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The scaffolded origami technique has provided an attractive tool for engineering nucleic acid nanostructures. This paper demonstrates scaffolded RNA origami folding in vitro in which all components are transcribed simultaneously in a single-pot reaction. Double-stranded DNA sequences are transcribed by T7 RNA polymerase into scaffold and staple strands able to correctly fold in high yield into the nanoribbon. Synthesis is successfully confirmed by atomic force microscopy and the unpurified transcription reaction mixture is analyzed by an in gel-imaging assay where the transcribed RNA nanoribbons are able to capture the specific dye through the reconstituted split Broccoli aptamer showing a clear green fluorescent band. Finally, we simulate the RNA origami in silico using the nucleotide-level coarse-grained model oxRNA to investigate the thermodynamic stability of the assembled nanostructure in isothermal conditions over a period of time.Our work suggests that the scaffolded origami technique is a valid, and potentially more powerful, assembly alternative to the single-stranded origami technique for future in vivo applications.Abstract Figure

Purple membranes (PM) are two-dimensional crystals formed by bacteriorhodopsin and a variety of lipids. The lipid composition and density in the cytoplasmic (CP) leaflet differ from those of the extracellular (EC) leaflet. A new way of differentiating the two sides of such asymmetric membranes using the phase signal in alternate contact atomic force microscopy is presented. This method does not require molecular resolution and is applied to study the stiffness and intertrimer lipid mobility in both leaflets of the PM independently over a broad range of pH and salt concentrations. PM stiffens with increasing salt concentration according to two different regimes. At low salt concentration, the membrane Young's normal modulus grows quickly but differentially for the EC and CP leaflets. At higher salt concentration, both leaflets behave similarly and their stiffness converges toward the native environment value. Changes in pH do not affect PM stiffness; however, the crystal assembly is less pronounced at pH > or = 10. Lipid mobility is high in the CP leaflet, especially at low salt concentration, but negligible in the EC leaflet regardless of pH or salt concentration. An independent lipid mobility study by solid-state NMR confirms and quantifies the atomic force microscopy qualitative observations.

Phase transitions in purple membrane have been a topic of debate for the past two decades. In this work we present studies of a reversible transition of purple membrane in the 50-60 °C range in zeptoliter volumes under different heating regimes (global heating and local heating). The temperature of the reversible phase transition is 52 ± 5 °C for both local and global heating, supporting the hypothesis that this transition is mainly due to a structural rearrangement of bR molecules and trimers. To achieve high resolution measurements of temperature-dependent phase transitions, a new scanning probe microscopy-based method was developed. We believe that our new technique can be extended to other biological systems and can contribute to the understanding of inhomogeneous phase transitions in complex systems.

At the nanoscale level biological membranes present a complex interface with the solvent. The functional dynamics and relative flexibility of membrane components together with the presence of specific ionic effects can combine to create exciting new phenomena that challenge traditional theories such as the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory or models interpreting the role of ions in terms of their ability to structure water (structure making/breaking). Here we investigate ionic effects at the surface of a highly charged extremophile membrane composed of a proton pump (bacteriorhodopsin) and archaeal lipids naturally assembled into a 2D crystal. Using amplitude-modulation atomic force microscopy (AM-AFM) in solution, we obtained sub-molecular resolution images of ion-induced surface restructuring of the membrane. We demonstrate the presence of a stiff cationic layer condensed at its extracellular surface. This layer cannot be explained by traditional continuum theories. Dynamic force spectroscopy experiments suggest that it is produced by electrostatic correlation mediated by a Manning-type condensation of ions. In contrast, the cytoplasmic surface is dominated by short-range repulsive hydration forces. These findings are relevant to archaeal bioenergetics and halophilic adaptation. Importantly, they present experimental evidence of a natural system that locally controls its interactions with the surrounding medium and challenges our current understanding of biological interfaces.

Bacteriorhodopsin (bR) is a haloarchaeal membrane protein that converts the energy of single photons into large structural changes to directionally pump protons across purple membrane. This is achieved by a complex combination of local dynamic interactions controlling bR biomechanics at the submolecular level, producing efficient amplification of the retinal photoisomerization. Using single molecule force spectroscopy at different salt concentrations, we show that tryptophan (Trp) residues use steric specific interactions to create a rigid scaffold in bR extracellular region and are responsible for the main unfolding barriers. This scaffold, which encloses the retinal, controls bR local mechanical properties and anchors the protein into the membrane. Furthermore, the stable Trp-based network allows ion binding to two specific sites on the extracellular loops (BC and FG), which are involved in proton release and lateral transport. In contrast, the cytoplasmic side of bR is mainly governed by relatively weak nonspecific electrostatic interactions that provide the flexibility necessary for large cytoplasmic structural rearrangements during the photocycle. The presence of an extracellular Trp-based network tightly enclosing the retinal seems common to most haloarchaeal rhodopsins, and could be relevant to their exceptional efficiency.

