Future Trends in DNA-based Nanotechnology

We build branched DNA species that can be joined using Watson-Crick base pairing to produce N-connected objects and lattices. We have used ligation to construct DNA topological targets, such as knots, polyhedral catenanes, Borromean rings and a Solomon's knot. Nanorobotics is a key area of application. We have made robust 2-state and 3-state sequence-dependent programmable devices and bipedal walkers. We have constructed 2-dimensional DNA arrays with designed patterns from many different motifs. We have used DNA scaffolding to organize active DNA components. We have used pairs of 2-state devices to capture a variety of different DNA targets. We have constructed a molecular assembly line using a DNA origami layer and three 2-state devices, so that there are eight different states represented by their arrangements. We have demonstrated that all eight products can be built from this system. We have self-assembled a 3D crystalline array and reported its crystal structure to 4 $\AA$ resolution. We can use crystals with two molecules in the crystallographic repeat to control the color of the crystals. Rational design of intermolecular contacts has enabled us to improve crystal resolution to better than 3 $\AA$. We can now do strand displacement in the crystals to change their color, thereby making a 3D-based molecular machine; we can visualize the presence of the machine by X-ray diffraction. The use of DNA to organize other molecules is central to its utility. Earlier, we made 2D checkerboard arrays of metallic nanoparticles, and have now organized gold particles in 3D. Most recently, we have ordered triplex components within the same lattice. Thus, structural DNA nanotechnology has fulfilled its initial goal of controlling the internal structure of macroscopic constructs in three dimensions. A new era in nanoscale control awaits us.

I'll discuss how to use DNA to construct and visualize nanoscale structures. We have invented a general framework to program DNA/RNA strands to self-assemble into structures with user-specified geometry or dynamics. By interfacing these nanostructures with other functional molecules, we have introduced digital programmability into diverse application areas, e.g. fabrication of inorganic nanoparticles with arbitrary prescribed shapes, robust DNA probes with near optimal binding specificity, RNA-based genetically encodable translation regulators with unprecedented dynamic range and orthogonality, and a highly multiplexed (10× demonstrated), precisely quantitative (>90% precision), and ultra-high resolution (sub-5 nm) optical imaging method. For more details of our work, see http://molecular.systems.

Super-resolution fluorescence microscopy is a powerful tool for biological research, but obtaining multiplexed images for a large number of distinct target species in whole cells and beyond remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, a variation of point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed super-resolution imaging that achieves sub-10-nm spatial resolution in vitro on synthetic DNA structures. We report a multiplexing approach (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-color super-resolution imaging in vitro on synthetic DNA structures as well as four-color two-dimensional imaging and three-color 3D imaging of proteins in fixed cells. Finally, we demonstrate whole cell imaging using DNA- and Exchange-PAINT and optical sectioning, now allowing DNA-based super-resolution imaging deep inside cells, away from the glass coverslip.

Due to its nanoscale dimensions and ability to self-assemble via specific base pairing, DNA is rapidly taking on a new aspect where it is finding use as a construction element for architecture on the nanoscale. Structural DNA nanotechnology has yielded architectures of exquisite complexity and functionality in vitro.1 However, till 2009, the functionality of such synthetic DNA-based devices in living organisms remained elusive.1 Work from my group the last few years has bridged this gap where, we have chosen architecturally simple, DNA-based molecular devices and shown their functionality in complex living environments.2,3 I will describe our recent work describing the design and application of a cell targetable icosahedral DNA nanocapsule4 to illustrate the potential of DNA based molecular devices as unique tools with which to interrogate living systems.5, References. [1] Chakraborty, K., et al.. Nucleic acid based nanodevices in biological imaging. Ann. Rev. Biochem. 2016 in press. [2] Surana, S., et al. Designing DNA nanodevices for compatibility with the immune system of higher organisms. Nat. Nanotechnol. 2015, 10, 741-747. [3] Modi, S., et al. A DNA nanomachine that maps spatial and temporal pH changes in living cells. Nat. Nanotechnol. 2009, 4, 325-330. [4] Bhatia, D. et al, A synthetic icosahedral DNA-based host–cargo complex for functional in vivo imaging Nature Communications, 2011, 2, 339. [5] Bhatia, D. et al Quantum-dot loaded, monofunctionalized DNA icosahedra for single particle tracking of endocytic pathways. Nat. Nanotechnol. 2016, 11,1112-1119.

Discrete plasmonic nanostructures have attracted significant attentions for cutting-edge research in material sciences, optical modulation, and biosensing, etc. In this talk, we will introduce our recent efforts in precise construction of discrete plasmonic nanostructures by employing proteins and DNA origamis as templates, in which the compositions and patterns, as well as the spatial configurations, of the plasmonic building blocks in the nanostructures are precisely controlled. Thereafter, new optical properties are observed and are quantitatively studies in these discrete plasmonic nanostructures.

DNA nanostructures and DNA origami can be used to gain insights into how biological systems work at the nanoscale. In particular we are interested in using protein-decorated nanostructures to look at how distances affect biological signaling. The basis for the technology will be described as well as results from both published and recent unpublished experiments. Interestingly, the cell outcomes on a functional level can be directly linked to small changes in nanoscale distributions of ligands.

DNA origami, in which a long scaffold strand is assembled with a large number of short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. Although diverse shapes in 2D are possible, the single-layer rectangle has proven the most popular, as it features fast and robust folding and modular design of staple strands for simple abstraction to a regular pixel surface. Here I will discuss the DNA-origami barrel architecture, built as stacked rings of double helices, that retains these appealing features, while extending construction into 3D. I will discuss how we can use these architectures to scaffold liposome assembly, and how they can serve as building blocks for nanocapsules for future therapeutic application.

