The encapsulation of molecular cargo within well-defined supramolecular architectures is highly challenging. Synthetic hosts are desirable because of their well-defined nature and addressability. Encapsulation of biomacromolecules within synthetic hosts is especially challenging because of the former's large size, sensitive nature, retention of functionality postencapsulation and demonstration of control over the cargo. Here we encapsulate a fluorescent biopolymer that functions as a pH reporter within synthetic, DNA-based icosahedral host without molecular recognition between host and cargo. Only those cells bearing receptors for the DNA casing of the host-cargo complex engulf it. We show that the encapsulated cargo is therefore uptaken cell specifically in Caenorhabditis elegans. Retention of functionality of the encapsulated cargo is quantitatively demonstrated by spatially mapping pH changes associated with endosomal maturation within the coelomocytes of C. elegans. This is the first demonstration of functionality and emergent behaviour of a synthetic host-cargo complex in vivo.
We report a pH-dependent conformational transition in short, defined homopolymeric deoxyadenosines (dA15) from a single helical structure with stacked nucleobases at neutral pH to a double-helical, parallel-stranded duplex held together by AH+-H+A base pairs at acidic pH. Using native PAGE, 2D NMR, circular dichroism (CD) and fluorescence spectroscopy, we have characterized the two different pH dependent forms of dA15. The pH-triggered transition between the two defined helical forms of dA15 is characterized by CD and fluorescence. The kinetics of this conformational switch is found to occur on a millisecond time scale. This robust, highly reversible, pH-induced transition between the two well-defined structured states of dA15 represents a new molecular building block for the construction of quick-response, pH-switchable architectures in structural DNA nanotechnology.
DNA Trojan horse: A DNA icosahedron (black, see scheme) held together with aptamers (red) was used to encapsulate molecular cargo such as fluorescent dextran (green). In the presence of a molecular trigger (gray hexagons), the aptamers fold back leading to opening of the icosahedron and simultaneous release of the encapsulated cargo.
With faithful sample preservation and direct imaging of fully hydrated biological material, cryoelectron tomography (cryo-ET) provides an accurate representation of molecular architecture of cells. However, detection and precise localization of macromolecular complexes within cellular environments is aggravated by the presence of many molecular species and molecular crowding. We developed a template-free image processing procedure for accurate tracing of complex networks of densities in cryo-electron tomograms, a comprehensive and automated detection of heterogeneous membranebound complexes and an unsupervised classification. Applying this procedure to tomograms of intact cells and isolated endoplasmic reticulum (ER), we detected and classified small protein complexes like the ER protein translocons, which were not detected by other methods before. This classification provided sufficiently homogeneous particle sets and initial references to allow subsequent de novo subtomogram averaging. Therefore the procedure presented allows a comprehensive detection and a structural analysis of complexes in their native state. In addition, we present structural evidence that different ribosome-free translocon species are present at the ER membrane, determine their 3D structure, and show that they have different localization patterns forming nanodomains.
MicroRNAs control gene expression either by RNA transcript degradation or translational repression. Expressions of miRNAs are highly regulated in tissues, disruption of which leads to disease. How this regulation is achieved and maintained is still largely unknown. MiRNAs that reside on clustered or polycistronic transcripts represent a more complex case where individual miRNAs from a cluster are processed with different efficiencies despite being cotranscribed. To shed light on the regulatory mechanisms that might be operating in these cases, we considered the long polycistronic primary miRNA transcript pri-miR-17-92a that contains six miRNAs with diverse functions. The six miRNA domains on this cluster are differentially processed to produce varying amounts of resultant mature miRNAs in different tissues. How this is achieved is not known. We show, using various biochemical and biophysical methods coupled with mutational studies, that pri-miR-17-92a adopts a specific three-dimensional architecture that poses a kinetic barrier to its own processing. This tertiary structure could create suboptimal protein recognition sites on the pri-miRNA cluster due to higher-order structure formation.
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