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.

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.

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.

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.

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.

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.