Veikko Linko

The enabled state of DNA nanotechnology

It is notoriously difficult to observe, let alone control, the position and orientation of molecules due to their small size and the constant thermal fluctuations that they experience in solution. Molecular self-assembly with DNA enables building custom-shaped nanometer-scale objects with molecular weights up to the megadalton regime. It provides a viable route for placing molecules and constraining their fluctuations in user-defined ways, thereby opening up completely new avenues for scientific and technological exploration. Here, we review progress that has been made in recent years toward the state of an enabled DNA nanotechnology.

Virus-Encapsulated DNA Origami Nanostructures for Cellular Delivery

DNA origami structures can be programmed into arbitrary shapes with nanometer scale precision, which opens up numerous attractive opportunities to engineer novel functional materials. One intriguing possibility is to use DNA origamis for fully tunable, targeted, and triggered drug delivery. In this work, we demonstrate the coating of DNA origami nanostructures with virus capsid proteins for enhancing cellular delivery. Our approach utilizes purified cowpea chlorotic mottle virus capsid proteins that can bind and self-assemble on the origami surface through electrostatic interactions and further pack the origami nanostructures inside the viral capsid. Confocal microscopy imaging and transfection studies with a human HEK293 cell line indicate that protein coating improves cellular attachment and delivery of origamis into the cells by 13-fold compared to bare DNA origamis. The presented method could readily find applications not only in sophisticated drug delivery applications but also in organizing intracellular reactions by origami-based templates.

Dielectrophoretic Trapping of DNA Origami

In this thesis three-dimensional tube-shaped DNA-origamis were dielectrophoretically trapped within lithographically fabricated nanoelectrodes. The origamis had been premade while the electrodes were fabricated specifically for these experiments with two different gapsizes, 150 nm and 400 nm. The aim of the work was to capture individual nanotubes in the gap between the electrodes by utilizing the dielectrophoretic forces present in the structure when a solution containing the origamis was put onto the electrodes and a voltage was applied. It was observed during the experiments that the success of the dielectrophoretic trapping depended strongly on the trapping conditions. This caused the trapping to be somewhat challenging and it was also noticed that the electrode structure with the 400 nm gap particularly required patience in order to produce good results, since the origamis to be trapped were of the same size as the gap between the electrodes making the successful trapping problematic. Despite this, a sufficient amount of trapped single nanotubes were produced.

DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices.

DNA molecules can be assembled into custom predesigned shapes via hybridization of sequence-complementary domains. The folded structures have high spatial addressability and a tremendous potential to serve as platforms and active components in a plethora of bionanotechnological applications. DNA is a truly programmable material, and its nanoscale engineering thus opens up numerous attractive possibilities to develop novel methods for therapeutics. The tailored molecular devices could be used in targeting cells and triggering the cellular actions in the biological environment. In this review we focus on the DNA-based assemblies - primarily DNA origami nanostructures - that could perform complex tasks in cells and serve as smart drug-delivery vehicles in, for example, cancer therapy, prodrug medication, and enzyme replacement therapy.

Ionic Permeability and Mechanical Properties of DNA Origami Nanoplates on Solid-State Nanopores

While DNA origami is a popular and versatile platform, its structural properties are still poorly understood. In this study we use solid-state nanopores to investigate the ionic permeability and mechanical properties of DNA origami nanoplates. DNA origami nanoplates of various designs are docked onto solid-state nanopores where we subsequently measure their ionic conductance. The ionic permeability is found to be high for all origami nanoplates. We observe the conductance of docked nanoplates, relative to the bare nanopore conductance, to increase as a function of pore diameter, as well as to increase upon lowering the ionic strength. The honeycomb lattice nanoplate is found to have slightly better overall performance over other plate designs. After docking, we often observe spontaneous discrete jumps in the current, a process which can be attributed to mechanical buckling. All nanoplates show a nonlinear current-voltage dependence with a lower conductance at higher applied voltages, which we attribute to a physical bending deformation of the nanoplates under the applied force. At sufficiently high voltage (force), the nanoplates are strongly deformed and can be pulled through the nanopore. These data show that DNA origami nanoplates are typically very permeable to ions and exhibit a number of unexpected mechanical properties, which are interesting in their own right, but also need to be considered in the future design of DNA origami nanostructures.

Characterization of the conductance mechanisms of DNA origami by AC impedance spectroscopy.

