Free-Standing DNA Origami Superlattice to Facilitate Cryo-EM Visualization of Membrane Vesicles.

Technological breakthroughs in cryo-electron microscopy (cryo-EM) methods open new perspectives for highly detailed structural characterizations of extracellular vesicles (EVs) and synthetic liposome-protein assemblies. Structural characterizations of these vesicles in solution under a nearly native hydrated state are of great importance to decipher cell-to-cell communication and to improve EVs' application as markers in diagnosis and as drug carriers in disease therapy. However, difficulties in preparing holey carbon cryo-EM grids with low vesicle heterogeneities, at low concentration and with kinetic control of the chemical reactions or assembly processes, have limited cryo-EM use in the EV study. We report a straightforward membrane vesicle cryo-EM sample preparation method that assists in circumventing these limitations by using a free-standing DNA-affinity superlattice for covering holey carbon cryo-EM grids. Our approach uses DNA origami to self-assemble to a solution-stable and micrometer-sized ordered molecular template in which structure and functional properties can be rationally controlled. We engineered the template with cholesterol-binding sites to specifically trap membrane vesicles. The advantages of this DNA-cholesterol-affinity lattice (DCAL) include (1) local enrichment of artificial and biological vesicles at low concentration and (2) isolation of heterogeneous cell-derived membrane vesicles (exosomes) from a prepurified pellet of cell culture conditioned medium on the grid.

Isothermal self-assembly of multicomponent and evolutive DNA nanostructures.

Thermal annealing is usually needed to direct the assembly of multiple complementary DNA strands into desired entities. We show that, with a magnesium-free buffer containing NaCl, complex cocktails of DNA strands and proteins can self-assemble isothermally, at room or physiological temperature, into user-defined nanostructures, such as DNA origamis, single-stranded tile assemblies and nanogrids. In situ, time-resolved observation reveals that this self-assembly is thermodynamically controlled, proceeds through multiple folding pathways and leads to highly reconfigurable nanostructures. It allows a given system to self-select its most stable shape in a large pool of competitive DNA strands. Strikingly, upon the appearance of a new energy minimum, DNA origamis isothermally shift from one initially stable shape to a radically different one, by massive exchange of their constitutive staple strands. This method expands the repertoire of shapes and functions attainable by isothermal self-assembly and creates a basis for adaptive nanomachines and nanostructure discovery by evolution.

Mechanical DNA Origami to Investigate Biological Systems.

The ability to self-assemble DNA nanodevices with programmed structural dynamics that can sense and respond to the local environment can enable transformative applications in fields including mechanobiology and nanomedicine. The responsive function of biomolecules is often driven by alterations in conformational distributions mediated by highly sensitive interactions with the local environment. In this review, the current state-of-the-art in constructing complex DNA geometries with dynamic and mechanical properties to enable a molecular scale force measurement is first summarized. Next, an overview of engineering modular DNA devices that interact with cell surfaces is highlighted detailing examples of mechanosensitive proteins and the force-induced dynamic molecular interaction on the downstream biochemical signaling. Finally, the challenges and an outlook on this promising class of DNA devices acting as nanomachines to operate at a low piconewton range suitable for a majority of biological effects or as hybrid materials to achieve higher tension exertion required for other biological investigations, are discussed.

Melanotransferrin is efficiently sorted on the surface of exosomes secreted by melanoma cells.

Cutaneous melanoma is the most lethal type of skin cancer. Early detection is crucial to improve the outcome of melanoma patients. The identification of noninvasive prognostic biomarkers for the follow-up of melanoma patients is still in demand for clinical use. We show here that exosomal melanotransferrin fulfills the biomarker characteristics required to meet this demand. Melanotransferrin is typically overexpressed in melanoma cells compared to other cell types - including cancer cells - and is efficiently sorted and secreted with nanovesicles, or so-called exosomes, due to its membrane-anchoring by a glycosylphosphatidylinositol. Melanotransferrin is exposed on the surface of exosomes and is accessible for antibody recognition. An ELISA was set up to quantify melanotransferrin after immobilization of nanovesicles through the exosomal constituent tetraspanins CD63. Melanotransferrin was detected using a low number of exosomes purified from melanoma cell line cultures, and melanotransferrin detection was abolished by phosphatidylinositol-specific phospholipase C treatment. This exosomal melanotransferrin ELISA was able to discriminate an equal number of assayed exosomes purified from two different melanoma cell lines (A-375 vs. SK-MEL-28). Moreover, plasma samples from patients with melanoma and noncancer disease were assayed using this ELISA and elevated levels of exosomal melanotransferrin were seen in the plasma of patients with melanoma. We propose that exosomal melanotransferrin should be assessed as a potential melanoma biomarker.

