Zev J. Gartner

Boron nitride nanotubes are noncytotoxic and can be functionalized for interaction with proteins and cells.

We report the discovery that boron nitride nanotubes (BNNTs), isosteres of CNTs with unique physical properties, are inherently noncytotoxic. Furthermore, we developed a biomemetic coating strategy to interface BNNTs with proteins and cells. Finally, we showed that BNNTs can deliver DNA oligomers to the interior of cells with no apparent toxicity. This work suggests that BNNTs may be superior to CNTs for use as biological probes and in biomaterials.

Expanding the Reaction Scope of DNA-Templated Synthesis

The translation of amplifiable information into chemical structure is a key component of nature×s approach to generating functional molecules. The ribosome accomplishes this feat by catalyzing the translation of RNA sequences into proteins. Developing general methods to translate amplifiable information carriers into synthetic molecules may enable chemists to evolve non-natural molecules in a manner analogous to the cycles of translation, selection, amplification, and diversification currently used by nature to evolve proteins. As an initial step towards this goal, we recently examined the generality of DNA-templated synthetic chemistry.[1, 2] We demonstrated the ability of DNA-templated synthesis to direct a modest collection of chemical reactions without requiring the precise alignment of reactive groups into DNA-like conformations. Indeed, the distance independence and sequence fidelity of DNA-templated synthesis allowed the simultaneous, one-pot translation of a model library of more than 1000 templates into the corresponding thioether products, one of which was enriched by in vitro selection for binding to the protein streptavidin and amplified by the polymerase chain reaction (PCR). The range of reactions known to be supported by DNAtemplated synthesis,[2] however, remains a tiny fraction of those used either by synthetic chemists or by nature to generate molecules with desired properties. Many reactions central to the construction of natural or synthetic molecules have yet to be developed in a DNA-templated format despite their known compatibility with water.[3] We describe here the development of several useful DNA-templated reactions, including the first reported DNA-templated organometallic couplings and carbon ± carbon bond forming reactions other than pyrimidine photodimerization.[4, 5] Collectively, these reactions represent an important additional step towards the in vitro evolution of non-natural synthetic molecules by enabling the DNA-templated construction of a much more diverse set of structures than has been previously achieved. We first investigated the ability of DNA-templated synthesis to direct reactions that require a non-DNA-linked activator, catalyst, or other reagent in addition to the principal reactants. To test the ability of DNA-templated synthesis to mediate such reactions without requiring structural mimicry of the DNA backbone, we performed DNA-templated reductive aminations between amine-linked template 1 and benzaldehydeor glyoxal-linked reagents (2 and 3) with millimolar concentrations of NaBH3CN at room temperature in aqueous solutions. Products formed efficiently when the template and reagent sequences were complementary. In contrast, control reactions in which the sequence of the reagent did not complement that of the template, or in which NaBH3CN was omitted, yielded no significant product (Table 1 and Figure 1). While DNA-templated reductive aminations to generate products closely mimicking the

Direct Cell Surface Modification with DNA for the Capture of Primary Cells and the Investigation of Myotube Formation on Defined Patterns

Previously, we reported a method for the attachment of living cells to surfaces through the hybridization of synthetic DNA strands attached to their plasma membrane. The oligonucleotides were introduced using metabolic carbohydrate engineering, which allowed reactive tailoring of the cell surface glycans for chemoselective bioconjugation. While this method is highly effective for cultured mammalian cells, we report here a significant improvement of this technique that allows the direct modification of cell surfaces with NHS-DNA conjugates. This method is rapid and efficient, allowing virtually any mammalian cell to be patterned on surfaces bearing complementary DNA in under 1 h. We demonstrate this technique using several types of cells that are generally incompatible with integrin-targeting approaches, including red blood cells and primary T-cells. Cardiac myoblasts were also captured. The immobilization procedure itself was found not to activate primary T-cells, in contrast to previously reported antibody- and lectin-based methods. Myoblast cells were patterned with high efficiency and remained undifferentiated after surface attachment. Upon changing to differentiation media, myotubes formed in the center of the patterned areas with an excellent degree of edge alignment. The availability of this new protocol greatly expands the applicability of the DNA-based attachment strategy for the generation of artificial tissues and the incorporation of living cells into device settings.

Chemically programmed cell adhesion with membrane-anchored oligonucleotides

Cell adhesion organizes the structures of tissues and mediates their mechanical, chemical, and electrical integration with their surroundings. Here, we describe a strategy for chemically controlling cell adhesion using membrane-anchored single-stranded DNA oligonucleotides. The reagents are pure chemical species prepared from phosphoramidites synthesized in a single chemical step from commercially available starting materials. The approach enables rapid, efficient, and tunable cell adhesion, independent of proteins or glycans, by facilitating interactions with complementary labeled surfaces or other cells. We demonstrate the utility of this approach by imaging drug-induced changes in the membrane dynamics of non-adherent human cells that are chemically immobilized on a passivated glass surface.

