7. Highlights. Highlight Title: Mechanically Functional DNA Nano-devices to Probe and Actuate Cellular Machinery Author Names: Carlos Castro

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1 7. Highlights University Name: The Ohio State University (OSU) Cooperative Agreement Number: NSEC-CANPBD Grant # Science Highlights: Highlight Title: Mechanically Functional DNA Nano-devices to Probe and Actuate Cellular Machinery Author Names: Carlos Castro Highlight Title: High Throughput Nanochannel Electroporation for Controlled Cell Reprogramming Author Name: L. James Lee Education Highlight: Highlight Title: High School Students Use the Ohio Supercomputer Center to Model Nanofluidics Author Name: Sherwin Singer Shared Facilities Highlight: Highlight Title: Asymmetric Flow Field-Flow Fractionation/Multi-Angle Light Scattering and cryo-tem for Exosome Characterization Author Names: Michael Paulaitis

2 Mechanically Functional DNA Nano-devices to Probe and Actuate Cellular Machinery DNA nano-machine components θ d Molecular force sensors 20 nm 20 nm Outcome: Directly probing biomolecular machinery in the context of cellular function will can enable novel insight into the molecular mechanisms that regulate cellular processes. Devices that fabricated at a similar lengthscale to the biomolecular systems of interest provide a unique advantage of examining function in a wide range of experimental assays including studying cells or biomolecules in environments similar to in vivo conditions. Researchers in the Automated Cell to Biomolecule Analysis (ACBA) platform team have made critical progress towards developing a panel of devices designed to probe physical and chemical interactions of biomolecules. These devices are assembled using a recently developed DNA-based self assembly approach termed DNA origami nanotechnology. In particular, our results have led to novel approaches to create DNA-based nanodevices with mechanical functionality, including actuated moving parts and deformable components with tunable stiffness. This work has been presented at international conferences and has led to a publication in ACS Nano and other publications in preparation. Through this work, we have also established collaborations with biophysicists and physician scientists that have led to additional NSF funding and NIH funding. Platform for molecular functionalization DNA origami platform streptavidin Need: Current state-of-the-art tools to probe physical behaviors of biomolecular machinery in the context of cellular function include force spectroscopy approaches, such as atomic force microscopy, which enable application and/or measurement of forces but require cumbersome instrumentation and experimental assays that are generally not well-suited for physiological environments (i.e. fibrous matrices). Furthermore, resolving dynamic behavior in the ~10-100nm range, where many important molecular processes occur is highly challenging. Mechanically functional DNA-based devices with dimensions in the nm range can open the door to investigating physical processes or actuating machinery of biomolecular complexes to understand or manipulate cellular processes, such as cell migration, with mechanical readouts or inputs. antibody DNA origami platform Impact/Benefits: This work will broaden the scope of DNA nanotechnology and has the potential to provide a set of robust nanodevices that can be applied in physiological cellular environments, such as fibrous matrices. In addition, the dimensions of these devices are on the size scale such that they could be directly injected into cells to study biomolecule function native environments.

3 High Throughput Nanochannel Electroporation for Controlled Cell Reprogramming Fig. A NEP platform Fig. B Total internal reflection fluorescence Microscope In vitro cell reprogramming studies Outcome: Researchers at CANPBD have recently developed a novel high throughput nanochannel electroporation (NEP) platform that can be used to controllably deliver a myriad of cargo (e.g., genes, oligonucleotides, drugs, probes) into a large cell population in a highly deterministic and benign manner. Such technology is currently being used in cutting-edge regenerative medicine research. Through collaborations with the Departments of Pathology and Surgery at OSU s College of Medicine, and the Department of Biomedical Engineering at Duke University, CANPBD researchers are working on the implementation of this technology in cell reprogramming/engineering applications, where complex combinations of genes need to be precisely (time- and dosage-wise) delivered into cells in order to attain uniformly-transfected/reprogrammed populations with minimum intercellular variations, which is not achievable by any of the existing transfection technologies. Besides fine-tuning and exploring alternative NEP-based transfection conditions for existing reprogramming models, this technology is also being used to screen large pools of candidate genes and develop new reprogramming models of interest to regenerative medicine applications and/or disease-on-a-dish studies. This high throughput NEP technology was established in the past year and has already led to a number of manuscripts in preparation which will be submitted to high quality journals, presentations at national conferences, and joint NIH proposals through collaborations with physician and medical scientists working on cell reprogramming. Fig. C In vivo engrafting and/or tissue/function regeneration studies Visualizing DNA in nanochannels Explanation: Our research on the use of NEP for cell reprogramming applications has been demonstrated on the development of induced neurons through direct reprogramming, where adult cells undergo nuclear reprogramming directly to the cell of interest without passing through a pluripotent stem cell fate with the potential to deliver specific cell-based therapies to patients and provide human experimental models for high throughput pharmaceutical development. Cleanroom (see Fig. A) and noncleanroom-based technologies have been implemented to fabricate 3D NEP devices that can be used to transfect large cell populations (~1 millions). Briefly, cells are directly plated on the surface of such devices and electro-transfected through arrayed nanochannels that precisely control the amount of genes delivered into the population at the single cell level, and minimize any potential cell damage caused by the porating electric field. Uniform NEP-based transfection leads to fast and efficient in vitro reprogramming into the target fate (see Fig. B). Ongoing animal studies are also focused on understanding the underlying mechanisms of in vivo engrafting and reprogramming of NEP-treated cells in both adult and embryonic tissue (see Fig. C). Moreover, the regenerative capabilities of these cells are being tested in mice under different clinically-relevant conditions, such as stroke. Impact/Benefits: This platform technology was developed to allow for controlled and benign delivery of a variety of cargo, including genes and small oligonucleotides, into different and often hard to transfect cell populations, which could be of interest in number of applications besides cell reprogramming and regenerative medicine. As such, this platform has the potential to support different research projects and/or facilities (e.g., GMP Manufacturing) where precise cell transfection/engineering is required. Contact: L. James Lee (lee.31@osu.edu)

