing, targeted drug delivery, and cancer immunotherapy. These highly programmable nanoarchitectures are assembled into any shape or size with nanoscale precision by folding a single-stranded DNA scaffold with short complementary oligonucleotides. The standard scaffold strand used to fold DNA origami nanocarriers is usually the M13mp18 bacteriophage’s circular single-stranded DNA genome with limited design flexibility in terms of the sequence and size of the final objects. However, with the recent progress in automated DNA origami design—allowing for increasing structural complexity—and the growing number of applications, the need for scalable methods to produce custom scaffolds has become crucial to overcome the limitations of traditional methods for scaffold production. Improved scaffold synthesis strategies will help to broaden the use of DNA origami for more biomedical applic...

The DNA origami technique has emerged as one of the most versatile bottom-up nanofabrication methods due to its ability to construct well-defined complex three-dimensional nanostructures and guide assembly of functional nanoscale objects with unprecedented precision, high yields, and controlled stoichiometry. Nonetheless, limited compatibility with biologically relevant fluids and typical solvents utilized in nanofabrication often restricts applications of DNA origami-based assemblies and devices. Here we present an approach for coating DNA origami structures with silica. By careful adjustment of experiment parameters, we achieved reproducible growth of ultrathin silica shell in solution without agglomeration or deformation of DNA origami structures. The silica-coated structures are stable in water and exhibit an increased resistivity to nuclease-mediated degradation. In addition, the coated structures preserve their structural integrity in polar organic solvents. We anticipate that our results will aid further advancement of DNA origami techniques as the nanofabrication method.

2019

DNA and peptides are two of the most commonly used biomolecules for building self-assembling materials, but few examples exist of hybrid nanostructures that contain both components. Here we report the modification of two peptides that comprise a coiled-coil heterodimer pair with orthogonal DNA handles in order to link DNA origami nanostructures bearing complementary strands into micrometer long one-dimensional arrays. We probed the effect of number of coils on self-assembly and demonstrated the formation of self-assembled structures through multiple routes, to form dimers and trimers, an alternating copolymer of two different origami bundles, and stepwise assembly of purified structures with coiled-coil conjugates. Our results demonstrate the successful merging of two distinct self-assembly modes to create hybrid bionanomaterials expected to have a range of potential applications in the future.

Nucleic Acids Research, 2013

Most previously reported methods for purifying DNA-origami nanostructures rely on agarose-gel electrophoresis (AGE) for separation. Although AGE is routinely used to yield 0.1-1 mg purified DNA nanostructures, obtaining >100 mg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extraction, desalting and concentration steps. Here, we present a readily scalable purification approach utilizing rate-zonal centrifugation, which provides comparable separation resolution as AGE. The DNA nanostructures remain in aqueous solution throughout the purification process. Therefore, the desired products are easily recovered with consistently high yield (40-80%) and without contaminants such as residual agarose gel or DNA intercalating dyes. Seven distinct three-dimensional DNA-origami constructs were purified at the scale of 0.1-100 mg (final yield) per centrifuge tube, showing the versatility of this method. Given the commercially available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for preparation of larger amounts (e.g. milligram quantities) of DNA nanostructures.

Nature Nanotechnology, 2009

The development of nanoscale electronic and photonic devices will require a combination of the high throughput of lithographic patterning and the high resolution and chemical precision afforded by self-assembly 1-4 . However, the incorporation of nanomaterials with dimensions of less than 10 nm into functional devices has been hindered by the disparity between their size and the 100 nm feature sizes that can be routinely generated by lithography. Biomolecules offer a bridge between the two size regimes, with sub-10 nm dimensions, synthetic flexibility and a capability for self-recognition. Here, we report the directed assembly of 5-nm gold particles into large-area, spatially ordered, two-dimensional arrays through the site-selective deposition of mesoscopic DNA origami 5 onto lithographically patterned substrates 6 and the precise binding of gold nanocrystals to each DNA structure. We show organization with registry both within an individual DNA template and between components on neighbouring DNA origami, expanding the generality of this method towards many types of patterns and sizes.

Nanoscale, 2018

DNA nanostructures hold immense potential to be used for biological and medical applications. However, they are extremely vulnerable towards salt depletion and nucleases, which are common under physiological conditions. In this contribution, we used chitosan and linear polyethyleneimine for coating and long-term stabilization of several three-dimensional DNA origami nanostructures. The impact of the degree of polymerization and the charge density of the polymer together with the N/P charge ratio (ratio of the amines in polycations to the phosphates in DNA) on the stability of encapsulated DNA origami nanostructures in the presence of nucleases and in low-salt media was examined. The polycation shells were compatible with enzyme- and aptamer-based functionalization of the DNA nanostructures. Additionally, we showed that despite being highly vulnerable to salt depletion and nucleolytic digestion, self-assembled DNA nanostructures are stable in cell culture media up to a week. This was...

2019

Intercalation of drug molecules into synthetic DNA nanostructures formed through self-assembled origami has been postulated as a valuable future method for targeted drug delivery. This is due to the excellent biocompatibility of synthetic DNA nanostructures, and high potential for flexible programmability including facile drug release into or near to target cells. Such favourable properties may enable high initial loading and efficient release for a predictable number of drug molecules per nanostructure carrier, important for efficient delivery of safe and effective drug doses to minimise non-specific release away from target cells. However, basic questions remain as to how intercalation-mediated loading depends on the DNA carrier structure. Here we use the interaction of dyes YOYO-1 and acridine orange with a tightly-packed 2D DNA origami tile as a simple model system to investigate intercalation-mediated loading. We employed multiple biophysical techniques including single-molecul...

Journal of the American Chemical Society, 2018

DNA origami is a promising molecular delivery system for a variety of therapeutic applications including cancer therapy, given its capability to fabricate homogeneous nanostructures whose physicochemical properties (size, shape, surface chemistry) can be precisely tailored. However, the correlation between DNA-origami design and internalization efficiency in different cancer cell lines remains elusive. We investigated the cellular uptake of four DNA-origami nanostructures (DONs) with programmed sizes and shapes in multiple human cancer cell lines. The cellular uptake efficiency of DONs was influenced by size, shape, and cell line. Scavenger receptors were responsible for the internalization of DONs into cancer cells. We observed distinct stages of the internalization process of a gold nanoparticle (AuNP)-tagged rod-shape DON, using high-resolution transmission electron microscopy. This study provides detailed understanding of cellular uptake and intracellular trafficking of DONs in ...