Program / Speakers

Local hike TBA, probably Echo Mountain or Millard Canyon, depending on weather and such.

Decision: Meet outside the Rock Auditorium (100 Broad Center) at 1pm. We will go to Millard Canyon. Expect approximately 3 hours.

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, using L-nucleotides, a Solomon's knot.

Nanorobotics is a key area of application. We have made robust 2-state and 3-state sequence-dependent devices and bipedal walkers. 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.

One of the major aims of DNA-based materials research is to construct complex material patterns that can be reproduced. We have built such a system from DNA origami; it has reached 9 generations of exponential growth directly and 24 generations (with no apparent limit) in punctuated steps.

Wenyan Liu's rule states that the best arrays in multidimensional DNA systems result when helix axes span each dimension. We have self-assembled a 2D crystalline origami array by applying this rule. We used the same rule to self-assemble a 3D crystalline array. We initially reported its crystal structure to 4 Å resolution, but rational design of intermolecular contacts has enabled us to improve the crystal resolution to better than 3 Å. We can use crystals with two molecules in the crystallographic repeat to control the color of the crystals. We can change the color of crystals by doing strand displacement of duplex DNA; we can also color the crystals using triplex formation. We are using the crystals to attempt to control the structure of other materials in 3D.

Diverse biological functions require specific lipid curvatures, sizes, and geometries. We have developed a method to reconstitute liposomes with controllable geometry within the cavity of a 3D DNA origami. To accomplish this, the DNA origami is functionalized with lipid molecules serving as nucleation sites, which can direct liposome formation. In this way, we have scaffolded the self-assembly of 75 nm diameter uniform liposomes and nanotubes.

The invention of DNA origami has provided a unique tool to control the precise positioning of molecules and materials relative to each other by self-assembly. The specificity of DNA interactions and our ability to synthesize artificial functionalized DNA sequences makes it the ideal material for controlling self-assembly of components attached to DNA sequences. In particular we are using DNA origami, for positioning of materials such as organic molecules, dendrimers, polymers and biomolecules. We have used DNA origami to image chemical reactions with single molecule resolution and as a readout display for molecular computing. 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 in new ways. The main focus of the presentation will be on a conjugated DNA-phenylene vinylene polymer and its self-assembly on DNA origami for studies of its dynamics and optical properties.

From the perspective of a molecular biologist the DNA origami method [1] was a true eye-opener, since it unequivocally demonstrated that biomolecules can be designed according to human ingenuity and creativity, and not only by evolutionary processes. Inspired by these possibilities we developed a software tool that could transform a graphics shape into a DNA origami and initially demonstrated its use by recreating a dolphin shape from our university logo [2]. The unexpected observation of flexible tails on DNA origami dolphins prompted us to worked further on developing mechanical nano-devices. Our initial device was a 3D DNA origami box with a lid that could be opened by DNA keys [3]. In this talk I will show our progress in prototyping mechanical nano-devices and applying them for diagnostic and therapeutic purposes. Recently, we have introduced the single-stranded RNA origami method [4] and demonstrated it by building RNA honeycombs using RNA polymerases. The ability to produce well-defined RNA nanostructures by RNA polymerases opens up the possibility of expressing RNA nanostructures inside cells where they might be used to organize cellular components and reprogram cellular processes for applications in synthetic biology.

[1] P.W.K. Rothemund. Nature 440, 297-302 (2006)
[2] E.S. Andersen et a l. Acs Nano 2, 1213-1218 (2008)
[3] E.S. Andersen et al. Nature 459, 73-75 (2009)
[4] C. Geary et al. Science 345, 799-804 (2014)

Viral genomes including that of HIV consist of up to thousands of bases of RNA that adopt complex tertiary folds that play a central biological role in their host infection and reproduction. The tertiary structures of viral genomes remain largely unknown because their high molecular weight and flexibility render them unsolvable by conventional structural techniques including crystallization and NMR. Here, I will present a new framework we are developing to map the 3D tertiary structure of such high molecular weight viral genomes using scaffolded DNA origami. To achieve this, we are leveraging a recent integrated computational-experimental approach we have developed to “print” from the top-down nearly arbitrary 3D scaffolded DNA origami assemblies with full control over 3D positioning of DNA base-pairs at the single nucleotide level.

I will present some musings and memories of the olden days before scaffolded origami changed DNA nanotech forever. We will go over recent developments from my group to reliably make much larger [1] and smaller [2] origami scaffolds. In light of the amazing progress of lithography’s march into the nanoscale, it is worth asking whether bottom-up fabrication of nanoelectronic devices via molecular assembly has any chance of commercial relevance. I will argue that DNA origami-templated structures in combination with three-dimensional integration on longer length scales still offers us hope of industrial bionanofabrication.