Programmed synthesis of three-dimensional tissues

Reconstituting tissues from their cellular building blocks facilitates the modeling of morphogenesis, homeostasis, and disease in vitro. Here, we describe DNA Programmed Assembly of Cells (DPAC) to reconstitute the multicellular organization of tissues having programmed size, shape, composition, and spatial heterogeneity. DPAC uses dissociated cells that are chemically functionalized with degradable oligonucleotide “velcro,” allowing rapid, specific, and reversible cell adhesion to other surfaces coated with complementary DNA sequences. DNA-patterned substrates function as removable and adhesive templates, and layer-by-layer DNA-programmed assembly builds arrays of tissues into the third dimension above the template. DNase releases completed arrays of microtissues from the template concomitant with full embedding in a variety of extracellular matrix (ECM) gels. DPAC positions subpopulations of cells with single-cell spatial resolution and generates cultures several centimeters long. We used DPAC to explore the impact of ECM composition, heterotypic cell-cell interactions, and patterns of signaling heterogeneity on collective cell behaviors.

Formation of targeted monovalent quantum dots by steric exclusion

Precise control over interfacial chemistry between nanoparticles and other materials remains a significant challenge limiting the broad application of nanotechnology in biology. To address this challenge, we use “Steric Exclusion” to completely convert commercial quantum dots (QDs) into monovalent imaging probes by wrapping the QD with a functionalized oligonucleotide. We demonstrate the utility of these QDs as modular and non-perturbing imaging probes by tracking individual Notch receptors on live cells.

Directing Otherwise Incompatible Reactions in a Single Solution by Using DNA‐Templated Organic Synthesis

General methods for translating amplifiable information carriers such as DNA into synthetic molecules may enable the evolution of non-natural molecules through iterated cycles of translation, selection, and amplification that are currently available only to proteins and nucleic acids. During the process of developing such a method, we recently discovered that DNA templates can sequence-specifically direct a broad range of chemical reactions without any apparent structural requirements.[1,2] The generality of DNA-templated synthesis together with appropriate linker and purification strategies enabled the first multistep small-molecule syntheses programmed by DNA templates,[3] which raised the possibility of using this approach to generate synthetic small-molecule libraries of useful complexity. DNA-templated synthesis[4±24] can generate products individually linked to oligonucleotides that both encode and direct their syntheses.[1±3] This feature may enable reaction modes useful for library construction that are not available through current synthetic approaches. Present synthesis methodology, for example, cannot differentiate functional groups of similar reactivity on different molecules within the same solution even though such differentiation would enable diversification to take place without the effort or constraints associated with spatial separation. Here we report that DNA oligonucleotides can simultaneously direct several different types of synthetic reactions within the same solution, even though the reactants involved would be cross-reactive and therefore incompatible under traditional synthesis conditions. Our findings represent a new mode of reaction made possible by DNA-templated synthesis and may enable the one-pot diversification of synthetic library precursors into products of multiple reaction types. The ability of DNA templates to mediate diversification by using different types of reaction without spatial separation was first tested by preparing three oligonucleotide templates of different DNA sequences (1a±3a) functionalized at their 5’-ends with maleimide groups and three oligonucleotide reagents (4a±6a) functionalized at their 3’-ends with an amine, thiol, or nitroalkane group, respectively. The DNA sequences of the three reagents each contained a different 10base annealing region that was complementary to ten bases COMMUNICATIONS

Directing the assembly of spatially organized multicomponent tissues from the bottom up

The complexity of the human body derives from numerous modular building blocks assembled hierarchically across multiple length scales. These building blocks, spanning sizes ranging from single cells to organs, interact to regulate development and normal organismal function but become disorganized during disease. Here, we review methods for the bottom-up and directed assembly of modular, multicellular, and tissue-like constructs in vitro. These engineered tissues will help refine our understanding of the relationship between form and function in the human body, provide new models for the breakdown in tissue architecture that accompanies disease, and serve as building blocks for the field of regenerative medicine.

A Mechanogenetic Toolkit for Interrogating Cell Signaling in Space and Time

Tools capable of imaging and perturbing mechanical signaling pathways with fine spatiotemporal resolution have been elusive, despite their importance in diverse cellular processes. The challenge in developing a mechanogenetic toolkit (i.e., selective and quantitative activation of genetically encoded mechanoreceptors) stems from the fact that many mechanically activated processes are localized in space and time yet additionally require mechanical loading to become activated. To address this challenge, we synthesized magnetoplasmonic nanoparticles that can image, localize, and mechanically load targeted proteins with high spatiotemporal resolution. We demonstrate their utility by investigating the cell-surface activation of two mechanoreceptors: Notch and E-cadherin. By measuring cellular responses to a spectrum of spatial, chemical, temporal, and mechanical inputs at the single-molecule and single-cell levels, we reveal how spatial segregation and mechanical force cooperate to direct receptor activation dynamics. This generalizable technique can be used to control and understand diverse mechanosensitive processes in cell signaling. VIDEO ABSTRACT.

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity.

Significance Differences in cell–cell interfacial energies can explain how multiple cell types sort into spatially organized tissues. However, this strategy of self-organization is not robust to heterogeneity or changes to the interfacial energies that drive correct cell positioning. Therefore, heterogeneous epithelial tissues such as the human mammary and prostate glands use a different strategy. First, disorganized aggregates form an adhesive interface at the tissue–ECM boundary that provides geometric constraints to self-organization. Second, only one cell type interacts appreciably with this interface. This strategy can explain how self-organization remains robust in vivo, provides generalizable rules for reconstituting tissues in vitro, and suggests how structure might break down during cancer progression. Developing tissues contain motile populations of cells that can self-organize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its self-organization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissue–ECM boundary, rather than by differential homo- and heterotypic energies of cell–cell interaction. Surprisingly, interactions with the tissue–ECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cell–cell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cell–cell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cell–ECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer.