Thom H. LaBean - US grants

CBET-0835794
Hall

DNA can be possibly used to construct tiny scaffolds with many potential applications including molecular medicine, electronics, sensors, computing and communications equipment, and consumer electronics. Two-dimensional nanostructures have already been devised, but further design and development has so far been limited to making incremental improvements using empirically derived guidelines.

This project intends to break these barriers using advanced modeling techniques, a multidisciplinary team of modelers and experimentalists, and a cyber-driven strategy to design complex, three-dimensional objects at nanometer size scales. First, modified natural and non-natural nucleosides (the components RNA) will be devised to increase the number of molecular tools available to create these structures and to provide accessible sites for attachment of functional components. Structures will be computed for these two- and three-dimensional DNA-based building blocks, with and without modified nucleosides. Stability will then be determined for the building blocks in solution, on planar surfaces, and/or with metallic nanoparticles. Ultimately, new, hybrid extended three-dimensional architectures will be designed and built using these findings. A key to success is testing and refining the modeling approaches by comparing computed results to the team's experimental results.


The education and outreach agenda includes a team mentoring program for a small group of women graduate and undergraduate students, individual outreach efforts to underrepresented groups, and training of four graduate students and four undergraduates. Cyber-enabled education is an especially interesting aspect of the project, where a website containing freely-available cyber-structure software and visuals will allow researchers to design their own DNA superstructures and will also allow community members, including grades 7-12, to learn more about DNA self-assembly.

Abstract
Non-Technical:
The major goal of this project is the development of reliable, bioinspired, molecular assembly protocols and materials for fabricating functional photonic (light-active) and electronic (electron-active) devices. Biological structures with nanometer-scale dimensions are able to self-assemble using principles of molecular recognition, principles that can now be harnessed for production of technologically useful materials, objects, and devices. Fabrication techniques that mimic biological nanometer-scale assembly strategies promise to transform modern electronics manufacturing by reducing the need for increasingly expensive lithographic fabrication equipment and facilities, decreasing reliance on toxic, rare-earth elements, and increasing the energy efficiency of producing and operating computational and communications devices. Besides advancing science and engineering issues critical to future device manufacturing processes, this project will provide educational and research training opportunities for students at the undergraduate, post-graduate and high school levels. This project will train students in the unique combination of biochemical and physical methods of modern nanotechnology that are relevant for industrial and academic careers. Through Project SEED (Summer Educational Experience for the Disadvantaged), high school students from disadvantaged economic backgrounds will participate in laboratory research during the summer months to become acquainted with the emerging field of nanoscience. The project coordinators will continue to actively recruit participants from demographic groups typically under-represented in the science-technology-engineering-mathematical (STEM) disciplines.
Technical:
Specific objectives of this three year project include: 1) development of metallic clusters for significant enhancement of Raman scattering signals by templating metal nanoparticles on DNA origami, then using these Raman-bright clusters as photonic devices for tagging and tracking specific cell-types during cell-sorting; 2) application of newly prototyped tetrahedral origami for functional 3D metal cluster assemblies including single electron transistors and chiral plasmonic devices; and 3) fabrication and testing of a 51 kilobase DNA origami for larger, oriented helical structures for photonic devices. Development of self-assembling systems for bottom-up fabrication has been a long sought-after goal of nanotechnology. The particular merit of this project stems from the convergence of recent advances in DNA-based self-assembly methods with new understanding of plasmonically coupled metal nanoparticle clusters for a wide range of optoelectronic applications. Intellectual merit of the project also derives from the interdisciplinary collaboration that couples a biochemist and a physicist on a productive team with a history of successful scientific research and educational activities. The project will lead to development of alternative, cheaper (most steps are in aqueous solution) and more versatile fabrication methods for composite bio-nano-devices. These nanostructures may be useful in the development of naturally biocompatible devices with strong potential for use in biomedical applications. Results from this study may provide major impacts to a range of applications, including electronic devices with decreased size, weight, power consumption, and heat generation for mobile sensing and ubiquitous computing.

