Carlos E. Castro - US grants

This Broadening Participation Research Initiation Grant in Engineering (BRIGE) provides funding for the development of a nanoscale device for fluorescence-based high throughput single molecule force spectroscopy. The device will be assembled using the recently developed nanotechnology DNA origami. DNA origami enables the construction of nanoscale objects with unprecedented geometric complexity via programmed molecular self-assembly. The device will comprise a stiff framework of bundles of double-stranded DNA, attachment points for two biomolecules, a flexible polymer force probe, and fluorescent molecules to act as a readout of the interaction between the two biomolecules of interest. The force probe will facilitate binding between the biomolecules, and subsequently apply a known force acting to rupture the interaction. The bond lifetime will be monitored using a fluorescence readout, which is amenable to high throughput data collection. Interaction lifetimes will be measured as a function of force to determine kinetic parameters that govern the molecular interaction. Initial proof-of-principle experiments will probe DNA and RNA base-pairing interactions. Ultimately, the device will be implemented to study protein-DNA, protein-RNA, and protein-protein systems.

Single molecule force spectroscopy studies have provided critical insight into the interactions that stabilize biomolecular machinery and regulate the function of cells. However, the widespread application of force spectroscopy has been hindered by the need for cumbersome and expensive equipment and low throughput data acquisition. If successful, this research will enable economical and high-throughput force spectroscopy studies of biomolecular machinery. The ultimate goal is to develop a device that can be packaged and distributed for widespread application on basic laboratory fluorescence microscopes. This award will also support the establishment of a biomolecular machinery short course to be offered to high school age students at summer engineering camps and the development of a research seminar course aimed to recruit underrepresented engineering students to pursue graduate level research.

This research award provides funding for the development of a systematic design framework for nanoscale machines and mechanisms using DNA origami nanotechnology. DNA origami enables construction of two and three-dimensional nanoscale shapes with unprecedented geometric complexity via molecular self-assembly. The majority of DNA origami research focuses on assembly of static 2D or 3D structures. The goal of this work is to incorporate functional dynamic parts with directed motion into the DNA origami design toolbox. To achieve this goal, a kinematics approach will be implemented to design DNA origami machines and mechanisms that are comprised of links and joints, similar to macroscale machines. A catalogue of various DNA origami links and joints will be designed and fabricated, and their mechanical properties will be characterized experimentally and by molecular simulation. These links and joints will be implemented in the design of prototype mechanisms for proof-of-principle studies. A new theory called projection kinematics will be developed to evaluate the three-dimensional motion of these prototype mechanisms from two-dimensional electron microscopy snapshots. To facilitate future application of DNA origami machines, a computer aided design and simulation program will be developed that facilitates conceptual mechanism design and automates the integration of DNA origami links and joints from the previously developed part catalogues.

If successful, this research will greatly advance the fields of DNA nanotechnology, nanoscale design, and nano-robotics. The machines and mechanisms developed in this work together with nanoactuators and controllers will enable design of future nanorobots for applications in numerous fields such as self-assembling computers and chemical compounds, nano-manufacturing, molecular transport in bio-reactors, and drug delivery. This work also opens the door for applying kinematic design to DNA nanotechnology. The computer design automation software developed in this research will greatly facilitate the widespread use of DNA origami mechanisms and machines.

DESCRIPTION (provided by applicant): Dynamic organization of the human genome into chromatin regulates transcription initiation and elongation. Defects in chromatin modifications, assembly, disassembly and remodeling result in misregulation of oncogenes, which are associated with numerous cancers including ovarian, bladder, prostate, and colorectal tumors. Prior research has identified the components involved in chromatin transcriptional regulation (CTR), including histone variants and post-translational modifications (PTMs), histone modification enzymes, and histone chaperone assembly factors. Remarkably, genetic, biochemical, structural, deep sequencing and single molecule studies have not fully revealed the mechanisms of CTR. Therefore, new technologies are required to probe currently inaccessible dynamics and structure of chromatin assemblies at the 10-100 nm length scale, which encompasses critical molecular events the regulate DNA processing. This research will address current technological gaps through the development of nanoscale tools that measure mesoscale (10- 100nm) structure and dynamics of chromatin at specific cancer-relevant modification and processing sites. Specifically, we will develop 1) DNA origami nanostructures with multiple antibodies that recognize distinct physiological and cancer-relevant combinations of chromatin marks (histone modifications/variants and genomic DNA processing sites) and 2) DNA origami displacement sensors to study site-specific mesoscale dynamics at gene regulation sites. The long-term goal of this work is to develop tools and methods to probe chromatin function and dynamics at cancer-relevant chromatin modifications and oncogene regulation sites in vivo. Within the scope of this exploratory research, we will focus on the devic development, in vitro proof-of- principle, and characterizations of chromatin assemblies. Future work will build on the tools and experimental framework established here to implement DNA origami devices to probe intracellular function of chromatin assemblies.