Membrane proteins are laterally coupled to the surrounding cell membrane through complex interactions that can modulate their function. Here, we directly observe and quantify the dynamics of functioning bacteriorhodopsin (bR) in its native membrane, a crystalline aggregate of bR trimers. We show that much of a monomer's isomerization energy is mechanically redistributed into the membrane, producing cooperative activity within the trimer while simultaneously generating functionally relevant long-range lateral pressure waves. Our results provide evidence of coordinated short and long-range effects in the cell membrane. © 2009 The Royal Society of Chemistry.

The common polymorphism at codon 129 in the human prion protein (PrP) has been shown in many studies to influence not only the pathology of prion disease but also the misfolding propensity of PrP. Here we used NMR, CD and atomic force microscopy in solution to investigate differences in beta-oligomer (beta(O)) formation and inter-oligomer interaction depending on the polymorphism at codon 129. NMR investigations assigned the observable amide resonances to the beta(O) N-terminal segments, showing that it is the core region of PrP (residues 127-228) that is involved in beta(O) formation. Atomic force microscopy revealed distinctive 1.8 x 15 x 15-nm disk-like structures that form stacks through inter-oligomer interactions. The propensity to form stacks and the number of oligomers involved depended on the polymorphism at codon 129, with a significantly lower degree of stacking for beta(O) with valine at position 129. This result provides evidence for conformational differences between the beta(O) allelic forms, showing that the core region of the protein including position 129 is actively involved in inter-oligomer interactions, consistent with NMR observations.

Solid-liquid interfaces play a fundamental role in surface electrochemistry(1), catalysis(2), wetting(3), self-assembly(4) and biomolecular functions(5). The interfacial energy determines many of the properties of such interfaces, including the arrangement of the liquid molecules at the surface of the solid. Diffraction techniques are often used to investigate the structure of solid-liquid interfaces(6), but measurements of irregular or inhomogeneous interfaces remain challenging. Here, we report atomic-and molecular-resolution images of various organic and inorganic samples in liquids, obtained with a commercial atomic force microscope operated dynamically with small-amplitude modulation. This approach uses the structured liquid layers close to the solid to enhance lateral resolution. We propose a model to explain the mechanism dominating the image formation, and show that the energy dissipated during this process is related to the interfacial energy through a readily achievable calibration curve. Our topographic images and interfacial energy maps could provide insights into important interfaces.

Natural surfaces are often structured with nanometre-scale domains, yet a framework providing a quantitative understanding of how nanostructure affects interfacial energy, italic gammaSL, is lacking. Conventional continuum thermodynamics treats italic gammaSL solely as a function of average composition, ignoring structure. Here we show that, when a surface has domains commensurate in size with solvent molecules, italic gammaSL is determined not only by its average composition but also by a structural component that causes italic gammaSL to deviate from the continuum prediction by a substantial amount, as much as 20% in our system. By contrasting surfaces coated with either molecular- (5 nm), we find that whereas the latter surfaces have the expected linear dependence of italic gammaSL on surface composition, the former show a markedly different non-monotonic trend. Molecular dynamics simulations show how the organization of the solvent molecules at the interface is controlled by the nanostructured surface, which in turn appreciably modifies italic gammaSL.

At the solid-liquid interface molecules of the liquid adopt a particular arrangement which depends primarily on the interfacial energy and geometry. At the nanoscale this molecular arrangement is of central importance in a wide range of fields from molecular biology to surface physics, heterogeneous catalysis and electronics. In biology the role of interfacial liquid is further emphasized by the soft nature of most biomolecules whose conformation and dynamics depends on the surrounding medium. This is the case for protein function and folding (1), self-assembly processes and bio-electronics where a complex interplay between surface-bound liquid molecules and ions strongly affects any motion.Using amplitude modulation atomic force microscopy (AM-AFM) operated in liquid and in a particular regime, it is possible to simultaneously image the topography of the surface-bound liquid while measuring its adhesion energy to the solid investigated. We have used this method to map the binding energy of water to gold nanoparticles coated with mixed ligands, self-assembled in controlled patterns (2, 3). Such functionalized nanoparticles can mimic the typical surface of proteins (hydrophobicity, charge, surface domains) while allowing careful control of the domains’ size and properties (2). Our results show that the average binding energy of water to the surface of the nanoparticles strongly depends on the spatial arrangement of the ligands molecules. The geometry as well as the size of the ligand domains both affect the local adhesion energy of the solvent in a non-linear fashion. Our findings provide experimental and quantitative insight into the inter-play between solvent and surfaces in nanoscale biophysical processes.(1) H. Frauenfelder et al, (2006), Proc. Natl. Acad. Sci. 103, 15469-15472.(2) A. Verma et al, (2008), Nature Mat. 7, 588-595.(3) A. Centrone et al, (2008), Proc. Natl. Acad. Sci. 105, 9886-9891.