Image recovery from unknown network mechanisms for DNA sequencing-based microscopy.

Imaging-by-sequencing methods are an emerging alternative to conventional optical micro- or nanoscale imaging. In these methods, molecular networks form through proximity-dependent association between DNA molecules carrying random sequence identifiers. DNA strands record pairwise associations such that network structure may be recovered by sequencing which, in turn, reveals the underlying spatial relationships between molecules comprising the network. Determining the computational reconstruction strategy that makes the best use of the information (in terms of spatial localization accuracy, robustness to noise, and scalability) in these networks is an open problem. We present a graph-based technique for reconstructing a diversity of molecular network classes in 2 and 3 dimensions without prior knowledge of their fundamental generation mechanisms. The model achieves robustness by obtaining an unsupervised sampling of local and global network structure using random walks, making use of minimal prior assumptions. Images are recovered from networks in two stages of dimensionality reduction first with a structural discovery step followed by a manifold learning step. By breaking the process into stages, computational complexity could be reduced leading to fast and accurate performance. Our method represents a means by which diverse molecular network generation scenarios can be unified with a common reconstruction framework.

Stochastic modeling of antibody binding predicts programmable migration on antigen patterns.

Viruses and bacteria commonly exhibit spatial repetition of surface molecules that directly interface with the host immune system. However the complex interaction of patterned surfaces with immune molecules containing multiple binding domains is poorly understood. We developed a pipeline for constructing mechanistic models of antibody interactions with patterned antigen substrates. Our framework relies on immobilized DNA origami nanostructures decorated with precisely placed antigens. The results revealed that antigen spacing is a spatial control parameter that can be tuned to influence antibody residence time and migration speed. The model predicts that gradients in antigen spacing can drive persistent, directed antibody migration in the direction of more stable spacing. These results depict antibody-antigen interactions as a computational system wherein antigen geometry constrains and potentially directs antibody movement. We propose that this form of molecular programmability could be exploited during co-evolution of pathogens and immune systems or in the design of molecular machines.

Tuning intercellular adhesion with membrane-anchored oligonucleotides.

Adhesive interactions between cells play an integral role in development, differentiation and regeneration. Existing methods for controlling cell-cell cohesion and adhesion by manipulating protein expression are constrained by biological interdependencies, e.g. coupling of cadherins to actomyosin force-feedback mechanisms. We use oligonucleotides conjugated to PEGylated lipid anchors (ssDNAPEGDPPE) to introduce artificial cell-cell adhesion that is largely decoupled from the internal cytoskeleton. We describe cell-cell doublets with a mechanical model based on isotropic, elastic deformation of spheres to estimate the adhesion at the cell-cell interface. Physical manipulation of adhesion by modulating the PEG-lipid to ssDNAPEGDPPE ratio, and conversely treating with actin-depolymerizing cytochalasin D, resulted in decreases and increases in doublet contact area, respectively. Our data are relevant to the ongoing discussion over mechanisms of tissue surface tension and in agreement with models based on opposing cortical and cohesive forces. PEG-lipid modulation of doublet geometries resulted in a well-defined curve indicating continuity, enabling prescriptive calibration for controlling doublet geometry. Our study demonstrates tuning of basic doublet adhesion, laying the foundation for more complex multicellular adhesion control independent of protein expression.

A computational framework for DNA sequencing microscopy.

We describe a method whereby microscale spatial information such as the relative positions of biomolecules on a surface can be transferred to a sequence-based format and reconstructed into images without conventional optics. Barcoded DNA "polymerase colony" (polony) amplification techniques enable one to distinguish specific locations of a surface by their sequence. Image formation is based on pairwise fusion of uniquely tagged and spatially adjacent polonies. The network of polonies connected by shared borders forms a graph whose topology can be reconstructed from pairs of barcodes fused during a polony cross-linking phase, the sequences of which are determined by recovery from the surface and next-generation (next-gen) sequencing. We developed a mathematical and computational framework for this principle called polony adjacency reconstruction for spatial inference and topology and show that Euclidean spatial data may be stored and transmitted in the form of graph topology. Images are formed by transferring molecular information from a surface of interest, which we demonstrated in silico by reconstructing images formed from stochastic transfer of hypothetical molecular markers. The theory developed here could serve as a basis for an automated, multiplexable, and potentially superresolution imaging method based purely on molecular information.

Publisher Correction: Binding to nanopatterned antigens is dominated by the spatial tolerance of antibodies.

In the Supplementary Information file originally published with this Article, the Supplementary references 48-62 were missing; the amended file has now been uploaded.

Binding to nanopatterned antigens is dominated by the spatial tolerance of antibodies.