PI: Castro, Carlos E.
Proposal Number: 1351159

Forces applied between cells and their environment play a critical role in cellular physiologic behavior, including cell spreading, rolling and migration. These cellular traction forces (CTF) are applied via membrane proteins that mediate physical communication of cells with the local environment. The proposed work aims to develop and implement a nanoscale molecular force sensor (NMFS) to directly measure the CTF transmission of single membrane proteins and protein complexes. This methodology will be developed and validated using two physiologically relevant processes as test beds. The proposed work has significant broader impacts on elucidating cellular function and on guiding the design of biomedical devices for applications such as cell sorting and biological sensing. The developed NMFS designs will be broadly shared to encourage widespread application of this technology. The PI will develop a biomolecular design and mechanics workshop to be offered through Ohio State University (OSU) outreach efforts extending to middle and high school students focusing on underrepresented populations. Furthermore, the PI will recruit underrepresented students to participate in both summer REU programs and upper level thesis research in his lab. A yearly project team consisting of multi-disciplinary 2nd and 3rd year students participating in an annual Biomolecular Design Competition will also be established. Finally, the PI has developed a Biomolecular Mechanics course in the Mechanical Engineering curriculum, which is offered as a technical elective. The research proposed here will be leveraged to include a laboratory component that will promote student education in the relevant principles and techniques. Overall, the PI will implement significant activities, with an emphasis on students from underrepresented populations, which integrate the research of this project with education and outreach.


Current approaches to measure CTF largely rely on monitoring substrate displacements and require complex mathematical algorithms and assumptions regarding the location of the forces (i.e. at focal adhesions) to determine CTF fields. While these approaches have provided useful insight into net cellular forces and cell-substrate interactions, the single molecule details of CTF, in particular in physiologically realistic processes, remain poorly understood. Furthermore, technology is lacking to measure forces transmitted by specific single membrane proteins. Recent evidence has shown that forces may play a critical role in the function of individual receptors, such as the B cell receptor, which uses mechanical energy to differentiate antigens of varying affinities. The proposed work aims to develop and implement an NMFS to directly measure the CTF transmission of single membrane proteins and protein complexes in two studies focused on the physiological processes of migration and B cell antigen detection. Specifically, the aims of the proposed work are to: 1) design, build and calibrate a NMFS capable of interacting with single membrane proteins and membrane protein complexes; 2) employ the NMFS to measure traction forces of 3T3 fibroblasts migrating on two-dimensional (2D) soft substrates; 3) employ the NMFS to measure traction forces of 3T3 fibroblasts migrating in a matrix of fibers; and 4) employ the NMFS to study the role of mechanical forces and antigen affinity during B cell antigen detection. This research will develop, calibrate, and implement a NMFS that is capable of measuring CTF of single membrane proteins and protein complexes. This single molecule direct measurement device will be implemented to make previously intractable measurements of CTF in the cellular processes of migration in 3D fibrous environments and antigen detection. Results are expected to reveal new molecular insights into force transmission of membrane proteins during critical biological processes. The NMFS will be constructed using the nanotechnology, scaffolded DNA origami, and will integrate functionalization for cellular interaction (i.e. RGD-integrin binding) and substrate interaction (i.e. biotin-streptavidin), springs with calibrated stiffness, and fluorescent dyes for Fluorescence Resonance Energy Transfer (FRET) deformation readouts. The device will be validated by performing two sets of experiments: 1) measuring CTF of fibroblasts on 2D substrates and in 3D fibrous environments, and 2) measuring force application of B cells during antigen detection. This work will combine live cell imaging, fluorescence microscopy, single molecule FRET, and DNA origami to achieve new insights into cellular processes mediated by single molecule attachments.

Understanding the physical properties of the genome (which is composed of DNA) is essential to determining how processes such as gene expression, DNA replication, and DNA repair are controlled. A wide range of organisms organize their genome by repeatedly wrapping the DNA into small spools called nucleosomes. As a gene is expressed, replicated or repaired the organization of the nucleosomes is rearranged, and this project will develop a new approach to enable the study of chromatin structural dynamics over the length of a gene, thereby enabling new physical insights into how genes are turned on and off. Interdisciplinary training will be provided in a wide range of fields from molecular biology and biochemistry to nanotechnology and single molecule biophysics. The researchers will integrate this project into outreach programs including a Minority Engineering Program, Women in Engineering, and the Masters to PhD Bridge Program, all of which serve to increase the diversity of the next generation of STEM researchers.

Chromatin structural dynamics control the accessibility of DNA to complexes that regulate transcription, repair DNA damage and replicate DNA. Conversion between open euchromatin that is accessible to DNA regulatory complexes and compact heterochromatin that is inaccessible is essential for genome regulation and processing. Currently, there is a lack of tools for probing critical events during gene regulation at specific DNA sequences (i.e. promoter regions) that span 10-100nm over multiple nucleosomes. To address this challenge, the principal investigators will: (1) develop nanoscale hinge devices using DNA origami nanotechnology to quantitatively measure mesoscale conformational dynamics of chromatin; and (2) determine the 10-100nm structural dynamics of H1 compacted chromatin. The conformational changes in these nano-hinges will be detected by transmission electron microscopy and single molecule fluorescence. The development and application of these single molecule methods will provide new tools and insight into the structural events that occur within chromatin as it converts between distinct functional states.

This project is funded jointly by the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Division of Materials Research in the Directorate for Mathematical and Physical Sciences.

DNA origami mechanisms are nanometer scale mechanical devices that are made of deoxyribonucleic acid (DNA) biological materials. For comparison, a human hair is approximately 80,000 to 100,000 nanometers wide. Self-assembled via the so called DNA base-pairing process, the motion of these tiny mechanisms can be controlled to accomplish a task similar to machines and robots in the macro world. This technique has the potential to revolutionize medicine or reduce resource consumption and environmental pollution in manufacturing processes. For example, DNA nano-robots could potentially be used for nano-manufacturing, for molecular transport in bioreactors, for targeting cancer cells for drug delivery, or even for repairing damaged tissue. However, the motion of these nano-mechanisms is extremely difficult to control due to significant thermal fluctuation in solution, random errors in the molecular self-assembly process, and variation in material and structure properties. This award supports the fundamental research to address these challenges by developing a novel robust-design methodology borrowed from macroscopic compliant-mechanism design. The computational design tools resulting from this research will enable us to effectively design DNA nano-machines that are more accurately controllable, and that can be used in medicine for improving public health.

Recent success in applying kinematic mechanism principles in design of DNA origami nano-mechanisms has now opened new significant challenges in quantifying and managing the mechanical behavior of these mechanisms. The goal of this award is to develop a robust design framework for compliant DNA origami mechanisms to improve motion control and enable a wider range of mechanical behavior of DNA nano-structures. This design framework incorporates synthesis of commonly used compliant joints, pseudo-rigid-body models, kinetostatic analysis, synthesis algorithms and statistical theory based method for uncertainty quantification and management. The computational tools and experimental processes resulting from this research will enable: (a) design of DNA origami mechanisms with broader scope of mechanical behavior, (b) characterization of the properties of these nano-mechanisms, and (c) uncertainty quantification and management of DNA origami structures.

