Bottom-Up Synthesis and Mechanical Behavior of Refractory Coatings Made of Carbon Nanotube-Hafnium Diboride Composites.

We use aligned carbon nanotube (CNT) forests as scaffolds to deposit hafnium diboride (HfB2) and fabricate millimeter-thick ultrahigh-temperature composite coating. HfB2 has a melting temperature of 3250 °C, which makes it an attractive candidate for applications requiring operation in extreme environments. Compared to typical refractory HfB2 processing, which requires temperatures exceeding 1500 °C, we use conformal HfB2 chemical vapor deposition (CVD) to coat CNT forests at a low temperature of 200 °C. During this process, nanometer-thin HfB2 films grow on the CNT surface and uniformly fill tall CNT forests, thus transforming nanometer film deposition to a scalable HfB2 coating technology. The conformal HfB2 coating process uses static (S-) CVD, where the precursor is fed into a closed system, enabling highly conformal coating and economically efficient utilization of the HfB2 precursor reaching 85%. The modulus and compressive strength of the composites are measured using flat-punch indentation of micropillars having various coating thickness. Filling the CNTs with HfB2 strengthens their node morphology and effectively enhances the mechanical properties. We study the nonlinear behavior of the material to extract a unique modulus value that describes the stress-strain response at any applied compression. At the highest HfB2 coating thickness of 45 nm, the solid fraction is increased from 2% for the bare CNTs to 36% for the composite; the modulus and strength reach 107 and 1.5 GPa, respectively. An analytical model is used to explain the mechanism of the measured structure-mechanical property scaling. Finally, the process is used to fabricate CNT-HfB2 films having 1.7 mm height, a centimeter square area, and only 5.8 × 10-6 nm/nm thickness gradient to demonstrate the potential for scalability.

A close-up look at the spliceosome, at last.

Major developments in cryo-electron microscopy in the past three or four years have led to the solution of a number of spliceosome structures at high resolution, e.g., the fully assembled but not yet active spliceosome (Bact), the spliceosome just after the first step of splicing (C), and the spliceosome activated for the second step (C*). Therefore 30 years of genetics and biochemistry of the spliceosome can now be interpreted at the structural level. I have closely examined the RNase H domain of Prp8 in each of the structures. Interestingly, the RNase H domain has different and unexpected roles in each of the catalytic steps of splicing.

Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency.

Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the "master regulator" of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome's catalytic core and demonstrate that prp8 alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, the biological relevance of which are unknown. We performed an extensive characterization of prp8 alleles that map to this extension and, using in vitro and in vivo reporter assays, show they fall into two functional classes associated with the two structures: those that promote error-prone/efficient splicing and those that promote hyperaccurate/inefficient splicing. Identification of global locations of endogenous splice-site activation by lariat sequencing confirms the fidelity effects seen in our reporter assays. Furthermore, we show that error-prone/efficient RH alleles suppress a prp2 mutant deficient at promoting the first catalytic step of splicing, whereas hyperaccurate/inefficient RH alleles exhibit synthetic sickness. Together our data indicate that prp8 RH alleles link splicing fidelity with catalytic efficiency by biasing the relative stabilities of distinct spliceosome conformations. We hypothesize that the spliceosome "toggles" between such error-prone/efficient and hyperaccurate/inefficient conformations during the splicing cycle to regulate splicing fidelity.

Single Molecule Cluster Analysis dissects splicing pathway conformational dynamics.

We report Single Molecule Cluster Analysis (SiMCAn), which utilizes hierarchical clustering of hidden Markov modeling-fitted single-molecule fluorescence resonance energy transfer (smFRET) trajectories to dissect the complex conformational dynamics of biomolecular machines. We used this method to study the conformational dynamics of a precursor mRNA during the splicing cycle as carried out by the spliceosome. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify the signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified an open conformation adopted late in splicing by a 3' splice-site mutant, invoking a mechanism for substrate proofreading. SiMCAn enables rapid interpretation of complex single-molecule behaviors and should prove useful for the comprehensive analysis of a plethora of dynamic cellular machines.

The energy landscape of glassy dynamics on the amorphous hafnium diboride surface.

Direct visualization of the dynamics of structural glasses and amorphous solids on the sub-nanometer scale provides rich information unavailable from bulk or conventional single molecule techniques. We study the surface of hafnium diboride, a conductive ultrahigh temperature ceramic material that can be grown in amorphous films. Our scanning tunneling movies have a second-to-hour dynamic range and single-point current measurements extend that to the millisecond-to-minute time scale. On the a-HfB2 glass surface, two-state hopping of 1-2 nm diameter cooperatively rearranging regions or "clusters" occurs from sub-milliseconds to hours. We characterize individual clusters in detail through high-resolution (<0.5 nm) imaging, scanning tunneling spectroscopy and voltage modulation, ruling out individual atoms, diffusing adsorbates, or pinned charges as the origin of the observed two-state hopping. Smaller clusters are more likely to hop, larger ones are more likely to be immobile. HfB2 has a very high bulk glass transition temperature Tg, and we observe no three-state hopping or sequential two-state hopping previously seen on lower Tg glass surfaces. The electronic density of states of clusters does not change when they hop up or down, allowing us to calibrate an accurate relative z-axis scale. By directly measuring and histogramming single cluster vertical displacements, we can reconstruct the local free energy landscape of individual clusters, complete with activation barrier height, a reaction coordinate in nanometers, and the shape of the free energy landscape basins between which hopping occurs. The experimental images are consistent with the compact shape of α-relaxors predicted by random first order transition theory, whereas the rapid hopping rate, even taking less confined motion at the surface into account, is consistent with ß-relaxations. We make a proposal of how "mixed" features can show up in surface dynamics of glasses.