Although repetitive patterns of antigens are crucial for certain immune responses, an understanding of how antibodies bind and dynamically interact with various spatial arrangements of molecules is lacking. Hence, we introduced a new method in which molecularly precise nanoscale patterns of antigens are displayed using DNA origami and immobilized in a surface plasmon resonance set-up. Using antibodies with identical antigen-binding domains, we found that all the subclasses and isotypes studied bind bivalently to two antigens separated at distances that range from 3 to 17 nm. The binding affinities of these antibodies change with the antigen distances, with a distinct preference for antigens separated by approximately 16 nm, and considerable differences in spatial tolerance exist between IgM and IgG and between low- and high-affinity antibodies.

Solution-Controlled Conformational Switching of an Anchored Wireframe DNA Nanostructure.

Self-assembled DNA origami nanostructures have a high degree of programmable spatial control that enables nanoscale molecular manipulations. A surface-tethered, flexible DNA nanomesh is reported herein which spontaneously undergoes sharp, dynamic conformational transitions under physiological conditions. The transitions occur between two major macrostates: a spread state dominated by the interaction between the DNA nanomesh and the BSA/streptavidin surface and a surface-avoiding contracted state. Due to a slow rate of stochastic transition events on the order of tens of minutes, the dynamic conformations of individual structures can be detected in situ with DNA PAINT microscopy. Time series localization data with automated imaging processing to track the dynamically changing radial distribution of structural markers are combined. Conformational distributions of tethered structures in buffers with elevated pH exhibit a calcium-dependent domination of the spread state. This is likely due to electrostatic interactions between the structures and immobilized surface proteins (BSA and streptavidin). An interaction is observed in solution under similar buffer conditions with dynamic light scattering. Exchanging between solutions that promote one or the other state leads to in situ sample-wide transitions between the states. The technique herein can be a useful tool for dynamic control and observation of nanoscale interactions and spatial relationships.

Technological complexity and the global dispersal of modern humans.

Anatomically modern humans (Homo sapiens) dispersed out of Africa roughly 120,000 years ago and again after 75,000 years ago. The early dispersal was geographically restricted to the Arabian Peninsula, Levant, and possibly parts of southern Asia. The later dispersal was ultimately global in scope, including areas not previously occupied by Homo. One explanation for the contrast between the two out-of-Africa dispersals is that the modern humans who expanded into Eurasia 120,000 years ago lacked the functionally and structurally complex technology of recent hunter-gatherers. This technology, which includes, for example, mechanical projectiles, snares and traps, and sewn clothing, provides not only expanded dietary breadth and increased rates of foraging efficiency and success in places where plant and animal productivity is low, but protection from cold weather in places where winter temperatures are low. The absence of complex technology before 75,000 years ago also may explain why modern humans in the Levant did not develop sedentary settlements and agriculture 120,000 years ago (i.e., during the Last Interglacial).

Sequence-specific nuclease-mediated release of cells tethered by oligonucleotide phospholipids.

Single-stranded oligonucleotide-conjugated lipids (ssDNA-PEG-lipids) that associate with the cell membrane confer to the cell an artificial adhesive capability via sequence-specific hybridization to complementary oligonucleotides, forming bonds of double stranded oligonucleotides (dsDNA). Such artificial tethers permit surface patterning of cells or controlled formation of cellular aggregates. However, the hybridization responsible for tethering cells to surfaces or to other cells is not trivially reversed under physiological conditions. In this study, we approach the unbinding of tethered cells by cleaving dsDNA bonds with restriction endonuclease BamHI or digesting bonds with the nonspecific nuclease Benzonase. The procedure was applied to CCRF-CEM cells bearing dsDNA suspended in isolation, cells tethered to glass substrates, and cells aggregated heterotypically with other ssDNA-bearing cells. Cells liberated from surfaces with BamHI could be flushed from flow chambers and viably recovered while the majority of cells not bearing enzyme recognition sequences were retained on the surface, and DNA-tethered cells could be nonspecifically recovered viably from surfaces after Benzonase treatment. Heterotypic aggregates of cells joined by recognition sequence DNA could be dispersed with 10 min exposure to BamHI while undispersed cells heterotypically aggregated with a control sequence remained. Likewise, 10 min exposure to Benzonase was sufficient to disperse aggregates independently of sequence. The potential to undo artificially engineered DNA-mediated adhesion offers new possibilities in the controlled arrangement of cells relative to other cells and in the study of membrane biophysics.

Manipulation of cell sorting within mesenchymal stromal cell-islet cell multicellular spheroids.