The physical properties of the genome play a central role in controlling essential processes, including gene expression, DNA repair, and DNA replication. All eukaryotic organisms, from yeast to humans, organize their genomic DNA by repeatedly wrapping it around DNA-associated proteins, called histones, into small spools known as nucleosomes. These nucleosomes, which are about a millionth of a centimeter (10 nanometers) in size, are strung like beads on a string to form long chromatin fibers that ultimately make up chromosomes. The spatial arrangement of these nucleosomes within the chromatin fibers continually changes, often through coordinated movements. However, current technologies are limited in quantifying these structural rearrangements, and this limitation has in turn limited our understanding of how chromatin controls gene expression. This project will monitor structural changes in chromatin as gene expression is switched between the 'on' and 'off' states by developing DNA-based nanometer-sized calipers to detect structural changes in chromatin at the 10 nanometer to 100 nanometer scale, which covers a critical range for events during the regulation of gene expression. Graduate and undergraduate students will receive training in molecular biology, biochemistry, DNA nanotechnology, and single molecule detection, which will position them to become significant contributors as the next generation of biotechnology scientists. The PIs will also work with outreach programs, including the OSU Minority Engineering Program, the Women in Engineering Program, and the Masters to Ph.D. Bridge Program, to improve diversity and interest in the fields of biotechnology and biophysics.

The conversion of chromatin between open euchromatin and compact heterochromatin is essential for the regulation of transcription and cellular differentiation. This conversion is regulated by histone post-translational modifications (PTMs) and chromatin architectural proteins that recognize these PTMs to dynamically control chromatin structure. However, the dynamic chromatin structural properties that regulate transcription are currently not well understood. These properties includes changes in the distance between nucleosomes, changes in nucleosome orientation, and the time for these structural changes to occur. To directly investigate chromatin structural dynamics, this project is using DNA origami nanotechnology to develop DNA-based nano-calipers that quantify 10 nm to 100 nm distance changes within large macromolecular complexes. The project will use this DNA nano-caliper design to: (i) determine the long range structural dynamics of heterochromatin that is formed by Heterochromatin Protein 1 as it interacts with the histone H3 PTM, lysine 9 trimethylation, and (ii) determine the long range structural and dynamic response of compacted heterochromatin to the binding of transcription activating complexes. This approach involves integrating PTM-containing chromatin molecules into DNA nano-calipers where each end is attached to opposite nano-caliper arms. The DNA nano-caliper conformational changes will then be detected separately with transmission electron microscopy to determine the distribution of chromatin conformations and single molecule fluorescence to determine the transition rates between distinct chromatin structural states. These studies will provide key distance and time measurements of the structural dynamics that occur during heterochromatin formation and heterochromatin decompaction as transcription activators bind to their target sites. More broadly, the development of this DNA origami nanotechnology will be widely applicable to the study of numerous biological complexes that undergo structural changes on the 10 nm to 100 nm length scale.

This project is jointly supported by the Genetic Mechanisms and the Molecular Biophysics Programs of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences, and by the Biomaterials Program of the Division of Materials Research in the Directorate for Physical and Mathematical Sciences.

ABSTRACT Dysregulation of vascular architecture and function is characteristic of a broad spectrum of pathologies, including inflammation, cardiovascular diseases, and cancer. Therefore, the ability to control angiogenesis and vessel remodeling has considerable therapeutic benefit. Blood vessels are lined with a monolayer of tightly joined and mechanically coupled endothelial cells (ECs) that form the barrier between blood and the surrounding tissue. In addition, it is well established that fluid mechanical stresses, such as ones associated with intravascular and transvascular flow, are interpreted by ECs to help form and remodel blood vessels. However, while numerous mechanotransducers in ECs have been proposed, a detailed, quantitative, and complete model of flow sensing by ECs that assists in developing a systematic pathway to controlling angiogenesis does not exist. Thus, there is a significant need for accurately engineered in vitro platforms to systematically study and develop a comprehensive model of the functional outcomes of fluid stresses on blood vessel architecture. Based on our preliminary data and previous discoveries, we hypothesize that intravascular shear stress and transvascular flow impart competing effects in controlling blood vessel remodeling leading to quantifiable changes in angiogenesis vascular permeability, and interendothelial ultrastructure. By thoroughly assessing these parameters, we believe that our approach will identify the biophysical signatures of dysregulated vessel architecture that are characteristic of vascular diseases. Moreover, our goal is to use these biophysical signatures to help design strategies for controlling pathological angiogenesis and vascular permeability. To meet this goal, we will use an integrated strategy in which 3-D microfluidic systems that allow control of physiological levels of pressure and flow conditions and the cell/matrix topology of intact blood vessels will be used in conjunction with high-resolution microscopy and force spectroscopy with nanoscale devices to determine the physical mechanisms by which fluid stresses control angiogenesis and vascular permeability. In Aim 1, we will quantify changes in blood vessel structure and function in response to fluid stresses. In Aim 2, we will measure changes in tension at EC junctions in response to fluid stresses. In Aim 3, we will develop approaches for suppressing angiogenesis and vascular permeability by stabilizing EC junctions. Completion of these studies will help establish a new paradigm for using cellular and subcellular biophysics for controlling angiogenesis and blood vessel remodeling.