DNA origami is a modular platform for the combination of molecular and colloidal components to create optical, electronic, and biological devices. Integration of such nanoscale devices with microfabricated connectors and circuits is challenging: large numbers of freely diffusing devices must be fixed at desired locations with desired alignment. We present a DNA origami molecule whose energy landscape on lithographic binding sites has a unique maximum. This property enables device alignment within +/-3.2 degrees on $SiO_2$. Orientation is absolute (all degrees of freedom are specified) and arbitrary (every molecule’s orientation is independently specified). The use of orientation to optimize device performance is shown by aligning fluorescent emission dipoles within microfabricated optical cavities. Large-scale integration is demonstrated via an array of 3,456 DNA origami with 12 distinct orientations, which indicates the polarization of excitation light.

RNA nanotechnology has the great potential to allow us to produce well-defined nanostructures and devices inside cells and thus open up a wide range of design opportunities in synthetic biology. To achieve this goal we need to understand the design principles of geometry, folding kinetics and topology that will allow us to genetically encode well-defined RNA nanostructures that self-assemble during the transcription process. We have recently introduced the single-stranded RNA origami method and validated the architecture by transcribing RNA tiles that assemble into lattices of different geometries. I will introduce new software tools that allow interactive design of RNA origami structures using a library of functional modules and new sequence design approaches that allow large structures to be designed. Also I will show our latest progress in developing larger three-dimensional RNA origami structures and functional RNA nanodevices with applications in biosensing and diagnostics.

We use DNA origami as a platform for biosensing applications. The modular design allows the combination of different fluorescence sensing effects in self-assembled nanosensing devices. We present data ranging form plasmonic fluorescence enhancement to single-molecule force sensing and single DNA detection.

The precise and controllable arrangement of nanoparticles (NPs) has been a significant challenge because of the difficulty in labeling the relatively uniform surface chemistry of the particles with different organization scaffoldings; this is especially true for anisotropic particles. To achieve differentiated surface chemistry on the NPs, we selectively blocked their surfaces with a diblock copolymer (polystyrene-b-polyacrylic acid) by tuning the surface energy of the different NPs, resulting in different accessibilities to their surfaces. Modification of the polymer-free region on the surface of the NPs with single-stranded DNA leads to controllable and site-specific functionalization. The directionality and arrangement of the DNA-functionalized NPs is thereby specific and programmable and allows for increased order and complexity. This method has been shown to work on both isotropic and anisotropic particles of a variety of sizes and shapes. To date, we have constructed a series of programmable encapsulated nanoparticles (PENPs) with different binding affinities based on various nanoparticles with different shapes (NS, 0-D sphere; NR, 1-D rod; 2-D prism; and 3-D cube), different compositions (Au, Pd) and different sizes (20-100 nm). These particles can have then been combined into several different assembly motifs such as spherical gold NPs (AuNSs) dimers with same- or different-sized monomers, AuNR-AuNS dimer, AuNRs dimer (end-to-end or side-by-side), AuNRs linear chain, (-AuNR-AuNS-)X linear chain, AuNS-AuNR-AuNS trimer, etc. Since this strategy has been demonstrated generally on metal NPs, it is certainly worth applying it to other types of NPs, such as quantum dots, metal oxides, and magnetic NPs. The specificity of the DNA hybridization could programmably link PENPs with different composition, chemical functionality, or electrical conductivity into well-defined sequences and orientations, allowing for more complex materials.

We are using DNA as a programmable tool for directing the self-assembly of molecules and materials. The unique specificity of DNA interactions and our ability to synthesize artificial functionalized DNA sequences makes it the ideal material for controlling self-assembly and chemical reactions of components attached to DNA sequences. In particular we are using DNA origami, large self-assembled DNA structures as a template for positioning of materials such as organic molecules, dendrimers and biomolecules. Recently, we have developed a method for DNA-templated conjugation of DNA to proteins such as antibodies that in turn allows us to immobilize these structures on origami. We have also used DNA origami to image chemical reactions with single molecule resolution and as a readout display for molecular computing, The main focus of the presentation will be on a recently prepared conjugated DNA-phenylene vinylene polymer and its self-assembly on DNA origami for studies of optical properties.

Structural DNA nanotechnology has brought nanometer-precision fabrication to a molecular biology lab. Advancing the method further requires methods for controlling self-assembly down to the atomic scale and programming functional motions. In this talk I will describe how microscopic simulations can be used to characterize the structure of large DNA origami objects and examine their function.

DNA based self-assembly has been developed to design and construct nanoscale architectures with sophisticated morphologies. The spatial addressability of DNA nanostructures and sequence-dependent recognition enable functional elements to be precisely positioned to achieve customized properties. We show that DNA origami nanostructures can guide the assembly of achiral metallic nanoparticles into chiral geometries to generate tunable Circular Dichroism (CD) response in the visible to near IR region. We demonstrate that DNA origami nanostructures can deliver not only anti-cancer drug for chemotherapy but also metal nanostructures for photoacoustic imaging and photothermal therapy in vivo.

The DNA double-strand recognition, as well as the ability to manipulate its structure open a multitude of ways to make it useful for molecular electronics. Step by step we improve the synthesized constructs and the measurement methods of single DNA-based molecules. I will present new DNA-based metalized molecules and report on our measurements of their energy level structure and transport properties.

This talk will cover our work on in vivo computation and low-cost diagnostics using programmable RNA devices.