Colocalization of islets with immunoprivileged cell types such as mesenchymal stromal cells (MSCs) is a potentially multifaceted and adaptive approach to islet protection. We attempted to colocalize MSCs with islets by creating single-celled suspensions of MSCs and cells from dissociated islets on top of arrays of round-bottomed wells. Segregation between islet-derived cells and MSCs was observed within 3 days. When ROCK inhibitor Y-27632-containing medium was used during the preparation of MSC/islet coaggregates, coaggregates sorted into core-shell structures with islet-derived cells occupying the exterior while MSCs occupied the core. Immunostaining revealed that MSC-derived regions transition from expression of N-cadherin, vimentin, and CD44 to expression of E-cadherin, while pan-cadherin staining indicated reallocation of cadherins to cell borders, and shear-based cohesion measurements pointed to increased cohesive strength. The switch suggests that MSC-islet cohesion improved due to the greater degree of cell-cell adhesive compatibility. Functional evaluation of MSC-islet coaggregates confirmed normal insulin secretory function and partial suppression of anti-CD3-activated splenocyte proliferation. These findings demonstrate that manipulation of cell-cell interactions can be harnessed to control spheroid architecture in MSC-islet coaggregates, and this study also provides the basis for future islet therapies.

Long term culture of cells patterned on glass via membrane-tethered oligonucleotides.

Oligonucleotide-based membrane inserts can be used as tethers to control attachment of cells to patterned surfaces without interfering with internal cytoskeletal modes of adhesion. Such control can be employed as a means for study of cell-cell interactions or side-by-side co-culture of different cell types without separation/sorting. While there is utility for cell patterning methods decoupled from natural cytoskeletal mechanisms, the consequences of maintaining this artificially induced state of attachment remains unexplored. We present a method for the 2-dimensional patterning of cells via hybridization of membrane-tethered single stranded oligonucleotides to complimentary single stranded oligonucleotides bound to optically transparent glass substrates which allowed us to characterize the long term culture of patterned HEK293 cells. Patterned substrates immersed in FBS-containing media are shown to permit the adsorption of adhesive serum proteins which allowed for the spreading and engagement of natural cytoskeletal adhesion modes in cells initially attached only through DNA hybridization. We show that the coexisting modes of attachment result in competition between membrane-bound tethers and natural cytoskeletal adhesion machinery as cells attempt to migrate away from their initial points of attachment. This competition ends in the escape of cells from their designated patterns and the 'winning out' of cytoskeletal migration forces over the affinity of lipid inserts for the cell membrane.

Assessing the spatial resolution of cellular rigidity sensing using a micropatterned hydrogel-photoresist composite.

The biophysical machinery that permits a cell to sense substrate rigidity is poorly understood. Rigidity sensing of adherent cells likely involves traction forces applied through focal adhesions and measurement of resulting deformation. However, it is unclear if this measurement takes place underneath single focal adhesions, over local clusters of focal adhesions, or across the length of the entire cell. To address this question, we developed a composite, chip-based material containing many arrays of 6.5 µm × 6.5 µm rigid adhesive islands, with an edge-edge distance of 8 µm, grafted onto the surface of a non-adhesive polyacrylamide hydrogel. This material is thus rigid within single islands while long-range rigidity is determined by the hydrogel. On soft gels, most NIH 3T3 cells spread only across two islands in a given dimension forming small stress fibers and focal adhesions. On stiff gels, cell spreading, stress fibers, and focal adhesions were indistinguishable from those on regular culture surfaces. We conclude that rigidity sensing is dictated by material compliance across the cell length and that responses to rigidity may be inhibited at any point when large substrate strain is encountered during spreading. Our finding may serve as a guideline for the design of biomaterials for tissue engineering.

Presence of pores and hydrogel composition influence tensile properties of scaffolds fabricated from well-defined sphere templates.

Sphere templating is an attractive method to produce porous polymeric scaffolds with well-defined and uniform pore structures for applications in tissue engineering. While high porosity is desired to facilitate cell seeding and enhance nutrient transport, the incorporation of pores will impact gross mechanical properties of tissue scaffolds and will likely be dependent on pore size. The goals of this study were to evaluate the effect of pores, pore diameter, and polymer composition on gross mechanical properties of hydrogels prepared from crosslinked poly(ethylene glycol) (PEG) and poly(2-hydroxyethyl methacrylate) (pHEMA). Sphere templates were fabricated from uncrosslinked poly(methyl methacrylate) spheres sieved between 53-63 and 150-180 µm. Incorporating pores into hydrogels significantly decreased the quasi-static modulus and ultimate tensile stress, but increased the ultimate tensile strain. For pHEMA, decreases in gel crosslinking density and increases in pore diameters followed similar trends. Interestingly, the mechanical properties of porous PEG hydrogels were less sensitive to changes in pore diameter for a given polymer composition. Additionally, pore diameter was shown to affect skeletal myoblast adhesion whereby many cells cultured in porous hydrogels with smaller pores were seen spanning across multiple pores, but lined the inside of larger pores. In summary, incorporation of pores and changes in pore diameter significantly affect the gross mechanical properties, but in a manner that is dependent on gel chemistry, structure, and composition. Together, these findings will help to design better hydrogel scaffolds for applications where gross mechanical properties and porosity are critical.