Arun Richard Chandrasekaran

Fabrication of a nanofibrous scaffold with improved bioactivity for culture of human dermal fibroblasts for skin regeneration

Engineering dermal substitutes with electrospun nanofibres have lately been of prime importance for skin tissue regeneration. Simple electrospinning technology served to produce nanofibrous scaffolds morphologically and structurally similar to the extracellular matrix of native tissues. The nanofibrous scaffolds of poly(L-lactic acid)-co-poly(ε-caprolactone) (PLACL) and PLACL/gelatin complexes were fabricated by the electrospinning process. These nanofibres were characterized for fibre morphology, membrane porosity, wettability and chemical properties by FTIR analysis to culture human foreskin fibroblasts for skin tissue engineering. The nanofibre diameter was obtained between 282 and 761 nm for PLACL and PLACL/gelatin scaffolds; expressions of amino and carboxyl groups and porosity up to 87% were obtained for these fibres, while they also exhibited improved hydrophilic properties after plasma treatment. The results showed that fibroblasts proliferation, morphology, CMFDA dye expression and secretion of collagen were significantly increased in plasma-treated PLACL/gelatin scaffolds compared to PLACL nanofibrous scaffolds. The obtained results prove that the plasma-treated PLACL/gelatin nanofibrous scaffold is a potential biocomposite material for skin tissue regeneration.

Functionalizing Designer DNA Crystals with a Triple-Helical Veneer†

DNA is a very useful molecule for the programmed self-assembly of 2D and 3D nanoscale objects.[1] The design of these structures exploits Watson–Crick hybridization and strand exchange to stitch linear duplexes into finite assemblies.[2–4] The dimensions of these complexes can be increased by over five orders of magnitude through self-assembly of cohesive single-stranded segments (sticky ends).[5,6] Methods that exploit the sequence addressability of DNA nanostructures will enable the programmable positioning of components in 2D and 3D space, offering applications such as the organization of nanoelectronics,[7] the direction of biological cascades,[8] and the structure determination of periodically positioned molecules by X-ray diffraction.[9] To this end we present a macroscopic 3D crystal based on the 3-fold rotationally symmetric tensegrity triangle[3,6] that can be functionalized by a triplex-forming oligonucleotide on each of its helical edges.

Post-Assembly Stabilization of Rationally Designed DNA Crystals†

This manuscript reports an effort to stabilize self-assembled DNA crystals. Owing to their weak inter-unit cohesion, self-assembled DNA crystals are fragile, which limits the potential applications of such crystals. To overcome this problem, another molecule was introduced, which binds to the cohesive sites and stabilizes the inter-unit interactions. The extra interactions greatly improve the stability of the DNA crystals. The original DNA crystals are only stable in solutions of high ionic strength (e.g., ≥1.2 M (NH4)2SO4); in contrast, the stabilized crystals can be stable at ionic strengths as low as that of a 0.02 M solution of (NH4)2SO4. The current strategy is expected to represent a general approach for increasing the stability of self-assembled DNA nanostructures for potential applications, for example, as structural scaffolds and molecular sieves.

Beyond the Fold: Emerging Biological Applications of DNA Origami

The use of DNA as a material for nanoscale construction has blossomed in the past decade. This is largely attributable to the DNA origami technique, which has enabled construction of nanostructures ranging from simple two‐dimensional sheets to complex three‐dimensional objects with defined curves and edges. These structures are amenable to site‐specific functionalization with nanometer precision, and have been shown to exhibit cellular biocompatibility and permeability. The DNA origami technique has already found widespread use in a variety of emerging biological applications such as biosensing, enzyme cascades, biomolecular analysis, biomimetics, and drug delivery. We highlight a few of these applications and comments on the prospects for this rapidly expanding field of research.

Nucleic Acid Nanostructures for Chemical and Biological Sensing

The nanoscale features of DNA have made it a useful molecule for bottom-up construction of nanomaterials, for example, two- and three-dimensional lattices, nanomachines, and nanodevices. One of the emerging applications of such DNA-based nanostructures is in chemical and biological sensing, where they have proven to be cost-effective, sensitive and have shown promise as point-of-care diagnostic tools. DNA is an ideal molecule for sensing not only because of its specificity but also because it is robust and can function under a broad range of biologically relevant temperatures and conditions. DNA nanostructure-based sensors provide biocompatibility and highly specific detection based on the molecular recognition properties of DNA. They can be used for the detection of single nucleotide polymorphism and to sense pH both in solution and in cells. They have also been used to detect clinically relevant tumor biomarkers. In this review, recent advances in DNA-based biosensors for pH, nucleic acids, tumor biomarkers and cancer cell detection are introduced. Some challenges that lie ahead for such biosensors to effectively compete with established technologies are also discussed.

