Complex multicomponent patterns rendered on a 3D DNA-barrel pegboard

DNA origami, in which a long scaffold strand is assembled with a many short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. However, currently the design and optimization of custom 3D DNA-origami shapes is a barrier to rapid application to new areas. Here we introduce a modular barrel architecture, and demonstrate hierarchical assembly of a 100 megadalton DNA-origami barrel of similar to 90nm diameter and similar to 250nm height, that provides a rhombic-lattice canvas of a thousand pixels each, with pitch of similar to 8nm, on its inner and outer surfaces. Complex patterns rendered on these surfaces were resolved using up to twelve rounds of Exchange-PAINT super-resolution microscopy. We envision these structures as versatile nanoscale pegboards for applications requiring complex 3D arrangements of matter, which will serve to promote rapid uptake of this technology in diverse fields beyond specialist groups working in DNA nanotechnology

Programmable biomolecular integration and dynamic behavior of DNA-based systems for development of biomedical nano-devices

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, 2019Cataloged from PDF version of thesis.Includes bibliographical references.Departing from the traditional role as a carrier of genetic information, DNA has emerged as an engineering material for construction of nano-devices. The advances in the field of DNA nanotechnology have enabled design and synthesis of DNA nanostructures of arbitrary shapes and manipulation of the nanostructures' conformations in a programmable way. DNA-based systems offer potential applications in medicine by manipulating the biological components and processes that occur at the nanometer scale. To accelerate the translation of DNA-based systems for medical applications, we identified some of the challenges that are hindering our ability to construct biomedical nano-devices and addressed these challenges through advances in both structural and dynamic DNA nanotechnology. First, we tested the stability of DNA nanostructures in biological environments to highlight the necessity of and path towards protection strategies for prolonged integrity of biomedical nano-devices. Then, we constructed a platform for robust 3D molecular integration using DNA origami technique and implemented the platform for a nanofactory capable of production of therapeutic RNA to overcome the challenges in RNA delivery. Moreover, we established a mechanism to drive DNA devices by changing temperature with prolonged dynamic behavior that was previously challenging to accomplish without special modification of DNA and/or equipment not readily available in a typical lab setting. Together, the progress made in this thesis bring us another step closer to realization of medical applications of DNA nanotechnology by focusing on the challenges in both structural and dynamic aspects of the technology.by Jaeseung Hahn.Ph. D. in Medical Engineering and Medical PhysicsPh.D.inMedicalEngineeringandMedicalPhysics Harvard-MIT Program in Health Sciences and Technolog

Addressing the Instability of DNA Nanostructures in Tissue Culture

DNA nanotechnology is an advanced technique that could contribute diagnostic, therapeutic, and biomedical research devices to nanomedicine. Although such devices are often developed and demonstrated using <i>in vitro</i> tissue culture models, these conditions may not be compatible with DNA nanostructure integrity and function. The purpose of this study was to characterize the sensitivity of 3D DNA nanostructures produced <i>via</i> the origami method to the <i>in vitro</i> tissue culture environment and identify solutions to prevent loss of nanostructure integrity. We examined whether the physiological cation concentrations of cell culture medium and the nucleases present in fetal bovine serum (FBS) used as a medium supplement result in denaturation and digestion, respectively. DNA nanostructure denaturation due to cation depletion was design- and time-dependent, with one of four tested designs remaining intact after 24 h at 37 °C. Adjustment of medium by addition of MgSO₄ prevented denaturation. Digestion of nanostructures by FBS nucleases in Mg²⁺-adjusted medium did not appear design-dependent and became significant within 24 h and when medium was supplemented with greater than 5% FBS. We estimated that medium supplemented with 10% FBS contains greater than 256 U/L equivalent of DNase I activity in digestion of DNA nanostructures. Heat inactivation at 75 °C and inclusion of actin protein in medium inactivated and inhibited nuclease activity, respectively. We examined the impact of medium adjustments on cell growth, viability, and phenotype. Adjustment of Mg²⁺ to 6 mM did not appear to have a detrimental impact on cells. Heat inactivation was found to be incompatible with <i>in vitro</i> tissue culture, whereas inclusion of actin had no observable effect on growth and viability. In two <i>in vitro</i> assays, immune cell activation and nanoparticle endocytosis, we show that using conditions compatible with cell phenotype and nanostructure integrity is critical for obtaining reliable experimental data. Our study thus describes considerations that are vital for researchers undertaking <i>in vitro</i> tissue culture studies with DNA nanostructures and some potential solutions for ensuring that nanostructure integrity and functions are maintained during experiments