Lipid-bilayer membranes form barriers to define the boundaries of a cell and its subcellular compartments. With the help of membrane-associating molecules, they undergo dramatic structural changes during the life cycle of cells and mediate complex reactions that are vital to cell division, growth and communication. Inspired by such elegance in nature, bioengineers and synthetic biologists have aspired to build artificial membranes to recapitulate the cellular membrane structure and dynamics. In addition, such in vitro preparations provide a complexity-reduced system for cell biologists and biophysicists to study functional interactions between membranes and their associating molecules. Despite the advanced chemical and physical methods that are now available to manipulate and observe lipid membranes, two technical challenges have hampered our ability to construct a completely artificial system that mimics natural membrane systems. First, it has been difficult to produce large quantities of mono-dispersed lipid vesicles with well-defined structure (size & shape). Second, it has been challenging to regulate the membrane dynamics (fusion, budding, etc.) in a programmable way. Here we present our progress in resolving these technical limitations. Our approach is to use self-assembled DNA nanostructures as templates to guide the assembly of lipid bilayers and transduce the programmable feature of the DNA nanostructures to the templated vesicles. Specifically, we show the assembly, arrangement, and remodeling of liposomes with designer geometry: all of which are exquisitely controlled by a set of modular, reconfigurable DNA nanocages. Unlike previous methods that rely on trial and error to tune vesicle shape, here the geometry of a liposome is directly programmed into its DNA cage. Tubular and toroidal shapes, among others, are transcribed from DNA cages to liposomes with high fidelity, giving rise to membrane curvatures present in cells yet previously difficult to construct in vitro. Moreover, the conformational changes of DNA cages drive membrane fusion and bending with predictable outcomes, opening up opportunities for the systematic study of membrane mechanics.

The advent of DNA microarray technology in the course of the human genome project in the late 1980s has led to the evolution of sophisticated DNA-functionalized solid substrates which are nowadays routine tools for fundamental and applied research in biology and medicine. However, the versatility of DNA biochips goes far beyond the established applications in genotyping and expression profiling, because their capability for highly parallel, site-directed immobilization of complementary nucleic acids can be harnessed to assemble complex surface architectures comprised of colloidal materials and proteins. While this approach enables novel sensor platforms for protein and small-molecule analysis, the full potential of DNA surfaces can be exploited by implementation of structural DNA nanotechnology. We have previously applied this DNA chip-based approach to investigate cell adhesion and the recruitment of transmembrane proteins in living cells. By combining DNA micro- and nanostructures, we have now developed “multiscale origami structures as interface for cells” (MOSAIC). This tool allows one to present ligands to living cells on surfaces with a full control over their stoichiometry and nanoscale orientation, thereby enabling to address fundamental questions in cell signalling which cannot be tackled by conventional technologies.

The stimuli-triggered reconfiguration of DNA nanostructures has been widely applied for developing DNA machines and supramolecular DNA assemblies. Recent research efforts have applied the switchable, stimuli-responsive DNAs to develop new functional nanomaterials applicable to DNA nanotechnology. These activities will be discussed with the following examples: 1. Oligomers of DNA origami tiles, cross-linked by stimuli-responsive bridges, were prepared. The programmed signal-triggered cleavage of the tiles and their isomerization will be introduced. The use of origami tiles as dynamic scaffolds for the organization of plasmonic nanoparticles will be presented. 2. The synthesis of stimuli-responsive DNA-based hydrogels will be described. The control over the stiffness of the hydrogels by external triggers will be addressed, and the use of the hydrogels as shape-memory matrices and materials, exhibiting triggered modulated mechanical properties, will be introduced. Specifically, the immobilization of stimuli-responsive hydrogels on surfaces and the use of the hydrogels to assemble ion-channels and ion-pumps will be discussed. 3. The synthesis of stimuli-responsive DNA-based micro- and nano-carriers, such as SiO2 nanoparticles, microcapsules and metal-organic framework nanoparticles, will be presented. The biomarker-triggered unlocking of drug-loaded DNA-capped carriers and their cytotoxicity toward cancer cells will be described. 4. DNA-based constitutional dynamic networks (CDNs) will be introduced as functional stimuli-responsive DNA assemblies. The over-expression and under-expression of the constituents of CDNs and the control over the catalytic functions and mechanical functions of signal-triggered CDNs will be discussed. Specifically, inter-communication between CDNs and CDNs of enhanced complexities that reveal feedback or oscillatory functions will be highlighted.