Robotic materials are an emerging class of materials that integrate actuation, sensing, communication, and computing functions. Examples include artificial skins with embedded electronics for sensing, and composites with adaptive texture morphing for camouflage. While there has been significant progress in developing macroscopic robotic materials, integrating robotic functions at the nano to microscale remains a challenge. DNA is as an excellent candidate for creating such robotic nanomaterials because it enables fabrication of nanostructures with unprecedented complex geometry and reconfigurability. This Designing Materials to Revolutionize and Engineer our Future (DMREF) award supports fundamental research to enable development of robotic DNA materials with sensing, communicating, and phase separating functions, integrating multidisciplinary expertise in DNA nanotechnology, single molecule measurements, and molecular modeling. The team will create DNA nanostructures that sense the local environment and assemble those structures into larger systems that transmit signals or exhibit collective behaviors. This will enable materials that change their structure, adapt their properties, or modify their environment in response to external triggers, which could have a range of applications in nanomanufacturing, biological sensing, energy harvesting or storage, lab-on-a-chip systems, and drug delivery. This project will also provide unique training opportunities to graduate and undergraduate students in DNA nanotechnology, molecular robotics, single-molecule measurements, and multi-scale modeling. The researchers will organize workshops to facilitate sharing of new materials design and modeling methods and develop curricula in robotic nanomaterials. Furthermore, this project's findings will be integrated into outreach programs in Central Ohio and North Carolina to generate interest in science and engineering from the next generation workforce.

Robotic materials that integrate sensing, actuation, and communication and processing of information at nano- to microscales can provide many technological benefits to society, with applications ranging from nanomanufacturing to medicine. This research investigates the forces, signals, and mechanisms that enable the development of DNA-based robotic materials that sense forces under various loading conditions, communicate signals over long distances, and exhibit phase separating functionalities. To achieve these functions, the team will leverage the well-defined nanoscale geometry and dynamic properties of DNA nanostructures and the specificity and programmability of DNA binding interactions. Multi-scale molecular modeling methods will guide the design of DNA devices with targeted structural, mechanical, and dynamic properties with feedback from single molecule characterization methods. The team will create materials via hierarchical self-assembly that leverage individual device properties and interactions between devices to expand sensing capabilities, transmit signals via propagated conformational changes, and exhibit collective behaviors driven by local interactions. This will provide a foundation to develop new and complex robotic materials from the nano- to micron-scale that operate at room temperature in aqueous environments.

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.

Numerous organisms from yeast to humans organize their genome by wrapping it repeatedly around histone proteins into nanoscale spools known as chromatin. Cells use the organization of chromatin to dictate whether a gene is actively expressed or turned off. Combining the ability to target a specific gene, visualize its location and structure, activate the gene, and detect gene expression in live cells would be a major technological advance in how genes are studied and controlled in living organisms, and lead to applications in many fields, including medicine, agriculture, energy and the environment. DNA nanotechnology, which uses well-understood folding properties of DNA to engineer nanoscale, biocompatible structures, is an emerging technology with the potential to combine these functions. A 5-PI team will apply bioengineering, cell biology, genetics, single molecule spectroscopy, super resolution microscopy and multi-scale molecular modeling to develop such DNA-based nanodevices that can also operate in live cells and be "switchable" to allow these functions to be triggered at will. The research will be integrated into university curricula, and will enable cross-disciplinary, collaborative and international training of graduate students. The PIs will also broaden participation of underrepresented students in STEM by creating open access standards-based videos and modules for use by K-12 teachers.