For some time now, ample interest has been directed to DNA as a functional or electrically operational molecule. This is due to the superior self-assembly properties that may give DNA a major role in future bottom-up fabrication processes. A striking example of DNA self-assembly techniques is DNA origami, which involves folding a long single-stranded DNA molecule with the help of short oligonucleotides, that is, staple strands. Furthermore, each of the staple strands can serve as a functionalization center (i.e., the template has individually addressable pixels with 6 6-nm spacing). This has further led to the idea that such origami could serve as a nanobreadboard for complex self-assembled nanoelectronic systems. Due to this, apart from the fundamental interest, the intriguing question of DNA electrical conductivity gains further importance: the electrical properties of such a nanobreadboard must be well understood before it can be exploited in nanotechnology. In this Communication, we measure the conductivity, and experimentally analyze the conductivity mechanisms, of single rectangular DNA origamis trapped and immobilized between nanoelectrodes by utilizing alternating-current impedance spectroscopy (AC-IS). The experiments show that the nature of the DNA origami conductivity is not purely Ohmic but that it is a combination of ionic diffusion and electronic conductivity, with a resistance

Custom-shaped metal nanostructures based on DNA origami silhouettes

The DNA origami technique provides an intriguing possibility to develop customized nanostructures for various bionanotechnological purposes. One target is to create tailored bottom-up-based plasmonic devices and metamaterials based on DNA metallization or controlled attachment of nanoparticles to the DNA designs. In this article, we demonstrate an alternative approach: DNA origami nanoshapes can be utilized in creating accurate, uniform and entirely metallic (e.g. gold, silver and copper) nanostructures on silicon substrates. The technique is based on developing silhouettes of the origamis in the grown silicon dioxide layer, and subsequently using this layer as a mask for further patterning. The proposed method has a high spatial resolution, and the fabrication yields can approach 90%. The approach allows a cost-effective, parallel, large-scale patterning on a chip with fully tailored metallic nanostructures; the DNA origami shape and the applied metal can be specifically chosen for each conceivable implementation.

DNA-based enzyme reactors and systems

During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme) cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications.

Dielectrophoretic trapping of multilayer DNA origami nanostructures and DNA origami-induced local destruction of silicon dioxide

DNA origami is a widely used method for fabrication of custom‐shaped nanostructures. However, to utilize such structures, one needs to controllably position them on nanoscale. Here we demonstrate how different types of 3D scaffolded multilayer origamis can be accurately anchored to lithographically fabricated nanoelectrodes on a silicon dioxide substrate by DEP. Straight brick‐like origami structures, constructed both in square (SQL) and honeycomb lattices, as well as curved “C”‐shaped and angular “L”‐shaped origamis were trapped with nanoscale precision and single‐structure accuracy. We show that the positioning and immobilization of all these structures can be realized with or without thiol‐linkers. In general, structural deformations of the origami during the DEP trapping are highly dependent on the shape and the construction of the structure. The SQL brick turned out to be the most robust structure under the high DEP forces, and accordingly, its single‐structure trapping yield was also highest. In addition, the electrical conductivity of single immobilized plain brick‐like structures was characterized. The electrical measurements revealed that the conductivity is negligible (insulating behavior). However, we observed that the trapping process of the SQL brick equipped with thiol‐linkers tended to induce an etched “nanocanyon” in the silicon dioxide substrate. The nanocanyon was formed exactly between the electrodes, that is, at the location of the DEP‐trapped origami. The results show that the demonstrated DEP‐trapping technique can be readily exploited in assembling and arranging complex multilayered origami geometries. In addition, DNA origamis could be utilized in DEP‐assisted deformation of the substrates onto which they are attached.

Defined-size DNA triple crossover construct for molecular electronics: modification, positioning and conductance properties

We present a novel, defined-size, small and rigid DNA template, a so-called B-A-B complex, based on DNA triple crossover motifs (TX tiles), which can be utilized in molecular scale patterning for nanoelectronics, plasmonics and sensing applications. The feasibility of the designed construct is demonstrated by functionalizing the TX tiles with one biotin-triethylene glycol (TEG) and efficiently decorating them with streptavidin, and furthermore by positioning and anchoring single thiol-modified B-A-B complexes to certain locations on a chip via dielectrophoretic trapping. Finally, we characterize the conductance properties of the non-functionalized construct, first by measuring DC conductivity and second by utilizing AC impedance spectroscopy in order to describe the conductivity mechanism of a single B-A-B complex using a detailed equivalent circuit model. This analysis also reveals further information about the conductivity of DNA structures in general.