We have designed, created, and tested anticoagulant drugs composed of 2������������������-F-modified RNA that self-fold into origami structures displaying aptamer handles that bind to and inhibit thrombin enzyme in the blood coagulation cascade. The RNA origami-based anticoagulant will be safer, provide more stable and predictable dosing as well as a highly specific antidote, compared to currently available intravenous anticoagulant drugs.

Cells are able to sense and react to changes in the mechanical properties of their microenvironment through a process known as mechanotransduction. Many studies have demonstrated that substrate elasticity directs a multitude of cellular responses such as attachment, migration, proliferation, and differentiation (1-4). However, most biological tissues exhibit non-linear elastic properties (1). Recent studies have shown that non-linear elastic substrates can impact cell spreading, adhesion, TGF�������� activation/signaling, and downstream myofibroblastic differentiation (6-11). However, few model material platforms have been described with controllable non-linear elastic properties that display time-dependent deformation in response to cell derived forces. The overarching objective of this project is to develop a novel material system to elucidate the role that non-linear elastic properties play in modulating cell adhesion, spreading, TGF�������� activation/signaling, and myofibroblastic differentiation. To that end, in this project we describe the use of colloidally stable microgel thin films with controllable non-linear elastic properties for investigating the role of material loss tangent on fibroblast behavior. We hypothesize that by varying the amount of internal crosslinker of each microgel particle, we can control the film������������������s viscoelasticity and modulate cellular mechanotransduction response, cell adhesion and migration through modulation of integrin and TGF�������� receptor clustering. The main objectives of are to 1) Determine how microgel film viscoelasticity influences fibroblast adhesion, spreading, migration, and myofibroblastic differentiation; 2) Analysis of TGF�������� receptor clustering, activation, and signaling as a function of microgel film viscoelasticity; 3) Examine the effect of pre-pattering of TGF�������� receptors via DNA origami on cell adhesion, spreading, migration and myofibroblastic differentiation.

DNA������������������s capacity for highly reliable and programmable molecular recognition has led to the birth of the field of DNA-based nanotechnology, also known as structural DNA nanotechnology. Researchers in this field develop materials and techniques for DNA-guided molecular and nano-scale self-assembly and have made remarkable recent progress, including the ordering of matter with unprecedented accuracy and parallelism, nano-scale organization of proteins and metal particles, as well as fascinating demonstrations of artificial molecular machines made of DNA. One problem limiting DNA nanotech������������������s translation from prototype demonstrations to useful applications has been the lack of a general purpose method for functionalizing DNA structures with polypeptides. The Cas9 protein from the CRISPR RNA-directed bacterial immune system offers a novel solution to this problem. Cas9 is a programmable recognition protein that, in its wild-type form acts as an endonuclease for cleaving non-self nucleic acid targets. In an engineered form, dCas9 has been mutated to bind DNA sequences (specified by an RNA targeting molecule) without cleavage of the DNA backbone. Like the molecular recognition elements that have facilitated the development of DNA-based nanotechnology, the interactions between dCas9 and its target DNA sequences are well understood, modular, and programmable. Consequently, dCas9 is ripe for development into a new family of molecular assembly tools with designed sequence specificities and the ability to act as a new smart glue for the programmed assembly of other nanomaterials including protein enzymes, affinity peptides, inorganic nanoparticles, and carbon nanotubes. The proposed project will combine DNA nanotech and dCas9 to develop complex, self-assembling, supramolecular constructs that can be rapidly retooled for a wide variety of real world applications. The overarching goal of the proposed project is to add the programmable recognition and binding functions of dCas9 to the growing toolbox of materials and methods available to DNA-based nanotech. Specifically, the project seeks to develop fusion proteins bearing mutant Cas9 domains to organize other polypeptides (including enzymes, ligands, inhibitors, in vitro selected peptides) on DNA nanostructures in programmed patterns.

This project will scale up production of modified RNA anticoagulant (provisional patent filed) for next stage testing prior to clinical trials. RNA anticoagulants are safer alternatives to small-molecules currently used during surgery and treatment of disseminated intravascular coagulopathy. The NCBC grant will advance the drug toward commercialization in North Carolina.

This project seeks to continue the pursuit of innovative designs for self-assembling DNA nanostructures and their application to novel bionanofabrication strategies for electronic and photonic devices. The major goal of the project is to develop reliable protocols for fabricating functional photonic and electronic nanostructures using bioinspired, molecular assembly methods. The majority of the steps in the nanostructure fabrication will be performed in aqueous solution using techniques developed by the PIs. In particular, fabrication of complex metallic nanostructures (e.g. single-electron transistors, SET) by DNA templating and chemical surface patterning will be further developed for photonic and electronic applications. In one of the proposed tasks, the SET nanoparticle island will be selectively attached to the ���������������DNA origami������������������ through molecular recognition; seed nanoparticles will be selectively attached and fused to form the electrodes; and the whole assembly will be docked to a chemically patterned SiO2 surface.