This International Research Experience for Students (IRES) activity links expert nano research groups at North Carolina State University and the University of Aarhus in Denmark for a program of interdisciplinary mentoring, academic education, and research-based learning for participating students and junior researchers from both countries. Principal Investigator (PI), Thomas H. LaBean, and U.S. colleagues will work with Danish partners, led by Kurt V. Gothelf, to manage a balanced, team-based approach for research training at the interface between complex DNA nanostructures and DNA-guided chemistry. This builds on a program of cooperation in education and research that features the melding of leading Aarhus conjugation chemistry for coupling diverse compounds to oligonucleotieds with the U.S. team's expertise in design and implementation of complex nanostructures. Overall, research efforts promise significant outcomes in molecular engineering with potential applications in molecular and nano-electronics, nanochemistry, biosensors, or other biomedical areas.

The educational value of the IRES rests with the students' exposure to the productive, cooperative research of the two leading institutions and involvement in their mentors' cutting-edge research. The U.S.-Danish IRES model involves post-doctoral mentors and diverse, research-ready high school students as well as U.S. graduate and undergraduate students. The PI's participation in Project SEED, supported by the American Chemical Society, assists with identification of appropriate high school students. For balance and heightened interaction, the program also engages junior Danish researchers, supported independently by the Danish National Research Foundation. Each of the junior participants will be assigned projects that are designed to contribute to program-wide research goals. Through planned cross-disciplinary teamwork, they will acquire knowledge of techniques in synthetic chemistry, DNA structural engineering, protein biochemistry, materials science, and molecular biology.

This U.S.-Danish IRES activity in molecular engineering fulfills the program objective of developing global scientists and engineers by enabling experts in the United States and Europe to combine complementary talents and share research and education resources in an area of strong mutual interest and competence. Broader impacts include the introduction of U.S. junior researchers and students to international leaders in nanotechnology through involvement in cutting-edge research. If successful, results will continue to improve our ability to use DNA self-assembly to organize nanomaterials and to program sophisticated nanochemistry. The students' early career experiences are expected to increase the probability of their establishing international collaborations in the future.

IRES: Vertically Integrated Team for Structural DNA NanoTech in Denmark

This project sponsors a vertically integrated team (diverse members from education levels spanning 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. Scientifically, the research project will advance the engineering of biological macromolecules (DNA and proteins) for formation of materials with their structures controlled on the nanometer length scale. The team leader will recruit participants from groups commonly underrepresented in science and engineering disciplines including minority and female students. This international research training experience will provide participants with unique opportunities to enlarge their scientific networks and to experience first-hand the benefits of cultural mixing. The project is important and deserves NSF funding, because it will develop molecular tools as well as scientific personnel capable of advancing nanoscience goals critical to the electronics and medical fields. US participants will benefit from significant scientific and social interactions with foreign students and faculty on-site in Denmark. The project will produce significant outcomes including cutting-edge research results and impactful research training experiences for participating American students. The senior investigators involved have received funding through the Danish National Research Foundation which primarily funds Danish students both in Denmark and on visits to the NCSU lab. IRES funding complements the Danish investment by allowing US students to participate in research at Aarhus.

The project's scientific focus will be on the development of self-assembling DNA nanostructures and on their application toward nanofabrication of electronic, photonic, and biomedical 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. Developing ways to control matter on the nanoscale by exploiting the same construction principles used by biology (i.e. molecular recognition) holds great promise for diverse nanofabrication and biomedical applications. Engineered supramolecular complexes comprised of DNA, polypeptides, and inorganic nanomaterials provide the right length scale, range of physicochemical interactions, and complexity of shapes for interfacing effectively with biological structures. As such, DNA nanotechnology represents a perfect approach for creating custom diagnostic and ?smart? therapeutic agents for medical applications. Likewise, these same principles and materials are ideal for developing bionanofabrication procedures to compete with lithography for generation of electronic devices and circuits. Pursuit of biomimetic materials science, including molecular engineering for bottom-up assembly of complex nanostructures with diverse chemical, biological, photonic, or electronic functions, requires highly interdisciplinary teams. This cross-disciplinary work will therefore provide opportunities for very broad-based educational experiences to the participants. The cross-pollination provided by IRES funded international travel will lead to molecular materials engineering advances which could impact many fields of science and technology through applications in low-power nanoelectronics, programmable nanochemistry, novel biosensors, and smart therapeutics for biomedicine.