[1] Alexandria N. Marchi, Ishtiaq Saaem, Briana N. Vogen, Stanley Brown, and Thomas H. LaBean (2014) Toward larger DNA origami, Nano Letters 14, 5740–5747 (doi: 10.1021/nl502626s).

[2] Stanley Brown, Jacob Majikes, Amanda Martínez Reyes, Tania Montserrat Girón, Hannah Fennell, Enrique C. Samano, Thomas H. LaBean (2015) An Easy-to-Prepare Mini-Scaffold for DNA Origami, Nanoscale 7, 16621-16624 (doi: 10.1039/C5NR04921K).

RNA is the punk-brother of DNA. While DNA plays by rules, RNA is more rebellious. The diverse structural features of RNA that make it a powerfully-functional molecule in biology also make it difficult to tame and rationally-design. I will tell a story of how the field of RNA design has developed over the past decade alongside DNA origami research.

A prerequisite to build advanced plasmonic architectures is the ability to precisely control the organization of metal nanoparticles in space. To this end, DNA origami represents an ideal construction platform owing to its unique sequence specificity and structural versatility. I will present sequentially a diverse set of DNA-assembled plasmonic nanostructures according to their characteristic optical properties. I will also discuss about the inevitable evolution from static to dynamic plasmonic systems along with the fast development of this inter-disciplinary field. Finally, possible future directions and perspectives on the challenges are elucidated.

DNA origami has proved to be a remarkably successful strategy, especially given how little we know about the details of the origami assembly process. In particular, the use of a long scaffold strand introduces conceptual complexities that distinguish origami assembly from the familiar process of assembly-through-nucleation of small components. In this talk I discuss exploration of the role of the long scaffold strand using two models of different levels of detail.

When done right, they tend to fold.

With the goal of presenting unpublished results and stimulating discussions, I will discuss several ongoing projects that are focused on both enriching the design space of DNA/RNA origami and understanding the fundamental processes of DNA self-assembly. I will first discuss our progress of constructing single stranded DNA origami with high crossing numbers and single stranded RNA origami based on paranemic crossovers, and the design tools that enabled them. I will also discuss some of the results we obtained on understanding thermodynamics and kinetics of DNA tile-based and/or DNA origami framed self-assembly.

This talk will raise a number of questions about DNA origami and DNA nanotechnology: How is our field similar or different than other emerging technology fields? What lessons might we draw from the trajectory of these fields? What is the mode in which we are doing science? It isn't hypothesis driven, and it isn't exactly engineering...what is it? How is DNA nanotechnology perceived by other scientists, in terms of the role it might play in their work? What realistic role might DNA nanotechnology play in their work? What educational role might DNA origami play in helping people believe in molecules?

The guiding design principle of nanofabrication by DNA self-assembly is that the target structure is the single most stable configuration; however, the pathway and kinetics of assembly, and in particular origami assembly, are poorly understood. The folding transition is cooperative, and there is a strong analogy with protein folding: both are governed by information encoded in polymer sequence. Misfolded structures are kinetic traps. The yield of well-folded DNA origami can be low: yield is improved by titration of cations or by following empirical design rules, but it is frequently necessary to separate well-folded origami from misfolded objects. We present an origami structure that is designed to reveal the assembly process. Our system has the unusual property of having a small set of distinguishable, well-folded shapes that represent discrete and approximately degenerate energy minima in a vast folding landscape. We obtain a high yield of well-folded origami structures, demonstrating the existence of efficient folding pathways. The distribution of shapes provides information about individual trajectories through the folding landscape. We show that the assembly pathway can be steered by rational design and 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. Expanding the rational design process to include the assembly pathway is the key to reproducible synthesis, which is essential if nucleic acid self-assembly is to continue its rapid development and become a reliable manufacturing technology.

Nanophotonics as a field has undergone rapid development since the 1980s with the promise of enabling devices for single-molecule study, nano-lasers and quantum information processing. The pace of development was also greatly assisted by the utilization of standard semiconductor processing techniques to realize many of the devices. While many proof-of-principle devices have been realized over the last three decades, development of functional devices have been limited due to challenges involving incorporating functional active components, like emitters, into microfabricated optical structures with high precision.

In this talk I will introduce our work on using DNA origami as a modular adaptor to load organic emitters into passive nanooptical resonators. The technique enables us to digitally control the number of emitters within the resonators as well as the location and orientation. In addition to being a research tool to study interesting optical phenomena like cavity-less lasing the technique can also potentially enable practical devices like integrated single photon sources as well as light gated ultrafast transistors.

