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.
Information in biological cells is received, transmitted, and processed by biomolecular machinery in large part via highly specific molecular interactions. The nature of these interactions (i.e. number, frequency, and timescale) can trigger and finely tune cellular processes such as migration or cell death upon binding of extracellular molecules to a receptor on the surface of a cell. The focus of this project was the development of a novel method to test the physical strength of these interactions, which plays a crucial role in their biological function. Typically these types of experiments require cumbersome and expensive instrumentation; and hence, they are only carried out in specialized research laboratories. Through this work, we designed, fabricated and demonstrated proof-of-principle studies for a device constructed from DNA via molecular self-assembly that is capable of forming an interaction between biomolecules, and then applying a force to test the strength of that interaction. The device consists of a stiff platform approximately 100 nanometers in size that comprises a bundle of 8 double-stranded DNA helices and a flexible single-stranded DNA linker that functions as the force generating component much like a spring. One of the binding partners of interest is immobilized on the platform and the other on the end of the linker. When the two molecules bind, the linker is stretched so that it exerts a force on the interaction. The formation and rupture of the bond is monitored by a fluorescent signal emitted from the device that changes color when the bond is intact. The output can be measured by bulk fluorescence or single molecule fluorescence methods. We tested the functionality of the device by demonstrating the ability to form and rupture DNA-DNA binding interactions over a range of applied forces. Our next steps include modifying the device to probe DNA-protein interactions that regulate gene processing. Ultimately, the technology developed can be applied to study a wide variety of molecular interactions. We have also leveraged our research results to engage a broad range of students in research at the interface of biology, engineering, and physics. In particular, this project supported two graduate students and two undergraduate students directly involved in the project. The research team developed a biomolecular design and assembly workshop for middle school and high school engineering summer camps at The Ohio State University. In addition, design challenges based on DNA self-assembly and molecular force measurements were developed and presented at a Columbus’ Knowledge is Power Program (KIPP) charter school. These design challenges were also placed online with supporting videos to facilitate broader use.
