Using light irradiation as a trigger, large-scale structural reconfiguration of DNA nanostructures is demonstrated. We incorporated photo-cleavable spacers at strategic locations within the short ...oligonucleotide strands connecting adjacent helices within a DNA origami sphere, and then used light to transform the sphere into two tethered hemispheres.
Abstract Antibodies have long served as vital tools in biological and clinical laboratories for the specific detection of proteins. Conventional methods employ fluorophore or horseradish ...peroxidase-conjugated antibodies to detect signals. More recently, DNA-conjugated antibodies have emerged as a promising technology, capitalizing on the programmability and amplification capabilities of DNA to enable highly multiplexed and ultrasensitive protein detection. However, the nonspecific binding of DNA-conjugated antibodies has impeded the widespread adoption of this approach. Here, we present a novel DNA-conjugated antibody staining protocol that addresses these challenges and demonstrates superior performance in suppressing nonspecific signals compared to previously published protocols. We further extend the utility of DNA-conjugated antibodies for signal-amplified in situ protein imaging through the hybridization chain reaction (HCR) and design a novel HCR DNA pair to expand the HCR hairpin pool from the previously published 5 pairs to 13, allowing for flexible hairpin selection and higher multiplexing. Finally, we demonstrate highly multiplexed in situ protein imaging using these techniques in both cultured cells and tissue sections.
Recent innovations in DNA nanofabrication allow the creation of intricately shaped nanostructures ideally suited for many biological applications. To advance the use of DNA nanotechnology for the ...controlled release of bioactive molecules, we report a general strategy that uses light to liberate encapsulated cargoes from DNA nanostructures with high spatiotemporal precision. Through the incorporation of a custom, photolabile cross-linker, we encapsulated cargoes ranging in size from small molecules to full-sized proteins within DNA nanocages and then released such cargoes upon brief exposure to light. This novel molecular uncaging technique offers a general approach for precisely releasing a large variety of bioactive molecules, allowing investigation into their mechanism of action, or finely tuned delivery with high temporal precision for broad biomedical and materials applications.
New storage technologies are needed to keep up with the global demands of data generation. DNA is an ideal storage medium due to its stability, information density and ease-of-readout with advanced ...sequencing techniques. However, progress in writing DNA is stifled by the continued reliance on chemical synthesis methods. The enzymatic synthesis of DNA is a promising alternative, but thus far has not been well demonstrated in a parallelized manner. Here, we report a multiplexed enzymatic DNA synthesis method using maskless photolithography. Rapid uncaging of Co
ions by patterned UV light activates Terminal deoxynucleotidyl Transferase (TdT) for spatially-selective synthesis on an array surface. Spontaneous quenching of reactions by the diffusion of excess caging molecules confines synthesis to light patterns and controls the extension length. We show that our multiplexed synthesis method can be used to store digital data by encoding 12 unique DNA oligonucleotide sequences with video game music, which is equivalent to 84 trits or 110 bits of data.
Human pluripotent stem cells (hPSCs) offer an unprecedented opportunity to model diverse cell types and tissues. To enable systematic exploration of the programming landscape mediated by ...transcription factors (TFs), we present the Human TFome, a comprehensive library containing 1,564 TF genes and 1,732 TF splice isoforms. By screening the library in three hPSC lines, we discovered 290 TFs, including 241 that were previously unreported, that induce differentiation in 4 days without alteration of external soluble or biomechanical cues. We used four of the hits to program hPSCs into neurons, fibroblasts, oligodendrocytes and vascular endothelial-like cells that have molecular and functional similarity to primary cells. Our cell-autonomous approach enabled parallel programming of hPSCs into multiple cell types simultaneously. We also demonstrated orthogonal programming by including oligodendrocyte-inducible hPSCs with unmodified hPSCs to generate cerebral organoids, which expedited in situ myelination. Large-scale combinatorial screening of the Human TFome will complement other strategies for cell engineering based on developmental biology and computational systems biology.
Hydrogels are being increasingly studied for use in various biomedical applications including drug delivery and tissue engineering. The successful use of a hydrogel in these applications greatly ...relies on a refined control of the mechanical properties including stiffness, toughness, and the degradation rate. However, it is still challenging to control the hydrogel properties in an independent manner due to the interdependency between hydrogel properties. Here it is hypothesized that a biodegradable polymeric crosslinker would allow for decoupling of the dependency between the properties of various hydrogel materials. This hypothesis is examined using oxidized methacrylic alginate (OMA). The OMA is synthesized by partially oxidizing alginate to generate hydrolytically labile units and conjugating methacrylic groups. It is used to crosslink poly(ethylene glycol) methacrylate and poly(N‐hydroxymethyl acrylamide) to form three‐dimensional hydrogel systems. OMA significantly improves rigidity and toughness of both hydrogels as compared with a small molecule crosslinker, and also controls the degradation rate of hydrogels depending on the oxidation degree, without altering their initial mechanical properties. The protein‐release rate from a hydrogel and subsequent angiogenesis in vivo are thus regulated with the chemical structure of OMA. Overall, the results of this study suggests that the use of OMA as a crosslinker will allow the implantation of a hydrogel in tissue subject to an external mechanical loading with a desired protein‐release profile. The OMA synthesized in this study will be, therefore, highly useful to independently control the mechanical properties and degradation rate of a wide array of hydrogels.
Oxidized methacrylic alginate (OMA), which presents multiple methacrylates and biodegradable units, can crosslink a variety of polymers to form hydrogels which show significant increase in mechanical strength, and tune degradation rates. OMA‐crosslinked hydrogels can control the release of vascular endothelial growth factor, leading to enhanced angiogenesis in vivo.
Utilization of neuropharmaceuticals for central nervous system(CNS) disease is highly limited due to the blood-brain barrier(BBB) which restricts molecules larger than 500Da from reaching the CNS. ...The development of a reliable method to bypass the BBB would represent an enormous advance in neuropharmacology enabling the use of many potential disease modifying therapies. Previous attempts such as transcranial catheter implantation have proven to be temporary and associated with multiple complications. Here we describe a novel method of creating a semipermeable window in the BBB using purely autologous tissues to allow for high molecular weight(HMW) drug delivery to the CNS. This approach is inspired by recent advances in human endoscopic transnasal skull base surgical techniques and involves engrafting semipermeable nasal mucosa within a surgical defect in the BBB. The mucosal graft thereby creates a permanent transmucosal conduit for drugs to access the CNS. The main objective of this study was to develop a murine model of this technique and use it to evaluate transmucosal permeability for the purpose of direct drug delivery to the brain. Using this model we demonstrate that mucosal grafts allow for the transport of molecules up to 500 kDa directly to the brain in both a time and molecular weight dependent fashion. Markers up to 40 kDa were found within the striatum suggesting a potential role for this technique in the treatment of Parkinson's disease. This proof of principle study demonstrates that mucosal engrafting represents the first permanent and stable method of bypassing the BBB thereby providing a pathway for HMW therapeutics directly into the CNS.
An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and several types of somatic cells. ...Among these, granulosa cells play a key role in follicle formation and support for oogenesis. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs), a method of generating granulosa cells has been elusive. Here, we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of hiPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology and may enable the development of therapies for female reproductive health.
Since the elucidation of its structure, DNA has been at the forefront of biological research. In the past half century, an explosion of DNA‐based technology development has occurred with the most ...rapid advances being made for DNA sequencing. In parallel, dramatic improvements have also been made in the synthesis and editing of DNA from the oligonucleotide to the genome scale. In this Review, we will summarize four different subfields relating to DNA technologies following this trajectory of smaller to larger scale. We begin by talking about building materials out of DNA which in turn can act as delivery vehicles in vivo. We then discuss how altering microbial genomes can lead to novel methods of production for industrial biologics. Next, we talk about the future of writing whole genomes as a method of studying evolution. Lastly, we highlight the ways in which barcoding biological systems will allow for their three‐dimensional analysis in a highly multiplexed fashion.
At the center of it all: DNA‐based technologies continue to be developed at a rapid pace. Spurred by advances in the DNA synthesis and editing, future application derived from DNA technologies ranging from designing new nanomaterials to designing new organisms are discussed.
Coronavirus disease 2019 (COVID‐19) manifests with high clinical variability and warrants sensitive and specific assays to analyze immune responses in infected and vaccinated individuals. Using ...Single Molecule Arrays (Simoa), we developed an assay to assess antibody neutralization with high sensitivity and multiplexing capabilities based on antibody‐mediated blockage of the ACE2‐spike interaction. The assay does not require live viruses or cells and can be performed in a biosafety level 2 laboratory within two hours. We used this assay to assess neutralization and antibody levels in patients who died of COVID‐19 and patients hospitalized for a short period of time and show that neutralization and antibody levels increase over time. We also adapted the assay for SARS‐CoV‐2 variants and measured neutralization capacity in pre‐pandemic healthy, COVID‐19 infected, and vaccinated individuals. This assay is highly adaptable for clinical applications, such as vaccine development and epidemiological studies.
Using Single Molecule Arrays (Simoa), we developed a simple neutralization assay based on antibody‐mediated blockage of the ACE2‐spike interaction. This assay can assess antibody neutralization with high sensitivity and multiplexing capabilities. We used this assay to profile neutralization capacity in patients infected with SARS‐CoV‐2 and vaccinated individuals and show that this assay is useful for various clinical applications.
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