Complex webs of cell signaling pathways make up the cell-to-cell communications networks responsible for everything from homeostasis, to wound healing, development, and immunity. Frequently these communications systems make use of direct contact between neighboring cells via specific nanometer-scale organization and presentation of cell surface receptors and the ligands with which they interact. This project seeks to employ the rich palette of tools provided by structural DNA nanotechnology to specifically organize, orient, and present biologically active molecules to living cells in order to understand, interrupt, and reprogram interactions involved in cell signaling. Specifically, the project will examine the spacing and multiplicity of presentation of the major histocompatibility complex (MHC) and its interaction with T-cell receptors on the surfaces of blood cells. DNA origami will be assembled with different architectures and patterns in order to test a range of hypotheses in cell signaling science that would be very difficult or have been impossible to test by other, less programmable molecular experimental methods.

This project will fund a vertically integrated team (members from all education levels from high school through post-doctoral) of 5 or 6 participants per year from North Carolina for travel to Aarhus University in Denmark to pursue month-long research projects in nanochemistry, molecular self-assembly, and DNA-based fabrication. The project's scientific focus will be on self-assembling DNA nanostructures and particularly their application to nanofabrication of electronic and photonic devices. The NCSU team has used DNA as a building material to implement molecular computers, and to organize proteins and metallic nanoparticles with molecular-scale precision, while the Aarhus team is expert in DNA-guided chemistry for programmed synthesis of very large organic molecules. The project will improve research training at the interface between complex DNA nanostructures and DNA-guided chemistry by continuing the established collaboration between these leading DNA nanotech groups. Production of significant outcomes including cutting-edge research results (12 publications over 2 previous funding cycles) and impactful research training experiences are only possible by maintaining this successful international collaboration. NCSU and Aarhus PIs have received funding through the Danish National Research Foundation which primarily funds Danish students both at home and on visits to the NCSU lab. IRES funding will complement the Danish investment by allowing US students to participate in research at Aarhus. US participants will benefit from significant foreign interaction and mentoring on-site in Denmark.

Biomemetic self-assembling materials using molecular recognition for programmed fabrication of templated electronics devices has the potential for generating disruptive technology for information processing, computer, and communications applications. We will experimentally and theoretically examine a system involving molecular assembly of functioning components into nodes that then further assemble into networks capable of displaying complex, emergent behaviors. Deterministic assembly at nanometer length-scales followed by stochastic assembly just below the micron scale will provide materials with exploitable electronic behaviors. Theoretical work will particularly focus on modeling and simulation of node function and network structure in order to approximate expected emergent properties. Network architectures will follow neuromorphic principles and will result in trainable circuits with potential capabilities including memory, logic, and complex signal processing.

The major goal of the proposed project is to develop a reliable methodology to fabricate nanoscale electronic structures based on biochemical techniques. Most of the steps in the nanostructure fabrication will be performed in solution using the techniques recently developed by the PIs. In particular, one of the major functional elements that will be studied here is the Single-Electron Transistor (SET) based on an individual metal nanoparticle specifically attached to SiO2 surface by biological or chemical patterning (e.g. anchored to DNA template). Assembly of a functional SET based on chemical surface patterning has recently been demonstrated in the PIs? laboratories. In one of the proposed approaches, the nanoparticle will be selectively attached to a DNA lattice through molecular recognition, and the DNA will be metallized to create interconnects. An SET having a central island of less than 10 nm will operate at room temperature, a target structure not achievable by existing lithographic methods.

The goals of this FRPD proposal project are two-fold – both for professional development and as seed funding to expand research in the LaBean group into a new area of inquiry. From the faculty professional development standpoint, LaBean intends to expand his training further from his roots in biochemistry and to delve deeper into experimental electronic measurements in order to gain practical understanding of the critical issues by training with Dr. David Gundlach at NIST. From the point of view of seed funding for a new research venture, laboratory supplies and graduate student salary are requested in order to gather initial data on the fabrication of electrically active DNA templated hydrogels and aerogels. The LaBean lab has pioneered the bottom-up fabrication of complex, programmable supramolecular structures using DNA-based self-assembly. Since joining the Material Science and Engineering department about two and a half years ago, the PI has looked for ways of advancing DNA nanotechnology into the realm of bulk materials. Now, by combining DNA origami and DNA hydrogels, the group will attempt to create new self-assembling molecular materials with great potential for future advances in 3D integration of electronic components. Such materials could find use in diverse areas all the way from sensors in electronic noses to neuromimetic hardware networks for computational and control circuits.