Bjoern Sander

Self-assembly of a nanoscale DNA box with a controllable lid

The unique structural motifs and self-recognition properties of DNA can be exploited to generate self-assembling DNA nanostructures of specific shapes using a ‘bottom-up’ approach. Several assembly strategies have been developed for building complex three-dimensional (3D) DNA nanostructures. Recently, the DNA ‘origami’ method was used to build two-dimensional addressable DNA structures of arbitrary shape that can be used as platforms to arrange nanomaterials with high precision and specificity. A long-term goal of this field has been to construct fully addressable 3D DNA nanostructures. Here we extend the DNA origami method into three dimensions by creating an addressable DNA box 42 × 36 × 36 nm3 in size that can be opened in the presence of externally supplied DNA ‘keys’. We thoroughly characterize the structure of this DNA box using cryogenic transmission electron microscopy, small-angle X-ray scattering and atomic force microscopy, and use fluorescence resonance energy transfer to optically monitor the opening of the lid. Controlled access to the interior compartment of this DNA nanocontainer could yield several interesting applications, for example as a logic sensor for multiple-sequence signals or for the controlled release of nanocargos.

GraFix: sample preparation for single-particle electron cryomicroscopy.

We developed a method, named GraFix, that considerably improves sample quality for structure determination by single-particle electron cryomicroscopy (cryo-EM). GraFix uses a glycerol gradient centrifugation step in which the complexes are centrifuged into an increasing concentration of a chemical fixation reagent to prevent aggregation and to stabilize individual macromolecules. The method can be used to prepare samples for negative-stain, cryo-negative-stain and, particularly, unstained cryo-EM.

Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes

Little is known about the higher‐order structure of prespliceosomal A complexes, in which pairing of the pre‐mRNAs splice sites occurs. Here, human A complexes were isolated under physiological conditions by double‐affinity selection. Purified complexes contained stoichiometric amounts of U1, U2 and pre‐mRNA, and crosslinking studies indicated that these form concomitant base pairing interactions with one another. A complexes contained nearly all U1 and U2 proteins plus ∼50 non‐snRNP proteins. Unexpectedly, proteins of the hPrp19/CDC5 complex were also detected, even when A complexes were formed in the absence of U4/U6 snRNPs, demonstrating that they associate independent of the tri‐snRNP. Double‐affinity purification yielded structurally homogeneous A complexes as evidenced by electron microscopy, and allowed for the first time the generation of a three‐dimensional structure. A complexes possess an asymmetric shape (∼260 × 200 × 195 Å) and contain a main body with various protruding elements, including a head‐like domain and foot‐like protrusions. Complexes isolated here are well suited for in vitro assembly studies to determine factor requirements for the A to B complex transition.

An Assembly Chaperone Collaborates with the SMN Complex to Generate Spliceosomal SnRNPs

Spliceosomal small nuclear ribonucleoproteins (snRNPs) are essential components of the nuclear pre-mRNA processing machinery. A hallmark of these particles is a ring-shaped core domain generated by the binding of Sm proteins onto snRNA. PRMT5 and SMN complexes mediate the formation of the core domain in vivo. Here, we have elucidated the mechanism of this reaction by both biochemical and structural studies. We show that pICln, a component of the PRMT5 complex, induces the formation of an otherwise unstable higher-order Sm protein unit. In this state, the Sm proteins are kinetically trapped, preventing their association with snRNA. The SMN complex subsequently binds to these Sm protein units, dissociates pICln, and catalyzes ring closure on snRNA. Our data identify pICln as an assembly chaperone and the SMN complex as a catalyst of spliceosomal snRNP formation. The mode of action of this combined chaperone/catalyst system is reminiscent of the mechanism employed by DNA clamp loaders.

Three-dimensional structure of a pre-catalytic human spliceosomal complex B

Major structural changes occur in the spliceosome during its transition from the fully assembled complex B to the catalytically activated spliceosome. To understand the rearrangement, it is necessary to know the detailed three-dimensional structures of these complexes. Here, we have immunoaffinity-purified human spliceosomes (designated BΔU1) at a stage after U4/U6·U5 tri-snRNP integration but before activation, and have determined the three-dimensional structure of BΔU1 by single-particle electron cryomicroscopy at a resolution of ∼40 Å. The overall size of the complex is about 370 × 270 × 170 Å. The three-dimensional structure features a roughly triangular body linked to a head domain in variable orientations. The body is very similar in size and shape to the isolated U4/U6·U5 tri-snRNP. This provides initial insight into the structural organization of complex B.

Localization of Prp8, Brr2, Snu114 and U4/U6 proteins in the yeast tri-snRNP by electron microscopy.

The U4/U6-U5 tri–small nuclear ribonucleoprotein (snRNP) is a major, evolutionarily highly conserved spliceosome subunit. Unwinding of its U4/U6 snRNA duplex is a central event of spliceosome activation that requires several components of the U5 portion of the tri-snRNP, including the RNA helicase Brr2, Prp8 and the GTPase Snu114. Here we report the EM projection structure of the Saccharomyces cerevisiae tri-snRNP. It shows a modular organization comprising three extruding domains that contact one another in its central portion. We have visualized genetically tagged tri-snRNP proteins by EM and show here that U4/U6 snRNP forms a domain termed the arm. Conversely, a separate head domain adjacent to the arm harbors Brr2, whereas Prp8 and the GTPase Snu114 are located centrally. The head and arm adopt variable relative positions. This molecular organization and dynamics suggest possible scenarios for structural events during catalytic activation.

Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo

Mitochondrial pre‐messenger RNAs in kinetoplastid protozoa are substrates of uridylate‐specific RNA editing. RNA editing converts non‐functional pre‐mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of ∼20S and ∼35–40S and present the three‐dimensional structures of both complexes by electron microscopy. The ∼35–40S complexes consist of a platform density packed against a semispherical element. The ∼20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The ∼20S editosomes contain an RNA‐binding site, which binds gRNA, pre‐mRNA and gRNA/pre‐mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.

Characterization of Tailor-Made Copolymers of Oligo(ethylene glycol) Methyl Ether Methacrylate and N,N-Dimethylaminoethyl Methacrylate as Nonviral Gene Transfer Agents: Influence of Macromolecular Structure on Gene Vector Particle Properties and Transfection Efficiency

Oligo(ethylene glycol) methyl ether methacrylates (OEGMA) of various chain lengths (i.e., 9, 23, or 45 EG units) and N,N-dimethylaminoethyl methacrylate (DMAEMA) were copolymerized by atom transfer radical polymerization (ATRP), yielding well-defined P(DMAEMA-co-OEGMA) copolymers with increasing OEGMA molar fractions (F(OEGMA)) but a comparable degree of polymerization (DP approximately 120). Increase of both F(OEGMA) and OEGMA chain lengths correlated inversely with gene vector size, morphology, and zeta potential. P(DMAEMA-co-OEGMA) copolymers prevented gene vector aggregation at high plasmid DNA (pDNA) concentrations in isotonic solution and did not induce cytotoxicity even at high concentrations. Transfection efficiency of the most efficient P(DMAEMA-co-OEGMA) copolymers was found to be >10-fold lower compared with branched polyethylenimine (PEI) 25 kDa. Although OEGMA copolymerization largely reduced gene vector binding with the cell surface, cellular internalization of the bound complexes was less affected. These observations suggest that inefficient endolysosomal escape limits transfection efficiency of P(DMAEMA-co-OEGMA) copolymer gene vectors. Despite this observation, optimized p(DMAEMA-co-OEGMA) gene vectors remained stable under conditions for in vivo application leading to 7-fold greater gene expression in the lungs compared with PEI. Tailor-made P(DMAEMA-co-OEGMA) copolymers are promising nonviral gene transfer agents that fulfill the requirements for successful in vivo gene delivery.

Self-Assembly of Ternary Insulin-Polyethylenimine (PEI)-DNA Nanoparticles for Enhanced Gene Delivery and Expression in Alveolar Epithelial Cells

Enhancing gene delivery and expression in alveolar epithelial cells could offer the opportunity for the treatment of acquired and inherited lung diseases. Here, we show that particle adsorption of human insulin (INS) is capable of increasing plasmid DNA (pDNA) delivery from polyethylenimine (PEI) nanoparticles specifically in alveolar epithelial cells. INS receptors were predominantly detected on alveolar but not on bronchial epithelial cells. INS was adsorbed on the surface of PEI gene vectors by spontaneous self-assembly resulting in ternary PEI-pDNA-INS nanoparticles. Surface adsorption was confirmed by particle size, surface charge, and fluorescence resonance energy transfer (FRET) measurements. INS adsorption significantly increased gene expression of PEI-pDNA nanoparticles up to 16-fold on alveolar epithelial cells but not on bronchial epithelial cells. This increased gene expression was INS receptor specific. Our results demonstrate that targeting INS receptor for gene delivery in alveolar epithelial cells represents a promising approach for enhanced gene delivery and expression.

Visualization of bionanostructures using transmission electron microscopical techniques

In the recent years, nanotechnology has rapidly evolved as promising toolbox for many applications, including sensing and drug delivery. Nanotechnology aims at forming man‐designed two‐dimensional and three‐dimensional structures in the nanometer scale using e.g., the self‐assembly properties of smaller building blocks such as DNA and RNA. The visualization and structural characterization of these nanostructures do not only provide evidence for the correct formation of the desired shapes, but can also contribute to a better understanding of their formation and functionality. Transmission electron microscopy offers the possibility to directly visualize the individual nanostructures. The vitrification of the sample by using the plunge‐freezing method and subsequent electron cryomicroscopy (cryo‐EM) provides in‐solution snapshots of the nanostructures under cryogenic conditions and thus preserves the close‐to‐native structure of the particles. Here, we describe the plunge‐freezing and other sample preparation protocols such as negative staining and cryo‐negative staining as well as the various imaging and image processing methods, including electron crystallography, electron tomography, and single‐particle EM. Typical example applications are provided together with a discussion of benefits and shortcomings of these approaches. We also discuss how deviations from an ideal symmetry and structural heterogeneity, in general, can limit the resolution. Finally, we suggest that nanotechnological approaches may not only offer new applications in the field of nanomaterial science and nanomedicine, but may also emerge as tools for structural biology and structure‐related biomedical research. Microsc. Res. Tech., 2011.