Folding of phosphodiester-linked donor-acceptor oligomers into supramolecular nanotubes in water.

Inspired by the automated synthesis of DNA on a solid support, the electron-rich dialkoxynaphthalene (DAN) donor and the electron-deficient naphthalene-tetracarboxylic diimide (NDI) acceptor, amphiphilic foldamers have been synthesised from their respective phosphoramidite building blocks. The folding of the phosphodiester-linked hexamer (DAN-NDI)3 revealed the formation of regular supramolecular nanotubes in water resulting from the self-assembly of multiple hexamers stabilized by donor/acceptor interactions and the solvophobic effect.

Modular Imaging Scaffold for Single-Particle Electron Microscopy.

Technological breakthroughs in electron microscopy (EM) have made it possible to solve structures of biological macromolecular complexes and to raise novel challenges, specifically related to sample preparation and heterogeneous macromolecular assemblies such as DNA-protein, protein-protein, and membrane protein assemblies. Here, we built a V-shaped DNA origami as a scaffolding molecular system to template proteins at user-defined positions in space. This template positions macromolecular assemblies of various sizes, juxtaposes combinations of biomolecules into complex arrangements, isolates biomolecules in their active state, and stabilizes membrane proteins in solution. In addition, the design can be engineered to tune DNA mechanical properties by exerting a controlled piconewton (pN) force on the molecular system and thus adapted to characterize mechanosensitive proteins. The binding site can also be specifically customized to accommodate the protein of interest, either interacting spontaneously with DNA or through directed chemical conjugation, increasing the range of potential targets for single-particle EM investigation. We assessed the applicability for five different proteins. Finally, as a proof of principle, we used RNAP protein to validate the approach and to explore the compatibility of the template with cryo-EM sample preparation.

Complex wireframe DNA nanostructures from simple building blocks.

DNA nanostructures with increasing complexity have showcased the power of programmable self-assembly from DNA strands. At the nascent stage of the field, a variety of small branched objects consisting of a few DNA strands were created. Since then, a quantum leap of complexity has been achieved by a scaffolded 'origami' approach and a scaffold-free approach using single-stranded tiles/bricks-creating fully addressable two-dimensional and three-dimensional DNA nanostructures designed on densely packed lattices. Recently, wireframe architectures have been applied to the DNA origami method to construct complex structures. Here, revisiting the original wireframe framework entirely made of short synthetic strands, we demonstrate a design paradigm that circumvents the sophisticated routing and size limitations intrinsic to the scaffold strand in DNA origami. Under this highly versatile self-assembly framework, we produce a myriad of wireframe structures, including 2D arrays, tubes, polyhedra, and multi-layer 3D arrays.

Programmable self-assembly of three-dimensional nanostructures from 10,000 unique components.

Nucleic acids (DNA and RNA) are widely used to construct nanometre-scale structures with ever increasing complexity, with possible application in fields such as structural biology, biophysics, synthetic biology and photonics. The nanostructures are formed through one-pot self-assembly, with early kilodalton-scale examples containing typically tens of unique DNA strands. The introduction of DNA origami, which uses many staple strands to fold one long scaffold strand into a desired structure, has provided access to megadalton-scale nanostructures that contain hundreds of unique DNA strands. Even larger DNA origami structures are possible, but manufacturing and manipulating an increasingly long scaffold strand remains a challenge. An alternative and more readily scalable approach involves the assembly of DNA bricks, which each consist of four short binding domains arranged so that the bricks can interlock. This approach does not require a scaffold; instead, the short DNA brick strands self-assemble according to specific inter-brick interactions. First-generation bricks used to create three-dimensional structures are 32 nucleotides long, consisting of four eight-nucleotide binding domains. Protocols have been designed to direct the assembly of hundreds of distinct bricks into well formed structures, but attempts to create larger structures have encountered practical challenges and had limited success. Here we show that DNA bricks with longer, 13-nucleotide binding domains make it possible to self-assemble 0.1-1-gigadalton, three-dimensional nanostructures from tens of thousands of unique components, including a 0.5-gigadalton cuboid containing about 30,000 unique bricks and a 1-gigadalton rotationally symmetric tetramer. We also assembled a cuboid that contains around 10,000 bricks and about 20,000 uniquely addressable, 13-base-pair 'voxels' that serves as a molecular canvas for three-dimensional sculpting. Complex, user-prescribed, three-dimensional cavities can be produced within this molecular canvas, enabling the creation of shapes such as letters, a helicoid and a teddy bear. We anticipate that with further optimization of structure design, strand synthesis and assembly procedure even larger structures could be accessible, which could be useful for applications such as positioning functional components.