Direct observation of molecular motions is one of the most fundamental issues for elucidating the physical properties of individual molecules and their reaction mechanisms. Atomic force microscopy (AFM) enables direct molecular imaging, especially for biomolecules in the physiological environment. We have developed AFM-based single-molecule observation systems for biomolecule imaging by employing DNA origami nanostructures and high-speed AFM.[1,2] Using this system, we have characterized the DNA structural changes and enzyme reactions in the DNA nanostructures. We also employed photochemical reactions to construct the mobile nanosystems and devices controlled by hybridization and dehybridization of DNA strands by photo-irradiation. Using the photoresponsive systems, we directly observed the dynamic assembly and disassembly of hexagonal origami structures on a lipid bilayer during high-speed AFM scanning.[3] We further employed a lipid-bilayer to observe the dynamic 2D array formation from cross-shaped, triangular, and hexagonal origami monomers.[4] For control of a linear molecular movement, a pyrene-attached DNA motor and the track were assembled on the DNA origami tile.[5] We observed the photo-induced movement of the motor on the DNA origami surface as similar to an enzyme-induced DNA motor. In addition, we constructed a photo-controllable rotator system on the DNA origami tile, and the rotary movement of the photoresponsive DNA nanostructure was observed by switching UV/Vis irradiation. These chemically controlled DNA nanosystems are expected to be applied for construction of mobile nanostructures and nanodevices. Also the high-speed AFM observation supports the detailed analysis of the movements of the target molecules and the morphology changes of the nanostructures at nanoscale resolution.

A hallmark of some of the most complex biological assembly processes, such as the replication and separation of genomic DNA or cytoskeletal reorganization is the precise control over the pathway of assembly. While the advent of DNA origami and other DNA-based assembly methods have made it possible to assemble objects of outstanding structure complexity, we know comparatively little about how to control assembly dynamics.

A self-assembly process generally begins with nucleation, the assembly of monomers into an ordered cluster than can continue to grow. I will describe how DNA origami can be used to control the nucleation of DNA nanostructures, and how this control makes it possible to engineer advanced functionality into assembly processes, including the ability to propagate and replicate complex information and the ability to assemble adaptive "networks" of 1-dimensional structures.

More generally, the precise control that DNA origami affords over DNA nanostructure nucleation presents an opportunity to understand basic facts about nucleation, a notoriously poorly described process that nonetheless is a ubiquitous chemical phenomenon. I'll describe some experiments that aim to deduce general principles about the process of nucleating DNA crystals that could be exploited to both better understand DNA nanostructure self-assembly and build new classes of materials.

Everybody knows that charged molecules such as DNA do not like to penetrate or go through lipid bilayer membranes, as their low-dielectric interior represents too large of an energetic barrier (the “Born energy”). In nature, membrane-bound biomolecules (ion channels, receptors, etc.) thus contain hydrophobic regions that can be inserted into the membrane more easily. Technological or medical applications that involve the transport of nucleic acids across lipid membranes require disruption of the membrane or appropriate packaging of the cargo.

With the advent of DNA origami the seemingly impossible has become possible. The origami technique allows us to construct macromolecular assemblies made from DNA that resemble the shape of naturally occurring membrane channels. These structures still do not like to reside in a lipid bilayer, but – using DNA staple strands appropriately functionalized with hydrophobic molecules – they can be forced to go through the membrane nevertheless.

In this talk, we will present an overview of a variety of DNA-based membrane channels that have been created with the DNA origami technique so far. These channels have been shown to conduct ionic current, they can be used as single molecule sensors for the translocation of single-stranded DNA, double-stranded DNA, and also small molecules. In addition, they can be used as artificial membrane channels in giant liposomes, which they can permeabilize when added from the outside, and – upon encapsulation – also in an “inside-out” configuration.

In cell biology it is widely accepted that the biophysical context of ligands and receptors has significant impact of downstream signaling, however the concept is poorly understood due to difficulties in controlling and analyzing the microenvironment on the nanoscale. I will talk about how we think that DNA-nanotechnology can be of great help in learning the tactile alphabet of cells by poking them with protein decorated DNA-origami ‘nano-calipers’. I will also briefly present our recent work on ‘3D-printing’ DNA origami wireframe structures, a method that provides a way to make origami more accessible to experiments in physiological salt conditions.

Understanding individual molecular information within a large biomolecular complex, while maintaining its native environment, represents a key challenge in biology. Recent advances in fluorescence super-resolution microscopy have shown images of sub-cellular features and synthetic nanostructures down to ~15 nm in size, but direct optical observation of each individual molecular targets (~5 nm) in a densely packed biomolecular cluster ("discrete molecular imaging", or DMI) has yet to be demonstrated. We will present systematic characterisation and optimisation of four technical requirements for meeting this challenge with localisation microscopy. We demonstrate our ability to achieve DMI with DNA-PAINT (point accumulation for imaging in nanoscale topography), a method that exploits programmable transient oligonucleotide hybridisation for super-resolution microscopy, on synthetic DNA nanostructures. In particular, we examined the effects of high photon count, high blinking statistics and appropriate blinking duty cycle on imaging quality, and reported a novel software-based drift correction method that achieves <1 nm residual drift (r.m.s.) over hours. We reported fluorescence imaging of a densely packed triangular lattice pattern with ~5 nm point-to-point distance, and analysed DNA origami structural offset with angstrom-level precision (<2 Å) from single-molecule studies. Combined with multiplexed exchange-PAINT imaging, we demonstrated an optical nano-display with 5x5 nm pixel size and three distinct colours, and with <1 nm cross-channel registration accuracy. Using oligonucleotide conjugated small labeling agents, this method could potentially allow direct optical visualisation of individual molecular components in diverse nanoscale systems, with up to angstrom-level precision, and opens up new possibilities for studying quantitative molecular features.

