Jennifer E. Padilla

Programmable in situ amplification for multiplexed imaging of mRNA expression

In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR). With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization.

Nanohedra: Using symmetry to design self assembling protein cages, layers, crystals, and filaments

A general strategy is described for designing proteins that self assemble into large symmetrical nanomaterials, including molecular cages, filaments, layers, and porous materials. In this strategy, one molecule of protein A, which naturally forms a self-assembling oligomer, An, is fused rigidly to one molecule of protein B, which forms another self-assembling oligomer, Bm. The result is a fusion protein, A-B, which self assembles with other identical copies of itself into a designed nanohedral particle or material, (A-B)p. The strategy is demonstrated through the design, production, and characterization of two fusion proteins: a 49-kDa protein designed to assemble into a cage approximately 15 nm across, and a 44-kDa protein designed to assemble into long filaments approximately 4 nm wide. The strategy opens a way to create a wide variety of potentially useful protein-based materials, some of which share similar features with natural biological assemblies.

A statistic for local intensity differences: robustness to anisotropy and pseudo-centering and utility for detecting twinning.

A new approach to analyzing macromolecular single-crystal X-ray diffraction intensity statistics is presented. Instead of considering reflections in resolution shells, differences between local pairs of reflection intensities are taken and normalized separately. When the two reflections to be compared (having intensities I(1) and I(2), respectively) are chosen appropriately, the behavior of the parameter L = (I(1) - I(2))/(I(1) + I(2)) is insensitive to phenomena that tend to confound traditional intensity statistics, such as anisotropic diffraction and pseudo-centering. The distributions and expected values for L take simple forms when the intensity data are from ordinary crystals or from perfectly twinned specimens. The robustness of the approach is demonstrated with examples using real proteins whose diffraction data appear aberrant by other methods of intensity analysis. The new statistic is better suited than other available methods for diagnosing perfect hemihedral twinning.

Designing supramolecular protein assemblies.

Many natural proteins self-assemble, either to fulfill their biological function or as part of a pathogenic process. Biological assembly phenomena such as amyloidogenesis, domain swapping and symmetric oligomerization are inspiring new strategies for designing proteins that self-assemble to form supramolecular complexes. Recent advances include the design of novel proteins that assemble into filaments, symmetric cages and regular arrays.

ASYNCHRONOUS SIGNAL PASSING FOR TILE SELF-ASSEMBLY: FUEL EFFICIENT COMPUTATION AND EFFICIENT ASSEMBLY OF SHAPES

In this paper we demonstrate the power of a model of tile self-assembly based on active glues which can dynamically change state. We formulate the Signal-passing Tile Assembly Model (STAM), based on the model of Padilla et al. [24] to be asynchronous, allowing any action of turning a glue on or off, attaching a new tile, or breaking apart an assembly to happen in any order. Within this highly generalized model we provide three new solutions to tile self-assembly problems that have been addressed within the abstract Tile Assembly Model and its variants, showing that signal passing tiles allow for substantial improvement across multiple complexity metrics. Our first result utilizes a recursive assembly process to achieve tile-type efficient assembly of linear structures, using provably fewer tile types than what is possible in standard tile assembly models. Our second system of signal-passing tiles simulates any Turing machine with high fuel efficiency by using only a constant number of tiles per computation step. Our third system assembles the discrete Sierpinski triangle, demonstrating that this pattern can be strictly self-assembled within the STAM. This result is of particular interest in that it is known that this pattern cannot self-assemble within a number of well studied tile self-assembly models. Notably, all of our constructions are at temperature 1, further demonstrating that signal-passing confers the power to bypass many restrictions found in standard tile assembly models.

Hierarchical self assembly of patterns from the Robinson tilings: DNA tile design in an enhanced Tile Assembly Model

We introduce a hierarchical self assembly algorithm that produces the quasiperiodic patterns found in the Robinson tilings and suggest a practical implementation of this algorithm using DNA origami tiles. We modify the abstract Tile Assembly Model (aTAM), to include active signaling and glue activation in response to signals to coordinate the hierarchical assembly of Robinson patterns of arbitrary size from a small set of tiles according to the tile substitution algorithm that generates them. Enabling coordinated hierarchical assembly in the aTAM makes possible the efficient encoding of the recursive process of tile substitution.

Signal transmission across tile assemblies: 3D static tiles simulate active self-assembly by 2D signal-passing tiles

The 2-handed assembly model (2HAM) is a tile-based self-assembly model in which, typically beginning from single tiles, arbitrarily large aggregations of static tiles combine in pairs to form structures. The signal-passing tile assembly model (STAM) is an extension of the 2HAM in which the tiles are dynamically changing components which are able to alter their binding domains as they bind together. In this paper, we examine the

A Signal-Passing DNA-Strand-Exchange Mechanism for Active Self-Assembly of DNA Nanostructures†

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