The application of DNA-based structures in electronic or optical devices requires the functionalization of the DNA. To date, this is mostly realized by integration of metallic nanoparticles or by direct metal deposition. The incorporation of rigid and stiff inorganic matter can cause deformation of the rather soft DNA structures. Therefore, it could be more attractive to build in electrical or optical functionality by functional polymers such as π-conjugated polymers (CPs). These organic (semi)-conductors are lightweight and flexible. They have been already applied in diverse electronic and optoelectronic devices. But so far, only few approaches have appeared to attach CPs on DNA origami structures1,2. The main issue of these approaches is the lack of CPs with a defined molecular order and orientation. Both have a tremendous influence on the physical functionalities of these polymers. Here, we used polythiophenes of the P3RT-type as CP elements. The applied polymerization of these polythiophenes, the so called Kumada catalyst-transfer polycondensation, follows a chain-growth mechanism. The resulting polymers are characterized by a narrow dispersity, adjustable molecular weights (MWs) and the possibility of in-situ introduction of start/end groups3. Furthermore, we established a block copolymer design to incorporate oligodeoxynucleotide (ODN) functionality for the attachment to DNA origami structures. The block copolymer design enables us to attach a defined number of polythiophene molecules to the DNA template. First, we prepared the semiconducting polythiophene, P3(EO)3T having oligoethylene glycol side chains to provide water-solubility. An amine starting group was introduced via a functionalized catalyst that gives access to click chemistry. Accordingly, the P3(EO)3T was bound to an end-modified, synthetic ODN yielding to a well-defined block copolymer P3(EO)3T-b-ODN. 1H-NMR spectroscopy, size exclusion chromatography and dynamic light scattering measurements confirmed the well-defined molecular structure of the P3(EO)3T polymer resulting in a reasonable degree of starting group functionalization, narrow dispersity, adjustable MW and a monomodal distribution of the polymer particles in solution. Furthermore, we could maintain the well-defined structure also for the block copolymer. HPLC and asymmetric flow field-flow fractionation measurements confirmed the monomodal distribution with a narrow dispersity. The block copolymer was then site-specifically attached to a rectangular 2D DNA origami, called pad. The pad bears 40 attachment sites arranged in four parallel lanes with 10 sites each. The resulting hybrid structure P3(EO)3T@pad was investigated by high-resolution AFM. Intriguingly, the polymer objects were arranged preferentially on the inner lanes indicating attractive interactions between the polymer chains, probably due to π-π-stacking. This assumption could be confirmed by the surfactant-induced break-up of these interactions resulting in increasing emission intensity. The presence of π-π-stacked polymer conjugates is required for any electronic functionality between the polymer molecules. 1. Wang, Z.-G., Liu, Q. & Ding, B., Chem. Mater. 26, 3364–3367 (2014). 2. Knudsen, J. B. et al., Nat. Nanotechnol. 10, 892–898 (2015). 3. Kiriy, A., Senkovskyy, V. & Sommer, M. Macromol. Rapid Commun. 32, 1503–1517 (2011).

Self-assembled DNA origami is an emerging tool for nanofabrication of well-designed programmable structures. One of the key assignments of DNA origami is an easy arrangement of fluorophores, nanoparticles or proteins with nanometer precision. This enables facile functional hybrid platforms with unprecedented control. Within billion years of evolution, natural photosynthetic antennas present in algae, bacteria and green plants evolved very efficient building blocks for artificial light harvesting. In order to take advantage of these building blocks, scientist have intensively investigated their structural and optical properties.[1] At the same time, great efforts have been made to integrate natural light-harvesting antennas with plasmonic nanostructures to improve their photostability and increase fluorescence efficiency.[2] In this contribution, we employ peridinin-chlorophyll-protein (PCP) complex to fabricate optical antennas based on a DNA origami pillar and metallic nanoparticles.[3-5] This is the first experimental realization of the system in which a photosynthetic protein is positioned in the nanoantenna hotspot in a controlled manner with nanometer precision. This approach shows promises for fabrication biohybrid photonic devices and sensors composed of DNA origami, metallic nanoparticles and naturally evolved photosynthetic complexes. IK acknowledges support by the Mobility Plus grant 1269/MOB/IV/2015/0 from the Polish Ministry of Science and Higher Education (MNiSW). [1] R. Croce, H. v. Amerongen, Nature Chemical Biology, 10, 492 (2014). [2] E. Wientjes, J. Renger, A. G. Curto, R. Cogdell, N. F. v. Hulst, Nature Communications, 5, 4236 (2014). [3] G. P. Acuna, F. M. Möller, P. Holzmeister, S. Beater, B. Lalkens, P. Tinnefeld, Science, 338, 506 (2012). [4] A. Puchkova, C. Vietz, E. Pibiri, B. Wuensch, M. Sanz Paz, G. P. Acuna, P. Tinnefeld, Nano Letters, 15, 8354 (2015). [5] C. Vietz, B. Lalkens, G. P. Acuna, P. Tinnefeld, New Journal of Physics, 18, 045012 (2016).

Selective configuration control of plasmonic nanostructures has remained challenging using both top-down and bottom-up approaches in the field of active plasmonics. Here we demonstrate the realization of DNA origami-based reconfigurable plasmonic metamolecules which can respond to a wide range of pH changes in a programmable manner (1). Such programmability allows for selective reconfiguration of different plasmonic metamolecule species coexisting in solution through simple pH tuning. Importantly, this approach enables discrimination of chiral plasmonic quasi-enantiomers as well as arbitrary tuning of chiroptical effects with unprecedented degrees of freedom. Our work outlines a new blueprint for implementation of advanced active plasmonic systems, in which individual structural species can be programmed to perform multiple tasks and functions. 1. Kuzyk, A. et al. “Selective control of reconfigurable chiral plasmonic metamolecules” Science Advances 2017. In press.

Biological systems have developed a number of mechanisms how to assemble inorganic matter with complex shapes, e.g. by using correspondingly formed biomolecular structures as templates. DNA nanotechnology has recently provided a wealth of techniques to fold DNA into well-defined two- and three-dimensional structures in a programmable manner. Here we explore, how we can use such complex structures to synthesize inorganic materials with programmable shapes. To this end we employ a previously established concept in which rigid three-dimensional DNA origami nanostructures are used as molds to dictate the final shape of metal particles that form by a seeded-growth procedure. We use individual molds as bricks to build extended and more complex mold structures. These support then the formation of extended metal structures, such as µm-long gold nanowires with much higher uniformity than obtained in previous DNA metallization procedures. Transport measurements confirm the electric conductivity of these structures. Using mold elements with different shapes more structural complexity can be introduced.