Neurovascular coupling under chronic stress is modified by altered GABAergic interneuron activity

Neurovascular coupling (NVC), the interaction between neural activity and vascular response, ensures normal brain function by maintaining brain homeostasis. We previously reported altered cerebrovascular responses during functional hyperemia in chronically-stressed animals. However, the underlying neuronal-level changes associated with those hemodynamic changes remained unclear. Here, using in vivo and ex vivo experiments, we investigate the neuronal origins of altered NVC dynamics under chronic stress conditions in adult male mice. Stimulus-evoked hemodynamic and neural responses, especially beta and gamma-band local field potential activity, were significantly lower in chronically-stressed animals, and the NVC relationship, itself, had changed. Further, utilizing acute brain slices, we discovered that the underlying cause of this change was dysfunction of neuronal nitric oxide synthase (nNOS)-mediated vascular responses. Using in situ hybridization (FISH) to check the mRNA expression of several GABAergic subtypes, we confirmed that only nNOS mRNA was significantly decreased in chronically-stressed mice. Ultimately, chronic stress impairs NVC by diminishing nNOS-mediated vasodilation responses to local neural activity. Overall, these findings provide useful information in understanding NVC dynamics in the healthy brain. More importantly, this study reveals that impaired nNOS-mediated NVC function may be a contributory factor in the progression of stress-related diseases. Copyright © 2019 Han et al.11Nsciescopu

An Antiherpesviral Host-Directed Strategy Based on CDK7 Covalently Binding Drugs: Target-Selective, Picomolar-Dose, Cross-Virus Reactivity

The repertoire of currently available antiviral drugs spans therapeutic applications against a number of important human pathogens distributed worldwide. These include cases of the pandemic severe acute respiratory coronavirus type 2 (SARS-CoV-2 or COVID-19), human immunodeficiency virus type 1 (HIV-1 or AIDS), and the pregnancy- and posttransplant-relevant human cytomegalovirus (HCMV). In almost all cases, approved therapies are based on direct-acting antivirals (DAAs), but their benefit, particularly in long-term applications, is often limited by the induction of viral drug resistance or side effects. These issues might be addressed by the additional use of host-directed antivirals (HDAs). As a strong input from long-term experiences with cancer therapies, host protein kinases may serve as HDA targets of mechanistically new antiviral drugs. The study demonstrates such a novel antiviral strategy by targeting the major virus-supportive host kinase CDK7. Importantly, this strategy focuses on highly selective, 3D structure-derived CDK7 inhibitors carrying a warhead moiety that mediates covalent target binding. In summary, the main experimental findings of this study are as follows: (1) the in vitro verification of CDK7 inhibition and selectivity that confirms the warhead covalent-binding principle (by CDK-specific kinase assays), (2) the highly pronounced antiviral efficacies of the hit compounds (in cultured cell-based infection models) with half-maximal effective concentrations that reach down to picomolar levels, (3) a particularly strong potency of compounds against strains and reporter-expressing recombinants of HCMV (using infection assays in primary human fibroblasts), (4) additional activity against further herpesviruses such as animal CMVs and VZV, (5) unique mechanistic properties that include an immediate block of HCMV replication directed early (determined by Western blot detection of viral marker proteins), (6) a substantial drug synergism in combination with MBV (measured by a Loewe additivity fixed-dose assay), and (7) a strong sensitivity of clinically relevant HCMV mutants carrying MBV or ganciclovir resistance markers. Combined, the data highlight the huge developmental potential of this host-directed antiviral targeting concept utilizing covalently binding CDK7 inhibitors