Angular reconstitution-based 3D reconstructions of nanomolecular structures from superresolution light-microscopy images.

Superresolution light microscopy allows the imaging of labeled supramolecular assemblies at a resolution surpassing the classical diffraction limit. A serious limitation of the superresolution approach is sample heterogeneity and the stochastic character of the labeling procedure. To increase the reproducibility and the resolution of the superresolution results, we apply multivariate statistical analysis methods and 3D reconstruction approaches originally developed for cryogenic electron microscopy of single particles. These methods allow for the reference-free 3D reconstruction of nanomolecular structures from two-dimensional superresolution projection images. Since these 2D projection images all show the structure in high-resolution directions of the optical microscope, the resulting 3D reconstructions have the best possible isotropic resolution in all directions.

Structural insights into adiponectin receptors suggest ceramidase activity.

Adiponectin receptors (ADIPORs) are integral membrane proteins that control glucose and lipid metabolism by mediating, at least in part, a cellular ceramidase activity that catalyses the hydrolysis of ceramide to produce sphingosine and a free fatty acid (FFA). The crystal structures of the two receptor subtypes, ADIPOR1 and ADIPOR2, show a similar overall seven-transmembrane-domain architecture with large unoccupied cavities and a zinc binding site within the seven transmembrane domain. However, the molecular mechanisms by which ADIPORs function are not known. Here we describe the crystal structure of ADIPOR2 bound to a FFA molecule and show that ADIPOR2 possesses intrinsic basal ceramidase activity that is enhanced by adiponectin. We also identify a ceramide binding pose and propose a possible mechanism for the hydrolytic activity of ADIPOR2 using computational approaches. In molecular dynamics simulations, the side chains of residues coordinating the zinc rearrange quickly to promote the nucleophilic attack of a zinc-bound hydroxide ion onto the ceramide amide carbonyl. Furthermore, we present a revised ADIPOR1 crystal structure exhibiting a seven-transmembrane-domain architecture that is clearly distinct from that of ADIPOR2. In this structure, no FFA is observed and the ceramide binding pocket and putative zinc catalytic site are exposed to the inner membrane leaflet. ADIPOR1 also possesses intrinsic ceramidase activity, so we suspect that the two distinct structures may represent key steps in the enzymatic activity of ADIPORs. The ceramidase activity is low, however, and further studies will be required to characterize fully the enzymatic parameters and substrate specificity of ADIPORs. These insights into ADIPOR function will enable the structure-based design of potent modulators of these clinically relevant enzymes.

Designing DNA Nanotube Liquid Crystals as a Weak-Alignment Medium for NMR Structure Determination of Membrane Proteins.

Thirty percent of the human proteome is composed of membrane proteins that can perform a wide range of cellular functions and communications. They represent the core of modern medicine as the targets of about 50 % of all prescription pharmaceuticals. However, elucidating the structure of membrane proteins has represented a constant challenge, even in the modern era. To date, only a few hundred high-resolution structural models of membrane proteins are available. This chapter describes the emergence of DNA nanotechnology as a powerful tool for the structural characterization of membrane protein using solution-state nuclear magnetic resonance (NMR) spectroscopy. Here, we detail the large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into a dilute liquid crystal to be used as a weak-alignment media in solution NMR structure determination of membrane proteins.

Regulation at a distance of biomolecular interactions using a DNA origami nanoactuator.

The creation of nanometre-sized structures that exhibit controllable motions and functions is a critical step towards building nanomachines. Recent developments in the field of DNA nanotechnology have begun to address these goals, demonstrating complex static or dynamic nanostructures made of DNA. Here we have designed and constructed a rhombus-shaped DNA origami 'nanoactuator' that uses mechanical linkages to copy distance changes induced on one half ('the driver') to be propagated to the other half ('the mirror'). By combining this nanoactuator with split enhanced green fluorescent protein (eGFP), we have constructed a DNA-protein hybrid nanostructure that demonstrates tunable fluorescent behaviours via long-range allosteric regulation. In addition, the nanoactuator can be used as a sensor that responds to specific stimuli, including changes in buffer composition and the presence of restriction enzymes or specific nucleic acids.

Mapping conformational heterogeneity of mitochondrial nucleotide transporter in uninhibited states.