Scaffolded DNA origami offers the ability to program nearly arbitrary structured DNA nanoparticle geometries on the 10 to 100 nanometer scale. Applications of these assemblies include programmed therapeutic delivery, metallic nanoparticle fabrication, designer excitonic circuits, and self-assembly of higher-order 2D and 3D crystalline materials. To facilitate the autonomous design of complex nanoparticle geometries for these preceding applications, we have developed fully automated, top-down sequence design algorithms for the synthesis of nearly arbitrary target geometric shapes based on scaffolded DNA origami. Anti-parallel (DX) or parallel (PX) crossover motifs may be used to program nanoparticles with either synthetic staple strands or single-stranded DNA scaffold alone. Nanoparticle edges may be composed of either two, six, or arbitrary alternative numbers of parallel helices, depending on the target size and mechanical properties desired. Asymmetric PCR is implemented to synthesize fully synthetic scaffold sequences on the 2 to 12kb scale without relying on viral genomes that contain protein-coding sequences. Folding assays are used together with 3D cryo-electron microscopic reconstructions to validate nanoparticle designs.

How to precisely control the shape and size of final assemblies, especially using same amphiphilic molecules and under the same environmental conditions, is always a challenge in molecular assembly. Inspired by the cytoskeletal/membrane protein/lipid bilayer system that determines the shape of eukaryotic cells, we proposed and ‘the Frame Guided Assembly’ (FGA) strategy to prepare heterovesicles with programmed geometry and dimensions. This method offers greater control over self-assembly: with same molecular system, the size of final assemblies could be tuned at 1 nm level and their shape could vary from spherical to cubic, and even given sized two dimensional sheets. Most importantly, the principle of the FGA could be applied to various materials such as bock copolymers, small molecules including surfactants and lipids, which is a general rule in self-assembly.

Information relay at the molecular level is an essential phenomenon in numerous chemical and biological processes, such as intricate signaling cascades. One key challenge in synthetic molecular self-assembly is to construct artificial structures that imitate these complex behaviors in controllable systems. We demonstrated prescribed, long-range information relay in an artificial molecular array assembled from modular DNA structural units. The dynamic DNA molecular array exhibits transformations with programmable initiation, propagation, and regulation. The transformation of the array can be initiated at selected units and then propagated, without addition of extra triggers, to neighboring units and eventually the entire array. The specific information pathways by which this transformation occurs can be controlled by altering the design of individual units, the connections between units, and the geometry of the array.

One of the great challenges in cellular studies is to develop a rapid and biocompatible analytical tool for single-cell analysis. We report a rapid, DNA nanostructure-supported aptamer pull-down (DNaPull) assay under convective flux in a micro-channel for analyzing contents of droplets with nano or pico-liter volumes. We have demonstrated that the scaffolded aptamer can greatly improve the efficiency of target molecules’ pull-down. The convective flux enables complete reaction in less than 5 min, which is an 18-fold improvement compared to purely diffusive flux (traditional model of stationary case). Significantly, the designed microfluidic device compartmentalizes live cells into nanoliter-sized droplets to present single-cell samples. As a proof of concept, we demonstrated that cellular molecules (ATP) from a discrete number of HNE1 cells (0, 1, 2, 3, 4, 5 cells) lysed inside nanoliter sized droplets can be quantified, which provides a new paradigm in biosensor design and will be valuable for single cell analysis.

DNA nanostructures featuring gold nanoparticles have been studied extensively for the past twenty years. This fruitful association stems from the fact that DNA nanotechnology and plasmonics are enabling technologies for one another: well-defined DNA architectures allow the design of gold nanostructures with unprecedented optical properties (such as optical sensors, resonators for surface enhanced Raman scattering and fluorescence, as well as chiral plasmonic structures); while surface plasmon resonances provide nanoscale distance information on a DNA template as well as local heat sources for DNA melting. In this talk, I will review some of our recent work on the controlled interaction between one or two fluorescent molecules and optical nanoantennas using DNA; and on the parallel colorimetric monitoring of nanoscale DNA conformation changes using plasmon resonance spectroscopy.

G. Gines, A. Zadorin, J.-C Galas, T. Fujii, A. Estevez-Torres, Y. Rondelez Information stored in the sequence of synthetic nucleic acids can be used in vitro to create complex reaction networks implementing precisely programmed chemical dynamics. While this is generally done using free-floating oligonucleotides in bulk solution, we report here the extension of this approach to program the local and individual chemical behavior of microscopic solid “agents”. The DNA-programmed agents possess multiple stable states, thus maintaining a simple form of memory, and communicate by emitting various orthogonal signals. They can this sense the behavior of neighboring agents and use these stimulus to decide their own action. We build on these elements to create collective behaviors involving thousands of different agents, for example retrieving information over long distances, or creating spatial patterns. The possibility to scale up the information-processing capability of DNA-encoded artificial systems could find applications where many individual clues need to be combined to reach a decision.

Membrane nanopores and ion channels are essential in cells and control the transport of molecular cargo across bilayers. Replicating biological channels with artificial, rationally designed nanostructures can open up new applications. I describe the generation of stable self-assembled DNA nanopores that insert into lipid bilayers to facilitate transport across the membranes. The DNA channels are composed of interlinked duplexes and carry lipid anchors to hold the negatively charged channels in the membrane(1,2,3). One DNA version mimics the function of biological ligand-gated ion channels where a DNA ligand can re-open the channel(2). The pore can also distinguish with high selectivity the transport of small-molecule cargo that differs by the presence of a positive or negative charge. The synthetic analogue may be used for controlled drug release and the building of cell-like networks. Related DNA channels show other hallmarks of the biological templates such as voltage-gating at high transmembrane potentials(2,3,4). The artificial pores can furthermore be programmed to function as cytotoxic agents by killing cancer cells via membrane-rupturing(5). The synthetic pores expand the range of other DNA nanostructures that mimic biological functions of membrane proteins to control bilayer and cell shape(6). References: (1) Nano Lett. 2013 13 2351; Angew. Chem. Int. Ed. 2013 52 12069. (2) Nat. Nanotechnol. 2016 11 152. (3) ACS Nano 2015 9 1117. (4) ACS Nano 2015 9 11209. (5) Angew. Chem. Int. Ed. 2014 53 12466; Nat. Chem. 2014 7 17. (6) Science 2016 352 890.