Quantitative fluctuation electron microscopy in the STEM: methods to identify, avoid, and correct for artifacts.

Fluctuation electron microscopy can reveal the nanoscale order in amorphous materials via the statistical variance in the scattering intensity as a function of position, scattering vector, and resolution. However, several sources of experimental artifacts can seriously affect the magnitude of the variance peaks. The use of a scanning transmission electron microscope for data collection affords a convenient means to check whether artifacts are present. As nanodiffraction patterns are collected in serial, any spatial or temporal dependence of the scattering intensity across the series can easily be detected. We present examples of the major types of artifact and methods to correct the data or to avoid the problem experimentally. We also re-cast the statistical formalism used to identify sources of noise in view of the present results. The present work provides a basis on which to perform fluctuation electron microscopy with a high level of reliability and confidence in the quantitative magnitude of the data.

Yeast-gene replacement using PCR products.

It is often useful to replace a small region of the yeast genome containing a gene of interest with a selectable marker. The selectable marker allows for easy identification of yeast cells that have successfully carried out the gene replacement, and functional consequences of the loss of that gene can then be assessed. The same technique can also be used for removing noncoding portions of the genome that may be of interest, such as promoters or 3' or 5' UTRs, and for introducing tags on the N- or C-termini of proteins (alternatively, see a marker-free yeast gene replacement method on Gene Knockouts, in vivo site-directed mutagenesis and Other Modifications Using the Delitto Perfetto System in Saccharomyces cerevisiae).

Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing.

The spliceosome is a dynamic ribonucleoprotein (RNP) machine that catalyzes the removal of introns during the two transesterification steps of eukaryotic pre-mRNA splicing. Here we used single-molecule fluorescence resonance energy transfer to monitor the distance of the 5' splice site (5' SS) and branch point (BP) of pre-mRNA in affinity-purified spliceosomes stalled by a mutation in the DExD/H-box helicase Prp2 immediately before the first splicing step. Addition of recombinant Prp2 together with NTP and protein cofactor Spp2 rearranges the spliceosome-substrate complex to reversibly explore conformations with proximal 5' SS and BP that accommodate chemistry. Addition of Cwc25, a small heat-stable splicing factor, then strongly biases this equilibrium toward the proximal conformation, promoting efficient first-step splicing. The spliceosome thus functions as a biased Brownian ratchet machine where a helicase unlocks thermal fluctuations subsequently rectified by a cofactor 'pawl', a principle possibly widespread among the many helicase-driven RNPs.

Three-dimensional self-assembled photonic crystals with high temperature stability for thermal emission modification.

Selective thermal emission in a useful range of energies from a material operating at high temperatures is required for effective solar thermophotovoltaic energy conversion. Three-dimensional metallic photonic crystals can exhibit spectral emissivity that is modified compared with the emissivity of unstructured metals, resulting in an emission spectrum useful for solar thermophotovoltaics. However, retention of the three-dimensional mesostructure at high temperatures remains a significant challenge. Here we utilize self-assembled templates to fabricate high-quality tungsten photonic crystals that demonstrate unprecedented thermal stability up to at least 1,400 °C and modified thermal emission at solar thermophotovoltaic operating temperatures. We also obtain comparable thermal and optical results using a photonic crystal comprising a previously unstudied material, hafnium diboride, suggesting that refractory metallic ceramic materials are viable candidates for photonic crystal-based solar thermophotovoltaic devices and should be more extensively studied.

Quantifying nanoscale order in amorphous materials via scattering covariance in fluctuation electron microscopy.

Fluctuation Transmission Electron Microscopy (FTEM) has a unique ability to probe topological order on the 1-3 nm length scale in diffraction amorphous materials. However, extracting a quantitative description of the order has been challenging. We report that the FTEM covariance, computed at two non-degenerate Bragg reflections, is able to distinguish different regimes of size vs. volume fraction of order. The covariance analysis is general and does not require a material-specific atomistic model. We use a Monte-Carlo approach to compute different regimes of covariance, based on the probability of exciting multiple Bragg reflections when a STEM nanobeam interacts with a volume containing ordered regions in an amorphous matrix. We perform experimental analysis on several sputtered amorphous thin films including a-Si, nitrogen-alloyed GeTe and Ge2Sb2Te5. The samples contain a wide variety of ordered states. Comparison of experimental data with the covariance simulation reveals different regimes of nanoscale topological order.