PI: LaBean, Thom
Proposal Number: 1603179

Complex webs of cell signaling pathways make up cell-to-cell communication networks responsible for everything from homeostasis (maintaining equilibrium within our body), to wound healing, to development, and to immunity. These communication systems make use of direct contact between neighboring cells through specific nanometer-scale organization and presentation of cell surface receptors and ligands. This project will use a unique molecular engineering tool, termed DNA origami (or DNA folding), to determine how genes that control the production of proteins on the outer membranes of cells (major histocompatibility complex or MHC) interact with T-cell receptors (TCR). The interaction of the MHC and the TCR is the first step in the T-cell activation process, which impacts important biomedical issues in normal immune function, cancer, and autoimmune disease. Thus, the work in this proposal has the potential to enable a powerful toolkit for performing fundamental biological studies on receptor dynamics and developing therapies for immunological disorders.

The goal of this project is to employ the rich palette of molecular engineering tools provided by structural DNA nanotechnology to specifically organize, orient, and present biologically active molecules to living cells in order to understand, facilitate, interrupt, and reprogram molecular interactions involved in cell signaling. Complex webs of cell signaling pathways make up the cell-to-cell communication networks responsible for everything from homeostasis, to wound healing, to development, and to immunity. These communication systems often make use of direct contact between neighboring cells via specific nanometer-scale organization and presentation of cell surface receptors and ligands they interact with. Specifically, the project will examine the spacing and multiplicity of presentation of the major histocompatibility complex (MHC) and its interaction with T-cell receptors (TCR) on the surfaces of living cells. The interaction of MHC and TCR is the first step in the complex biochemical cascade involving T-cell activation that impacts important biomedical issues in normal immune function, cancer, and autoimmune disease. DNA origami, a subset of DNA nanotechnology, will be assembled with designed architectures, patterns, and structural reinforcement in order to test a range of hypotheses in cell signaling science that would be very difficult or impossible to test by other, less programmable experimental methods. Novel basic science can now be pursued as well as testing of possible therapeutic strategies using DNA origami molecular assemblies displaying proteins, ligands, receptors, aptamers, small molecules, and other cell effectors. The combination of well-engineered experimental set-ups for probing individual cell-cell interactions allied with the programmable molecular recognition platforms of DNA-based nanostructures represents a new intellectual frontier with significant long-term potential in understanding biology and affecting human health. The program will lead to development of naturally biocompatible molecular organizers for a variety of biological and biomedical applications. Results from this study may provide major advances in understanding important events in T-cell activation and in cell-to-cell communications in general. Future extensions of this project could involve application of molecular materials and methods developed here toward other cell signaling pathways critical to understanding and improving human health. Graduate students will gain valuable opportunities for education and research training. The PI has consistently involved undergraduate, high school, and under-represented students in research programs and will redouble efforts to do so on this project

For the production of electronic devices like computers and smart phones, conventional lithographic techniques provide error-free circuits, but their development in critical directions is limited by their inherently two-dimensional nature. On the other hand, biological structures are inherently three-dimensional, however their error-prone self-assembly procedures require biological systems to utilize error-tolerant architectures. This project will mimic biology using materials that self-assemble by molecular recognition for programmed fabrication of electronic devices. Such fabrication methods could potentially generate revolutionary technology in information processing, computer, and communications applications. This research could increase the speed and energy efficiency of some computations and decrease the cost of their production. The project will provide interdisciplinary research training at the graduate, undergraduate, and postdoc levels. Participation by under-represented groups will be pursued and encouraged at all levels including through Project SEED for high school students.

By applying techniques of affinity peptide-based assembly and structural DNA nanotechnology to create bioinspired materials with macro-scale dimensions and nano- scale feature resolution, this project will generate unique, electronically-active, materials with integral networks that are expected to display interesting non-linear behaviors. These materials have potential for use in novel three-dimensionally integrated circuits with extremely-high connectivity that are predicted to display useful electrical behaviors and will be developed for computation, communications, signal processing, and control applications.