The initial proposal catalyzing the rapid development of DNA nanotechnology was to arrange periodic DNA frameworks to host guest molecules for crystal structure analysis. Despite enormous efforts and great successes, placing guest molecules in designed DNA crystals remains a challenging goal. Ned Seeman and Chengde Mao reported a 3D DNA crystal based on the "tensegrity triangle", where three DNA duplexes are interconnected in a self-restricting over-under, over-under, over-under fashion. By adopting this design principle, we here present a tensegrity triangle design based on DNA origami that crystallizes into three dimensional, micrometer-scale assemblies. TEM and SEM analysis confirmed periodic assembly on the micrometer scale and surprisingly good order over longer distances. One of the advantages of using our comparatively large and rigid DNA origami building blocks lies in the possibility to host up to 30 nm large guest molecules such as gold nanoparticles of different sizes. SAXS measurement showed excellent scattering peaks from the DNA origami crystals and the corresponding peaks for Au nanoparticle precisely arranged on the guest molecule positions. I will also present a method to study force interactions between biomolecules. Well-established techniques such as atomic force microscopy and magnetic or optical tweezers are usually applied to investigate protein folding or biopolymer elasticity. In contrast to those techniques, our nanoscopic DNA origami-based single-molecule force spectroscopy device has no physical connection to the macroscopic world such as a micrometer-sized bead or a cantilever. We exploit the entropic elasticity of single-stranded DNA to apply tension on a system mounted on the device and single-molecule Förster Resonance Energy Transfer (smFRET) is used as a readout to study two dynamic systems under different tensions: the transition behavior of a Holliday junction and the bending of a DNA promotor sequence induced by the TATA-binding protein (TBP). We are able to generate reliable single-molecule force spectroscopy data in the piconewton range in a high throughput fashion. In the future, our DNA origami force spectrometer will be employed to study a wide variety of DNA interacting biomolecules.

Colloidal nanoparticles offer a range of interesting optical and electronic effects. A prominent example is the localized surface plasmon resonance (LSPR) of metal nanoparticles due to resonant excitations of vibrations of the particles’ free-electron cloud by light. Due to the LSPR, plasmonic nanoparticles provide excellent means for controlling electromagnetic near-fields at optical frequencies, which has led to a broad range of applications in various field such as surface enhanced spectroscopy, light harvesting or photonics. While much research is dedicated to understanding nanoparticle synthesis and tailor their LSPR on the single particle level [1], ordering particles on surfaces opens another powerful avenue towards optical and electronic functionality. Plasmonic particles can couple locally, altering their LSPR, but as well collective long range phenomena can give rise to novel effects. In this context, methods for ordering particles that are scalable to macroscopic areas are of great interest. We discuss, how biomimetic approaches can contribute to solving this technological challenge. In particular we focus on controlled wrinkling as a versatile means for surface patterning and its application in template assisted self assembly [2]. We discuss the underlying physico-chemical effects and perspectives for applications in Surface Enhanced Raman Spectroscopy and Photonics.   References [1] Mayer, M.; et al. Nano Letters 2015, 15, 5427-5437. [2] Tebbe, M.; et al. Faraday Discussions 2015, 181, 243-260; Hanske, C.; et al. Nano Letters 2014, 14 (12), 6863 - 6871. Pazos-Perez, N.; et al. Chemical Science 2010, 1 (2), 174-178.

Guillermo P. Acuna†, Carolin Vietz†, Izabela Kaminska†, Qingshan Wei┴, Aydogan Ozcan┴ and Philip Tinnefeld† † Institute for Physical & Theoretical Chemistry – NanoBioScience, and LENA (Laboratory for Emerging Nanometrology), and BRICS (Braunschweig Integrated Center for Systems Biology), Braunschweig University of Technology, Rebenring 56, 38106 Braunschweig, Germany ┴ Bioengineering Department, University of California, Los Angeles, Los Angeles, CA, 90095, USA Email: g.acuna@tu-bs.de In this contribution, we will show that DNA origami based [1] optical antennas can outperform their lithographic counterparts in terms of fluorescence enhancement and single molecule detection at elevated concentrations. We demonstrate that an enhancement factor of 5000 and single molecule detection at 25 µM can be achieved [2] with 100 nm gold based dimer antennas [3]. Furthermore, by incorporating silver nanoparticles , a fluorescence enhancement over a broader spectrum in the visible range can be obtained. We will show that dimer antennas based on 80 nm Ag nanoparticles can lead to a strong enhancement in the blue, green and red spectral region [4]. We will exploit this enhancement for the detection of single molecules with low-tech instrumentation such as a smartphones. This approach will enable the development of affordable point of care diagnostic devices [5] for the detection of low-abundant pathogens at an early stage in an environment of substantial background without an amplification step. [1] P. W. Rothemund, Nature 440, (2006). [2] A. Puchkova et al., Nano Letters 15, (2015). [3] C. Vietz, et al., New J. of Phys. 18, (2016). [4] C. Vietz, et al., submitted (2017). [5] Wei, et al., submitted (2017).

DNA has high degree of programmability as well as have ability to self-assemble into complex and dynamic shapes. In this seminar, I will introduce my work in computational design compiler for DNA nano structures, including the geometric theory, systems and experimental results. I will present how to guarantee a mathematical concreteness. I will also demonstrate the distributed systems we built and will finally show the experimental results.