Recent advances in genetic and epigenetic methods have enabled chromatin engineering technologies that 1) target genes to 2) visualize chromatin structure, 3) activate target genes, and 4) detect gene-specific transcription. However, current tools (including super-resolution imaging, chromatin conformation capture and genome engineering with CRISPR/Cas9) are usually only able to accomplish one of these functions at a time, giving single-channel, static views of the players and processes at a specific transcription site. This project will leverage DNA nanotechnology to leapfrog current technologies for probing and engineering genome and epigenome functions. A team of five PIs will develop multi-functional DNA origami (DO) nanodevices that combine targeting, functional modifications and RNA detection onto a single platform, operate in live cell nuclei, and are "switchable", allowing for real-time detection and for functions to be triggered by endogenous or external signals. The outcomes will serve as a foundation for future automated devices that target and regulate genome and epigenome functions, including gene expression, and serve as diverse new toolsets for science, engineering, and medical applications.

This award is co-funded by the Genetic Mechanisms cluster in the Division of Molecular and Cellular Biosciences in the Biological Sciences Directorate, the Emerging Frontiers in Research and Innovation program in the Division of Emerging Frontiers and Multidisciplinary Activities in the Engineering Directorate, and the Chemistry of Life Processes program in the Division of Chemistry in the Mathematics and Physical Sciences Directorate.

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.

DNA nanotechnology enables the design of dynamic nano-scale devices that can exhibit complex motion, reconfiguration, and transfer forces similar to macroscopic machines. These tiny constructs are also easily modified to incorporate biomolecules or nanoparticles, making them highly promising as tools to perform mechanical testing at a molecular scale or to create shape transforming materials. However, realizing the functional potential of such DNA-based devices or materials requires a robust approach for the rapid and precise control of their motion. To address these challenges, dynamic DNA-based devices and materials will be integrated with magnetic actuation platforms to enable robust and cost-effective approaches to test mechanical properties of biomolecules and DNA-based nanomaterials whose shape and properties can be magnetically controlled in real-time. These devices and materials can have broad applications in fields including biophysics, nanomanufacturing, biosensing, and nanorobotics. In addition, the research project will provide inter-disciplinary training of graduate and undergraduate students in fields including magnetism, biophysics, DNA nanotechnology, and nanomaterials. The work will be translated into broader science and engineering workforce training through outreach activities for high- and middle-school students and teachers in topics of physics, engineering and biology (e.g. magnetism, mechanics, DNA) with hands-on classroom and laboratory projects that demonstrate DNA self-assembly and magnetic actuation of DNA devices.

DNA-based self-assembly is a powerful method to create nano-constructs of molecular systems. To achieve the full potential of these systems rapid temporal (sub-second) and high spatial (10-50 nm) control has remained a challenge. This work will integrate design and hierarchical assembly of DNA devices and materials with a domain-wall based magnetic tweezers platform to enable advanced controlled actuation of DNA constructs. These magnetic tweezers allow for simultaneous control of multiple magnetic particles with force and motion inputs in multiple directions and at multiple locations. The study will thus leverage magnetically driven DNA constructs to develop force spectroscopy platforms with a focus on testing effects of applied forces on molecular interactions, in particular protein-DNA interactions. The devices will enable application of forces in multiple directions (e.g. tension and compression), which is highly challenging with other methods, while the DNA structures provide precise control over relative positioning and orientation of molecular samples. Furthermore, materials will be developed based on hierarchical assembly of DNA nanostructures where domain-wall magnetic tweezers will drive expansion, collapse, or rotation of materials and/or underlying components. Geometrical parameters and actuation forces will be guided by finite element simulations to render novel tailored DNA structural metamaterials with tunable properties such as negative Poisson's ratio or drastic shape changes that can be controlled via user-defined or computer controlled magnetic fields. Outreach and education efforts will engage students and high school teachers through the Ohio State University Translating Engineering to K through 8 program. Portable magnetic actuation systems will be developed to allow these high school teachers and students to interact with nanoscale systems and demonstrate associated basic concepts.

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.