4 High School Students Use the Ohio Supercomputer Center to Model Nanofluidics Outcome: Under the guidance of NSEC faculty and students, four gifted high school students learned how molecular dynamics simulations can be used to predict and understand fluid dynamics in nanometer-scale devices. They learned to carry the project forward for design of the model to analysis of results, and organize their results in a coherent presentation. Students became aware of potential medical and societal benefits of nanotechnology-based biomedical devices, and how modeling can help improve device function. The students took a tour of a fabrication facility where they saw realworld application of the processes they modeled on the supercomputer center. Impact/Benefits: The mission of the Ohio Supercomputer Center Summer Institute and the NSEC at Ohio State is inspire gifted high school students to pursue careers in science and engineering. Some of the student feedback included, Besides teaching me a lot about nanofluidics, it has given me the skills to approach difficult projects, it was a great experience that taught me about both supercomputing and researching and presenting topics At their final presentation, members of a four member nanofluidics team of gifted high school students explain the motivation for their research (top), and the technical details of the modeling (bottom). Explanation: A CANPBD faculty member and graduate student has guided a team of gifted high school students to perform realistic molecular modeling of fluid flow in nanostructures. The student experienced the same type of openended problem solving they are likely to encounter as future scientists and engineers., The Ohio State University, Center for Affordable Nanoengineering of Polymeric Biomedical Devices

5 Asymmetric Flow Field-Flow Fractionation/Multi-Angle Light Scattering and cryo-tem for Exosome Characterization Asymmetric Flow Field-Flow Fractionation/Multi-Angle Light Scatting Asymmetric Flow Field-Flow Fractionation Cryo-TEM images of Cell-Secreted Exosomes Exosome-Lipoplex Nanoparticle Fusion Outcome: CANPBD researchers have created a shared facility for characterizing nanoparticle sizes, size distributions, and vesicle morphologies of exosomes derived from living cells, tumors, blood, or other bodily fluids. This facility integrates multiple experimental probes asymmetric flow field-flow fractionation, multiangle light scattering, and transmission electron microscropy at cryogenic temperatures to enable accurate, quantitative determinations of exosome concentrations, in addition to the above biophysical characterizations, that are critical to developing nanotechnologies for measuring the chemical composition of biomolecules encapsulated in the vesicles. These capabilities are currently being applied in several CANPBD projects to develop non-invasive assays for tumor-derived exosomes in blood or other bodily fluids as biomarkers for the early detection of cancers. For example, the efficiency of tethered cationic lipolex nanoparticle arrays for capturing exosomes and identifying exosomal mirna signatures of cancer is being evaluated using these techniques. This facility is also being used by CANPBD researchers to measure changes in exosomal mirna profiles in response to treatments that can inhibit intracellular signaling pathways critical for proliferation, development, and cell survival in support of applications of the tethered liposome nanoparticle array assay in clinical settings. The shared facility has already contributed to numerous research publications over the past year in high quality scientific and medical journals. Asymmetric flow field-flow fractionation/multi-angle light scattering and cryo-tem characterizations of cell-secreted exosomes have also been reported at national and international conferences. Nanoparticle characterization capabilities provided by this facility have also become an integral component of NSF and NIH proposals submitted by NSEC faculty over the past several years, and have led to collaborations with medical researchers and clinicians focused on leukemias, lung cancer, and thyroid cancer. Explanation: Asymmetric flow field-flow fractionation/multi-angle light scattering circumvents the inherent limitation of other light scattering techniques in analyzing polydisperse samples by first fractionating the vesicle populations based on particle size. Since the resulting fractions have essentially monodisperse size distributions, accurate determinations of exosome number densities are possible. In addition, each fraction of the sample can be collected separately to directly visualize vesicle morphologies by cryo-tem, and analyzed further for protein, lipid, and/or nucleic acid compositions. Thus, asymmetric flow field-flow fractionation/ multi-angle light scattering can be used as a separation technique in addition to a biophysical characterization tool in conjunction with many current assay technologies under development in CANPBD. Impact/Benefits: This shared facility has been established to enable quantitative assessments of CANPBD technologies for simultaneously isolating and analyzing exosomes derived from living cells, tumors, blood, or other bodily fluids in the early detection of cancers. Accurate quantitative measurements of exosome number densities in an isolation and analysis processes are unique to the fractionation/light scattering experimental techniques that are currently available to CANPBD researchers. Several collaborations within CANPBD are now applying these techniques to build reliable, cost effective devices for simultaneous exosome capture and exosomal mirna analyses in clinical settings.