Recent progress in structural DNA nanotechnology has opened up numerous opportunities to use well-defined DNA nanostructures in a variety of bioapplications [1,2]. Using customized DNA structures as smart biochemical nanodevices [3] and targeting cells with DNA-based drug delivery vehicles [4] are arguably some of the most intriguing implementations. In order to enhance the cellular delivery and to improve the stability of DNA nanostructures in biological environment, we have tested different DNA origami coating strategies. Previously, we have demonstrated how 2D DNA origamis can be encapsulated with virus capsid proteins to increase transfection rates [5]. Recently, we have created polymer-origami- and protein-origami-complexes by combining 3D DNA origamis with cationic block-copolymers [6] and modular protein-dendron conjugates [7]. We have observed that the albumin-dendron–based coating of origamis can improve their transfection to HEK293 cells, increase the stability against endonucleases and attenuate activation of immune cells. In addition, we have studied how active enzymes could be delivered into HEK293 cells in vitro when they are attached to a tubular DNA origami [8,9]. We employed bioluminescent enzymes as a cargo and monitored the activity of these delivered enzymes from a cell lysate [9]. The results show that the enzymes stay intact and retain activity in the transfection process. Owing to the modularity of DNA origami, these proposed techniques could become applicable for advanced drug-delivery and therapeutics. [1] V. Linko, H. Dietz*, Curr. Opin. Biotechnol. 24, 555–561 (2013). [2] V. Linko*, M. A. Kostiainen*, Nat. Biotechnol. 34, 826–827 (2016). [3] V. Linko*, S. Nummelin, L. Aarnos, K. Tapio, J. J. Toppari, M. A. Kostiainen*, Nanomaterials 6, 139 (2016). [4] V. Linko, A. Ora, M. A. Kostiainen*, Trends Biotechnol. 33, 586–594 (2015). [5] J. Mikkilä, A.-P. Eskelinen, E. H. Niemelä, V. Linko, M. J. Frilander, P. Törmä, M. A. Kostiainen*, Nano Lett. 14, 2196–2200 (2014). [6] J. K. Kiviaho, V. Linko, A. Ora, T. Tiainen, E. Järvihaavisto, J. Mikkilä, H. Tenhu, Nonappa, M. A. Kostiainen*, Nanoscale 8, 11674–11680 (2016). [7] H. Auvinen, H. Zhang, Nonappa, A. Kopilow, E. H. Niemelä, S. Nummelin, A. Correia, T. Mäkelä, H. A. Santos, V. Linko*, M. A. Kostiainen*, Submitted for publication (2017). [8] V. Linko, M. Eerikäinen, M. A. Kostiainen*, Chem. Commun. 51, 5351–5354 (2015). [9] A. Ora, E. Järvihaavisto, H. Zhang, H. Auvinen, H. A. Santos, M. A. Kostiainen*, V. Linko*, Chem. Commun. 52, 14161-14164 (2016).

DNA nanotechnology offers unique control over matter on the nanoscale. We extend the DNA origami (1) and the single-stranded tile method (2) to cover a range of wireframe truss structures composed of equilateral triangles, which use less material per volume than standard multiple-helix bundles. From a flat truss design, we folded tetrahedral, octahedral, or irregular dodecahedral trusses by exchanging few connector strands. Other than standard origami designs, the trusses can be folded in low-salt buffers that make them compatible with cell culture buffers. The structures also have defined cavities that may in the future be used to precisely position functional elements such as metallic nanoparticles or enzymes. Our graph routing program and a simple design pipeline will enable other laboratories to make use of this valuable and potent new construction principle for DNA-based nanoengineering (Agarwal et al, Nano Lett. 2016, 2108-2113). Finally, a method will be described to fill single-stranded regions in these trusses with a gap filling polymerase to rigidify them.

DNA is not only a wonderful material for nanoscale construction, its hybridization or hydrolysis can be used to provide energy for synthetic molecular machinery. With DNA it is possible to design and build three-dimensional scaffolds, to attach molecular components to them with sub-nanometre precision – and then to make them move. I shall describe our work on DNA origami assembly pathways and the use of synthetic molecular machinery to control covalent chemical synthesis. Assembly Pathways[1]: DNA origami is a robust molecular assembly technique by which a single-stranded DNA template is folded by annealing with hundreds of short ‘staple’ strands[2]. There is a strong analogy with protein folding: both are governed by information encoded in polymer sequence[3]. We present an origami structure that has the unusual property of having a small set of distinguishable, well-folded shapes that represent discrete and approximately degenerate energy minima among 10$^{23}$ disordered alternative staple configurations. We obtain a high yield of well-folded structures, demonstrating that efficient folding pathways exist[4], and show that the assembly pathway can be steered by rational design. We identify similarities to protein folding: assembly is highly cooperative; reversible bond-formation is important in recovering from transient misfoldings; and the early formation of long-range connections can be very effective in forcing particular folds. Programmable Synthesis[5]: We report the programmed synthesis of peptides and unnatural oligomers using synthetic molecular machinery made from DNA to control and record the formation of covalent bonds[6]. DNA-templated covalent bond formation is controlled by an autonomous cascade of DNA hybridization reactions which brings reactants together in a controlled sequence defined by a reconfigurable molecular program. The sequence of assembly reactions, and thus the structure, of each oligomer synthesized is recorded in a DNA molecule which enables this information to be recovered by PCR amplification followed by DNA sequencing. We apply this technique to the programmed synthesis of two families of oligomers with different backbone chemistries: peptide, with the potential for biomimetic synthesis, and Wittig, creating bio-orthogonal linkages. The system can also be programmed to achieve combinatorial assembly, opening up the possibility of applications in drug discovery. ________________________________ 1. Dunn, K. E., Dannenberg F. et al., Nature 525, 82–86 (2015) 2. Rothemund, P. W. K. Nature 440, 297-302 (2006) 3. Anfinsen C. B. Science 181, 223-230 (1973) 4. Levinthal, C. Univ. Illinois Bull. 67, 22-24 (1969) 5. W. Meng et al., Nature Chem. (2016) doi:10.1038/nchem.2495 6. X. Li & D. R. Liu, Angew. Chem. Int. Ed. 43, 4848-4870 (2004)