Topological Linkage of DNA Tiles Bonded by Paranemic Cohesion

Catenation is the process by which cyclic strands are combined like the links of a chain, whereas knotting changes the linking properties of a single strand. In the cell, topoisomerases catalyzing strand passage operations enable the knotting and catenation of DNA so that single- or double-stranded segments can be passed through each other. Here, we use a system of closed DNA structures involving a paranemic motif, called PX-DNA, to bind double strands of DNA together. These PX-cohesive closed molecules contain complementary loops whose linking by Escherichia coli topoisomerase 1 (Topo 1) leads to various types of catenated and knotted structures. We were able to obtain specific DNA topological constructs by varying the lengths of the complementary tracts between the complementary loops. The formation of the structures was analyzed by denaturing gel electrophoresis, and the various topologies of the constructs were characterized using the program Knotilus.

Addressable configurations of DNA nanostructures for rewritable memory

Abstract DNA serves as natures information storage molecule, and has been the primary focus of engineered systems for biological computing and data storage. Here we combine recent efforts in DNA self-assembly and toehold-mediated strand displacement to develop a rewritable multi-bit DNA memory system. The system operates by encoding information in distinct and reversible conformations of a DNA nanoswitch and decoding by gel electrophoresis. We demonstrate a 5-bit system capable of writing, erasing, and rewriting binary representations of alphanumeric symbols, as well as compatibility with ‘OR’ and ‘AND’ logic operations. Our strategy is simple to implement, requiring only a single mixing step at room temperature for each operation and standard gel electrophoresis to read the data. We envision such systems could find use in covert product labeling and barcoding, as well as secure messaging and authentication when combined with previously developed encryption strategies. Ultimately, this type of memory has exciting potential in biomedical sciences as data storage can be coupled to sensing of biological molecules.

Shear Dependent LC Purification of an Engineered DNA Nanoswitch and Implications for DNA Origami

As DNA nanotechnology matures, there is increasing need for fast, reliable, and automated purification methods. Here, we develop UHPLC methods to purify self-assembled DNA nanoswitches, which are formed using DNA origami approaches and are designed to change conformations in response to a binding partner. We found that shear degradation hindered LC purification of the DNA nanoswitches, removing oligonucleotides from the scaffold strand and causing loss of function. However, proper choice of column, flow rate, and buffers enabled robust and automated purification of DNA nanoswitches without loss of function in under a half hour. Applying our approach to DNA origami structures, we found that ∼400 nm long nanotubes degraded under the gentlest flow conditions while ∼40 nm diameter nanospheres remained intact even under aggressive conditions. These examples show how fluid stresses can affect different DNA nanostructures during LC purification and suggest that shear forces may be relevant for some applications of DNA nanotechnology. Further development of this approach could lead to fast and automated purification of DNA nanostructures of various shapes and sizes, which would be an important advance for the field.

Triplex-forming oligonucleotides: a third strand for DNA nanotechnology

Abstract DNA self-assembly has proved to be a useful bottom-up strategy for the construction of user-defined nanoscale objects, lattices and devices. The design of these structures has largely relied on exploiting simple base pairing rules and the formation of double-helical domains as secondary structural elements. However, other helical forms involving specific non-canonical base-base interactions have introduced a novel paradigm into the process of engineering with DNA. The most notable of these is a three-stranded complex generated by the binding of a third strand within the duplex major groove, generating a triple-helical (‘triplex’) structure. The sequence, structural and assembly requirements that differentiate triplexes from their duplex counterparts has allowed the design of nanostructures for both dynamic and/or structural purposes, as well as a means to target non-nucleic acid components to precise locations within a nanostructure scaffold. Here, we review the properties of triplexes that have proved useful in the engineering of DNA nanostructures, with an emphasis on applications that hitherto have not been possible by duplex formation alone.

DNA-based construction at the nanoscale: emerging trends and applications

The field of structural DNA nanotechnology has evolved remarkably-from the creation of artificial immobile junctions to the recent DNA-protein hybrid nanoscale shapes-in a span of about 35 years. It is now possible to create complex DNA-based nanoscale shapes and large hierarchical assemblies with greater stability and predictability, thanks to the development of computational tools and advances in experimental techniques. Although it started with the original goal of DNA-assisted structure determination of difficult-to-crystallize molecules, DNA nanotechnology has found its applications in a myriad of fields. In this review, we cover some of the basic and emerging assembly principles: hybridization, base stacking/shape complementarity, and protein-mediated formation of nanoscale structures. We also review various applications of DNA nanostructures, with special emphasis on some of the biophysical applications that have been reported in recent years. In the outlook, we discuss further improvements in the assembly of such structures, and explore possible future applications involving super-resolved fluorescence, single-particle cryo-electron (cryo-EM) and x-ray free electron laser (XFEL) nanoscopic imaging techniques, and in creating new synergistic designer materials.