One of the less well understood aspects of membrane transporters is the dynamic coupling between conformational change and substrate transport. NMR approaches are used herein to investigate conformational heterogeneity of the GTP/GDP carrier (GGC) from yeast mitochondria. NMR residual dipolar coupling (RDC) analysis of GGC in a DNA-origami nanotube liquid crystal shows that several structured segments have different generalized degrees of order (GDO), thus indicating the presence of conformational heterogeneity. Complete GDO mapping reveals asymmetry between domains of the transporter and even within certain transmembrane helices. Nucleotide binding partially reduces local structural heterogeneity, and the substrate binds to multiple sites along the transport cavity. These observations suggest that mitochondrial carriers in the uninhibited states are intrinsically plastic and structural plasticity is asymmetrically distributed among the three homologous domains.

Disruption of helix-capping residues 671 and 674 reveals a role in HIV-1 entry for a specialized hinge segment of the membrane proximal external region of gp41.

HIV-1 (human immunodeficiency virus type 1) uses its trimeric gp160 envelope (Env) protein consisting of non-covalently associated gp120 and gp41 subunits to mediate entry into human T lymphocytes. A facile virus fusion mechanism compensates for the sparse Env copy number observed on viral particles and includes a 22-amino-acid, lentivirus-specific adaptation at the gp41 base (amino acid residues 662-683), termed the membrane proximal external region (MPER). We show by NMR and EPR that the MPER consists of a structurally conserved pair of viral lipid-immersed helices separated by a hinge with tandem joints that can be locked by capping residues between helices. This design fosters efficient HIV-1 fusion via interconverting structures while, at the same time, affording immune escape. Disruption of both joints by double alanine mutations at Env positions 671 and 674 (AA) results in attenuation of Env-mediated cell-cell fusion and hemifusion, as well as viral infectivity mediated by both CD4-dependent and CD4-independent viruses. The potential mechanism of disruption was revealed by structural analysis of MPER conformational changes induced by AA mutation. A deeper acyl chain-buried MPER middle section and the elimination of cross-hinge rigid-body motion almost certainly impede requisite structural rearrangements during the fusion process, explaining the absence of MPER AA variants among all known naturally occurring HIV-1 viral sequences. Furthermore, those broadly neutralization antibodies directed against the HIV-1 MPER exploit the tandem joint architecture involving helix capping, thereby disrupting hinge function.

DNA nanotubes for NMR structure determination of membrane proteins.

Finding a way to determine the structures of integral membrane proteins using solution nuclear magnetic resonance (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling-based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this you need a weak-alignment medium that is detergent-resistant and it has thus far been difficult to obtain such a medium suitable for weak alignment of membrane proteins. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dilute liquid crystals for application as weak-alignment media in solution NMR structure determination of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400-nm-long six-helix bundles, each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable positive charge as well as modulation of molecular alignment, toward collection of linearly independent restraints, can be introduced by reducing the negative charge of DNA nanotubes using counter ions and small DNA-binding molecules. This detergent-resistant liquid-crystal medium offers a number of properties conducive for membrane protein alignment, including high-yield production, thermal stability, buffer compatibility and structural programmability. Production of sufficient nanotubes for four or five NMR experiments can be completed in 1 week by a single individual.

Low concentration of a Gd-chelate increases the signal-to-noise ratio in fast pulsing BEST experiments.

Despite numerous developments in the past few years that aim to increase the sensitivity of NMR multidimensional experiments, NMR spectroscopy still suffers from intrinsic low sensitivity. In this report, we show that the combination of two developments in the field, the Band-selective Excitation Short-Transient (BEST) experiment [Schanda et al., J. Am. Chem. Soc., 128 (2006) 9042] and the addition of the nonionic paramagnetic gadolinium chelate gadodiamide into NMR samples, enhances the signal-to-noise ratio. This effect is shown here for four different proteins, three globular and one unfolded, of molecular weights ranging from 6.5 kDa to 40 kDa, using 2D BEST HSQC and 3D BEST triple resonance sequences. Moreover, we show that the increase in signal-to-noise ratio provided by the gadodiamide is higher for peak resonances with lower than average intensity in BEST experiments. It is interesting to note that these residues are on average the weakest ones in those experiments. In this case, the gadodiamide-mediated increase can reach a value of 60% for low and 30% for high molecular weight proteins respectively. An investigation into the origin of this "paramagnetic gain" in BEST experiments is presented.

Two design strategies for enhancement of multilayer-DNA-origami folding: underwinding for specific intercalator rescue and staple-break positioning.