Molecular engineering approaches guided by simple and powerful mathematical principles have revolutionized technology and deepened our understanding of natural algorithms in many ways. A critical challenge is to scale up the complexity of nanoscale structures by taking advantage of the inherent stochasticity of molecular systems, while maintaining sufficient control with embedded deterministic rules. Here we show that the principle of non-deterministic Truchet tiling can be applied to provide a simple solution for creating complex nanoscale patterns that have combinatorial diversity and programmable features. As an example, we constructed patterns of random mazes with distinct emergent properties and with sizes of up to several microns, each self-assembled from thousands of square DNA origami tiles that are labeled with simple local patterns. We further demonstrated precise control of maze complexity by creating DNA origami arrays with unprecedented yield and designed sizes ranging from 4 to 25 tiles in each assembly, and showed the generality of our approach using arrays of triangular DNA origami tiles. The nanoscale mazes that we created are examples of “programmable disorder” in two-dimensional molecular structures. These structures could be used to test the robustness of molecular machines against a variety of operating environments with increasing complexity. Broadly speaking, by attaching proteins, metal nanoparticles, and organic dyes to origami arrays with combinatorial patterns of programmable features, our approach could enable efficient screening of functional molecular devices and advance nanoscale fabrication. Importantly, our work highlights the need for better understanding of programmable disorder and how it can be more generally applied in engineered molecular systems to enable solutions for problems that simultaneously demand complexity, diversity, and efficiency -- much like the algorithms we see in nature that exploit a sophisticated blend of deterministic and random processes.

A major goal of our lab is programming functional dynamic behavior of DNA origami nanostructures. We have focused on two approaches. First, taking inspiration from macroscopic machines and mechanisms, we have developed a modular design approach to combine DNA origami "joints" with rotational and linear degrees of freedom to achieve DNA origami mechanisms with complex and coordinated motion. We are currently working to enhance strand-based actuation and developing new actuation mechanisms for these DNA origami mechanisms. Secondly, we have recently developed an approach to engineer thermally driven structural dynamics. In this approach we incorporate energy barriers comparable to thermal energy and demonstrate structure entropic design parameters (i.e. tuning the number of accessible configuration) to control both equilibrium and kinetic parameters. We further demonstrated that these structural dynamics are sensitive to femtoNewton scale depletion forces, which is a result of the low energy barriers and relatively large conformational changes.

DNA origami has proven a powerful method for custom nanofabrication. Although diverse shapes in 2D and 3D are possible, one simple shape has remained the most popular for subsequent applications: the single-layer rectangle. One reason is that custom patterns can be created on the surface of the rectangle by functionalizing existing staple strands, without altering the underlying design. The rectangle has many attractive features, such as fast and robust folding and high yield. Modular design of staple strands enables simple abstraction to a regular pixel surface. We would like to address the question: can we design a set of generic 3D origami that can be used for many applications? We have designed a family of architectures, DNA barrels built as stacked rings of double helices, that retain the appealing features of the 2D rectangle, but extend construction into 3D, to provide 3D nanoscale pegboards for future nanoconstruction.

We use the DNA origami method [1] for the fabrication of functional self-assembled nanoscopic objects and materials [2]. By offering attachment sites for active nano-components on these DNA objects, we have realized complex and nanometer-precise assemblies of fluorophores and plasmonic nanoparticles [3].

Currently we are exploring plasmonic nanoantennas made by DNA origami that can be used as reliable and efficient probes for surface enhanced Raman spectroscopy (SERS). The nanoantennae are built up by pairs of gold nanoparticles on DNA origami templates at separation distances between 9 nm and 4 nm in order to achieve plasmonic coupling and the formation of strong plasmonic ‘hot spots’ [4].

In recent, unpublished experiments we studied 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 – particularly DNA – elasticity. Here we present a nanoscopic DNA origami based single-molecule force spectroscopy device without any physical connection to a micrometer-sized bead or cantilever. We exploit the entropic elasticity of single-stranded DNA to apply tension on a system mounted on the device [5] 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. Our DNA origami force spectrometer can in principle be employed with a wide variety of DNA interacting biomolecules.