Direct writing of sub-5 nm hafnium diboride metallic nanostructures.

Sub-5 nm metallic hafnium diboride (HfB(2)) nanostructures were directly written onto Si(100)-2 × 1:H surfaces using ultrahigh vacuum scanning tunneling microscope (UHV-STM) electron beam induced deposition (EBID) of a carbon-free precursor molecule, tetrakis(tetrahydroborato)hafnium, Hf(BH(4))(4). Scanning tunneling spectroscopy data confirm the metallic nature of the HfB(2) nanostructures, which have been written down to lateral dimensions of ∼2.5 nm. To our knowledge, this is the first demonstration of sub-5 nm metallic nanostructures in an STM-EBID experiment.

Fluctuation transmission electron microscopy: detecting nanoscale order in disordered structures.

The structures of many disordered materials are not ideally random, but contain structural order on the scale of 1-3 nm. However, such nanoscale order, called medium-range order, cannot be detected by conventional diffraction methods in most cases. Fluctuation transmission electron microscopy (FTEM) has the capability to detect medium-range order in disordered materials based on statistical analysis of nanodiffraction patterns or dark-field images from TEM. FTEM has been successful in demonstrating the theoretically predicted development of nanoscale nuclei in amorphous chalcogenides, as well as in revealing the subtle effect of different preparation routes on the medium-range order in amorphous semiconductors and metals. The fluctuation principle can also be applied to study structural order on longer length scales in polymers and other disordered materials using X-rays or visible light. Further advances in theory and practice of FTEM will greatly increase our understanding of amorphous structures and nucleation phenomena.

Preparation of fluorescent pre-mRNA substrates for an smFRET study of pre-mRNA splicing in yeast.

The spliceosome is a complex small nuclear (sn)RNA-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. For each splicing event, the spliceosome is assembled de novo on a pre-mRNA substrate and a complex series of assembly steps leads to the active conformation. To comprehensively monitor pre-mRNA conformational dynamics during spliceosome assembly, we developed a strategy for single-molecule FRET (smFRET) that utilizes a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5' and 3' splice sites. In this chapter, we describe the identification of Ubc4 pre-mRNA that is efficiently spliced in vitro and the methods we have developed for the chemical synthesis of fluorescent Ubc4 pre-mRNA for smFRET.

Conformational dynamics of single pre-mRNA molecules during in vitro splicing.

The spliceosome is a complex small nuclear RNA (snRNA)-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. The chemical steps are isoenergetic, yet splicing requires at least eight RNA-dependent ATPases responsible for substantial conformational rearrangements. To comprehensively monitor pre-mRNA conformational dynamics, we developed a strategy for single-molecule FRET (smFRET) that uses a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5' and 3' splice sites. During splicing in vitro, we observed a multitude of generally reversible time- and ATP-dependent conformational transitions of individual pre-mRNAs. The conformational dynamics of branchpoint and 3'-splice site mutants differ from one another and from wild type. Because all transitions are reversible, spliceosome assembly appears to be occurring close to thermal equilibrium.

Lanthanide N,N-dimethylaminodiboranates: highly volatile precursors for the deposition of lanthanide-containing thin films.

New lanthanide CVD precursors of stoichiometry Ln(H(3)BNMe(2)BH(3))(3) have been prepared, where Ln = La, Ce, Pr, Nd, Sm, Gd, Tb, Dy, Ho, Er, Tm, and Lu. The ligand is N,N-dimethylaminodiboranate, a new kind of multidentate borohydride. The structures of the Ln(H(3)BNMe(2)BH(3))(3) complexes are highly dependent on the size of the lanthanide ions: the coordination number decreases from Pr (CN = 14) to Sm (CN = 13) to Er (CN = 12) corresponding with the decrease in ionic radii. The Ln(H(3)BNMe(2)BH(3))(3) complexes are highly volatile and sublime at temperatures as low as 65 degrees C in vacuum. These complexes are useful CVD precursors; for example, Y(H(3)BNMe(2)BH(3))(3) has been used to deposit Y(2)O(3) on silicon at 300 degrees C by CVD using water as a coreactant.

Observation of the role of subcritical nuclei in crystallization of a glassy solid.

Phase transformation generally begins with nucleation, in which a small aggregate of atoms organizes into a different structural symmetry. The thermodynamic driving forces and kinetic rates have been predicted by classical nucleation theory, but observation of nanometer-scale nuclei has not been possible, except on exposed surfaces. We used a statistical technique called fluctuation transmission electron microscopy to detect nuclei embedded in a glassy solid, and we used a laser pump-probe technique to determine the role of these nuclei in crystallization. This study provides a convincing proof of the time- and temperature-dependent development of nuclei, information that will play a critical role in the development of advanced materials for phase-change memories.