Single-layer DNA origami is an efficient method for programmable self-assembly of nanostructures approximating almost any desired two-dimensional shape from ~5 MDa of DNA building material. In this method, a 7 kilobase single "scaffold" strand is assembled with hundreds of oligodeoxyribonucleotide "staple" strands to form a parallel array of double helices. Multiple layers of such DNA sheets also can be designed to assemble into a stack, enabling construction of solid three-dimensional shapes with considerably greater mechanical rigidity than two-dimensional shapes; however, the folding yield often is much lower and the required folding times are much longer. Here we introduce two strategies for designing multi-layer DNA origami that demonstrate potential for boosting assembly yield: (1) individual base pairs can be inserted between crossovers, allowing for greater bowing of helices at positions away from crossovers and therefore reduced electrostatic repulsion. At the same time, this underwinding of double helices increases a destabilizing torsional strain energy but then also increases affinity for intercalators, and binding of such intercalators can relieve this stress. We also have exploited this enhanced affinity for intercalators to PEGylate the surface of the nanostructures in a noncovalent fashion using PEG-tris-acridine. (2) Positioning of staple-strand breaks in the DNA origami such that each staple strand includes a 14 nucleotide (nt) continuous segment that binds to a complementary 14 nt continuous segment of the scaffold can greatly improve folding yields.

Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor.

The vasopressin type 2 (V2R) receptor belongs to the class of G-protein coupled receptors. It is mainly expressed in the membrane of kidney tubules, where it is activated by the extracellular arginine vasopressin. In men, inactivating and activating mutations cause nephrogenic diabetes insipidus and the nephrogenic syndrome of inappropriate antidiuresis respectively. Like most GPCRs, V2R's third intracellular loop (V2R-i3) is involved in the binding and activation of its major effector, the GαS protein. We overexpressed the V2R224₋274 fragment corresponding to V2R-i3 as a fusion protein with thioredoxin A at the N-terminus and a hexahistidine tag between the two proteins. Recombinant V2R-i3 was designed to harbor N- and C-terminal cysteines, in order to introduce a disulfide bond between N- and C-terminal extremities and hence reproduce the hairpin fold presumably present in the full-length receptor. The fusion protein was produced as inclusion bodies in Escherichia coli and purified by nickel affinity chromatography under denaturing conditions. After a refolding step, thioredoxin and hexahistidine tags were specifically cleaved with the tobacco etch virus protease. The hydrolysis yield, initially very low, increased up to 80% thanks to optimization of buffers and refolding methods. The cleaved fragment, V2224₋274, devoid of any tag, was then eluted with low imidazole concentrations in a second nickel affinity chromatography in denaturing conditions. The final yield was sufficient to prepare a ¹5N-¹³C labeled NMR sample suitable for triple resonance experiments. We assigned all NMR resonances and confirmed the correct peptide sequence. As expected, the peptide forms a hairpin stabilized by a disulfide bond between its N- and C-terminal parts, thus mimicking its native structure in the full-length receptor. This study may provide a strategy for producing and studying the structure/function relationship of GPCR fragments.

Structure of the third intracellular loop of the vasopressin V2 receptor and conformational changes upon binding to gC1qR.

The V2 vasopressin receptor is a G-protein-coupled receptor that regulates the renal antidiuretic response. Its third intracellular loop is involved in the coupling not only with the GalphaS protein but also with gC1qR, a potential chaperone of G-protein-coupled receptors. In this report, we describe the NMR solution structure of the V2 i3 loop under a cyclized form (i3_cyc) and characterize its interaction with gC1qR. i3_cyc formed a left-twisted alpha-helical hairpin structure. The building of a model of the entire V2 receptor including the i3_cyc NMR structure clarified the side-chain orientation of charged residues, in agreement with literature mutagenesis reports. In the model, the i3 loop formed a rigid helical column, protruding deep inside the cytoplasm, as does the i3 loop in the recently elucidated structure of squid rhodopsin. However, its higher packing angle resulted in a different structural motif at the intracellular interface, which may be important for the specific recognition of GalphaS. Moreover, we could estimate the apparent K(d) of the i3_cyc/gC1qR complex by anisotropy fluorescence. Using a shorter and more soluble version of i3_cyc, which encompassed the putative site of gC1qR binding, we showed by NMR saturation transfer difference spectroscopy that the binding surface corresponded to the central arginine cluster. Binding to gC1qR induced the folding of the otherwise disordered short peptide into a spiral-like path formed by a succession of I and IV turns. Our simulations suggested that this folding would rigidify the arginine cluster in the entire i3 loop and would alter the conformation of the cytosolic extensions of TM V and TM VI helices. In agreement with this conformational rearrangement, we observed that binding of gC1qR to the full-length receptor modifies the intrinsic tryptophan fluorescence binding curves of V2 to an antagonist.