NON-TECHNICAL

Even with very advanced lithographic techniques, it is difficult or impossible for human engineering to reach down and control matter in the nanometer length-scale. However, biological systems utilize the ability of molecules to recognize and bind to one another for self-assembling very complex structures with feature sizes down in the nanometer range. Taking inspiration from the rich diversity of functional architectures observed in biology and making use of new discoveries in engineered biomaterials, this project will expand basic understanding of biomaterials and their programmable assembly. Successful completion of the research will, in the short-term, enable significant progress in nano-scale self-assembly using proteins and nucleic acids. In the long-term, this will provide the needed technology for implementation of ever-smaller devices for computing, communications, and sensing applications; implantable medical devices and biosensors; and programmable, artificial molecular machines. This project will provide training opportunities for postgraduate and postdoctoral students in cutting-edge molecular engineering and bionanotechnology. Using NSF's supplemental funding mechanisms, high school and undergraduate students will be attracted to the project to participate research and to train in the emerging field of nanoscience. This project will expand our scientific understanding of nanometer-scale phenomena and materials as well as to improve our ability to design and engineer new functional materials on this length-scale. The results of this project could impact future application areas in the sustainable fabrication of electronic devices and new approaches to medical diagnostics and therapeutics.


TECHNICAL

DNA's capacity for highly reliable and programmable molecular recognition has led to 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 precision and parallelism, nano-scale organization of proteins and metal particles, as well as fascinating demonstrations of artificial molecular machines. One problem that has limited DNA nanotech's translation from prototype demonstrations to commercially viable applications has been the lack of a general purpose, rapidly-reprogrammable method for functionalizing DNA structures with polypeptides. The Cas9 protein from the CRISPR RNA-directed bacterial immune system is used here as a novel solution to this problem. Cas9 is a programmable protein that acts as an endonuclease for cleaving non-self nucleic acid targets. In an engineered form, dCas9 has been mutated to bind a DNA sequence (specified by an RNA molecule) without cleavage of the targeted DNA. The dCas9 protein, guide RNA, and its target DNA sequences are well understood, modular, and programmable. Consequently, dCas9 is perfect for use in the development of 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 overarching goal of the 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 ligands, in vitro selected affinity peptides, enzymes, and inhibitors) on DNA nanostructures in programmed patterns for biomedical applications as well as in the bionanofabrication of electronic and photonic devices.

Cells are able to sense and react to changes in the mechanical properties of their microenviroment through a process known as mechanotransduction. The effect of the surrounding environment affects cell attachment, migration, proliferation, and differentiation. However, most tissues that include cells do not behave in a simple, elastic fashion. In fact, their properties are generally non-linear and viscoelastic -- meaning that they vary as the tissue is stretched and based on how quickly the force or stretch is applied. Significant research effort is being conducted into the best ways to get cells to develop into healthy tissues for tissue replacement or enhancement. Understanding how cells respond to their surroundings is key to being able to design systems that will optimize these engineered tissues. This project will focus on the effect of the viscoelastic properties of a substrate -- its ability to dissipate energy based on how quickly the force is applied -- on the clustering of a cellular receptor for a common growth factor (TGF-Beta) as well as how cells respond downstream to signals that are generated. In addition to the scientific impact, the project team will focus on training students in a multidisciplinary environment so that they can in the future work effectively on interdisciplinary teams to tackle complex problems at the interface of engineering, materials science, and biomedical science.

In order to investigate the nonlinear, viscoelastic substrate variation on fibroblast behavior, three objectives have been established. First, to determine how the viscoelasticity of a developed microgel, colloidal thin film influences fibroblast adhesion, spreading, migration, and myofibroblastic differentiation. Second, to analyze TGF-Beta receptor clustering, activation, and signaling as a function of microgel film viscoelasticity. And, finally, to examine the effect of pre-patterning TGF-Beta receptors on the substrate via DNA origami on cell adhesion, spreading, migration, and myofibroblastic differentiation. The studies will provide new insights into how cells respond to changes in viscoelasticy in their microenvironment.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.