We describe an approach to bottom-up fabrication that allows integration of the functional diversity of proteins into designed three-dimensional structural frameworks. A set of custom staple proteins based on transcription activator–like effector proteins folds a double- stranded DNA template into a user-defined shape. Each staple protein is designed to recognize and closely link two distinct double-helical DNA sequences at separate positions on the template. We present design rules for constructing megadalton-scale DNA-protein hybrid shapes; introduce various structural motifs, such as custom curvature, corners, and vertices; and describe principles for creating multilayer DNA-protein objects with enhanced rigidity. We demonstrate self-assembly of our hybrid nanostructures in one-pot mixtures that include the genetic information for the designed proteins, the template DNA, RNA polymerase, ribosomes, and cofactors for transcription and translation.

One of the challenges of single molecule experiments in solution is the ability to trap and manipulate one or even multiple molecules against the erratic Brownian motion. The Brownian fluctuations are fueled by thermal energy and increase in strength with increasing temperature. Therefore, it is at first glance counterintuitive to confine Brownian fluctuations with the help of elevated temperatures. In thermal nonequilibrium, however, temperature gradients induce thermo-phoretic and thermo-osmotic drifts, which provide the means for single particle manipulation in solution. Here we describe experiments which use optically heated metal nanostructures to create dynamical temperature profiles in solution. These temperature profiles induce thermo-phoretic drift fields that act as effective potentials for objects suspended in liquid. Combined with optical feedback mechanisms, such effective potentials can be shaped to store and manipulate single or even a well-defined number of multiple objects in a small observation volume. The developed thermophoretic trapping system therefore paves the way for extended single molecule studies in solution or even well controlled bi- or multi molecular interaction studies.

A synthetic RNA aptamer, called Spinach, has been recently proposed as an alternative to Green Fluorescent Protein for real-time imaging, detection of cellular metabolites and for tracking dynamic bio-processes. Such RNA GFP-mimic aptamer is able to specifically bind a synthetic copy of the naturally occurring GFP fluorophore displaying GFP-like luminescent properties. Here we demonstrate for the first time a supramolecular antibody-templated assembly of the Spinach aptamer. To do this we have employed two antigen-tagged RNA strands that, upon binding to a specific target antibody, are co-localized in a confined space and re-assemble into the functional Spinach aptamer yielding a measurable fluorescence signal emission as a function of the antibody concentration. We have demonstrated the generality of our approach using two different antigen/antibody systems and found that both platforms are sensitive, specific and selective enough to work in crude cellular extracts.

Nanophotonics has the goal to arrange optical components on the nanoscale such that they interact with light in a defined and controllable manner. This way novel optical properties can be generated with the goal to study light-matter interactions at the nanoscale and to develop optics-based applications such as logical devices that are based on photonic instead of electronic interactions. Material candidates are metals, semiconductors or dielectrics which are usually structured with top-down lithography. In particular metallic structures have proven to be employable for the generation of novel optical effects due to the strong interactions of surface plasmons with electromagnetic waves at frequencies in the visible range. Notably metal nanoparticles and nanorods of sizes ranging from a few to tens or even hundreds of nanometers can support localized surface plasmons. In this tutorial I will introduce some of the basic concepts of plasmonics and review examples where DNA nanotechnology enabled new experimental studies. By employing self-assembly methods for the arrangement of plasmonic particles in space, several inherent limitations that result from the use of conventional lithography methods, such as limited spatial resolution and the difficulty to build bulk materials, can been overcome. In particular the applicability of DNA nanostructuring to arrange optical components in ways that were not achievable before opens exciting routes for future nanophotonic research.

Conventional top-down nanofabrication, over the last six decades, has enabled almost all the complex electronic and optical devices that form the foundation of our society. Parallely, self-assembly processes within naturally occurring materials have been extensively studied to enable bottom-up synthesis of nanostructures like quantum dots and carbon nanotubes with technologically relevant properties unattainable by any top-down method. While both these nanofabrication approaches have independently matured, ongoing efforts to create ``hybrid nanostructures" combining both strategies, has been fraught with technical challenges. The main roadblock is the absence of a scalable method to organize components built bottom-up within top-down nanofabricated structures. In this talk, I will introduce a directed self-assembly technique that utilizes DNA origamis of defined shapes as modular adaptors to position and orient bottom-up nanocomponents within top-down nanofabricated devices. I will also be discussing the utility of this technique to realize functional nanophotonics devices. Specifically, I will discuss results demonstrating the deterministic organization of discrete emitters inside photonic crystal cavities (PCCs) to map the modes of PCCs using an epifluorescence microscope, create ultra-small 2D polarimeter and enable optimized light-matter interaction within optical resonators. Finally, I will conclude by presenting my vision of how DNA origami and it's integration with top-down nanofabrication can enable a wide variety of functional electronic, optical and biomedical devices. In particular, I will discuss how DNA origami can enable sub-10nm semiconductor features, ultra-stable lasers, better solar cells and high-throughput tools for single-molecule biophysics.