Highly birefringent TiO2 nanocylinders : characterization and application in the optical torque wrench

Recent advances in optical tweezers have enabled the direct manipulation and measurement of optical torque using light spin momentum transferred to trapped birefringent particles. This powerful technique, termed Optical Torque Wrench (OTW), relies on trapping of quartz (SiO2) microcylinders which have become a standard and convenient probe for single-molecule studies. Here, we explore an alternative photonic probe based on rutile (TiO2) which has almost thirty-fold larger birefringence compared to quartz particles. By employing this promising material to fabricate rutile nanocylinders whose sizes can be easily tuned, we significantly enhance the accessible range of optical torques and angular frequencies in the OTW. In future, these novel photonic probes will allow us to study not only slowly processing torque-generating biological systems, as the genome processing machinery, but also fast rotating motors, including ATP-synthase and the bacterial flagellar motor

Trapping of highly birefringent rutile nanocylinders in the optical torque wrench

The optical torque wrench (OTW) is a powerful technique to measure the torsional properties of different biomolecules, including DNA, DNA- processing protein complexes and rotary motors. To date, quartz has proven to be a convenient birefringent material out of which to synthesize the micron-sized particles essential for this technique. However, the relatively low birefringence of quartz, which limits the maximal torque that can be applied in OTW, hampers the study of certain biological systems. A more attractive material is rutile, which has a thirty-fold higher birefringence. To date, however, the application of rutile in the trapping has been restricted due to its high refractive index, which results in low trapping efficiency. Here, we have employed finite element method calculations to determine the optimal dimensions of sub-micron-sized rutile cylinders for tight stable optical trapping. Using these calculations as a guideline, we have designed and devel- oped a nanofabrication protocol that allows us to produce rutile cylinders with the desired sizes at high yield. We have characterized the fabricated cylinders in the OTW setup and quantified both their linear and angular trapping proper- ties. In addition, we demonstrate full translational and rotational control of these functionalized cylinders tethered to individual DNA molecules for use in single-molecule applications

Spatiotemporal Distribution Of Different Extracellular Polymeric Substances And Filamentation Mediate Xylella Fastidiosa Adhesion And Biofilm Formation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Microorganism pathogenicity strongly relies on the generation of multicellular assemblies, called biofilms. Understanding their organization can unveil vulnerabilities leading to potential treatments; spatially and temporally-resolved comprehensive experimental characterization can provide new details of biofilm formation, and possibly new targets for disease control. Here, biofilm formation of economically important phytopathogen Xylella fastidiosa was analyzed at single-cell resolution using nanometer-resolution spectro-microscopy techniques, addressing the role of different types of extracellular polymeric substances (EPS) at each stage of the entire bacterial life cycle. Single cell adhesion is caused by unspecific electrostatic interactions through proteins at the cell polar region, where EPS accumulation is required for more firmly-attached, irreversibly adhered cells. Subsequently, bacteria form clusters, which are embedded in secreted loosely-bound EPS, and bridged by up to ten-fold elongated cells that form the biofilm framework. During biofilm maturation, soluble EPS forms a filamentous matrix that facilitates cell adhesion and provides mechanical support, while the biofilm keeps anchored by few cells. This floating architecture maximizes nutrient distribution while allowing detachment upon larger shear stresses; it thus complies with biological requirements of the bacteria life cycle. Using new approaches, our findings provide insights regarding different aspects of the adhesion process of X. fastidiosa and biofilm formation.5Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2010/51748-7, 2010/18107-8, 2010/50712-9]CNPq [479486/2012-3]CNPq [573913/2008-0]FAPESP [08/57906-3

Surface Physicochemical Properties At The Micro And Nano Length Scales: Role On Bacterial Adhesion And Xylella Fastidiosa Biofilm Development.

The phytopathogen Xylella fastidiosa grows as a biofilm causing vascular occlusion and consequently nutrient and water stress in different plant hosts by adhesion on xylem vessel surfaces composed of cellulose, hemicellulose, pectin and proteins. Understanding the factors which influence bacterial adhesion and biofilm development is a key issue in identifying mechanisms for preventing biofilm formation in infected plants. In this study, we show that X. fastidiosa biofilm development and architecture correlate well with physicochemical surface properties after interaction with the culture medium. Different biotic and abiotic substrates such as silicon (Si) and derivatized cellulose films were studied. Both biofilms and substrates were characterized at the micro- and nanoscale, which corresponds to the actual bacterial cell and membrane/ protein length scales, respectively. Our experimental results clearly indicate that the presence of surfaces with different chemical composition affect X. fastidiosa behavior from the point of view of gene expression and adhesion functionality. Bacterial adhesion is facilitated on more hydrophilic surfaces with higher surface potentials; XadA1 adhesin reveals different strengths of interaction on these surfaces. Nonetheless, despite different architectural biofilm geometries and rates of development, the colonization process occurs on all investigated surfaces. Our results univocally support the hypothesis that different adhesion mechanisms are active along the biofilm life cycle representing an adaptation mechanism for variations on the specific xylem vessel composition, which the bacterium encounters within the infected plant.8e7524

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA: LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies. (C) 2012 Elsevier B.V. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo - FAPESP (Sao Paulo, Brazil)Laboratorio de Espectroscopia e Calorimetria (LEC), Laboratorio Nacional de Biociencias - LNBio (Campinas, Brazil

Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the ‘‘backtracked’’ state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity

Fluorine-based dry etching and selective surface functionalization of titanium dioxide nanostructures

Titanium dioxide (TiO2) exhibits exceptional properties such as a rapid electron transport, high dielectric constant, wide band gap, high refractive index, and large birefringence. Further, it is a photocatalyst and highly biocompatible. It is widely implemented in electronic and photonic applications like solar cells, sensors, transistors, memory devices, and force transducers. However, its use at the nanoscale has been limited by challenges in fabrication. The most common bottom-up fabrication methods lack fine dimensional control and structural uniformity. To overcome these limitations, plasma etching-based top-down fabrication is preferred in general but TiO2 suffers from the difficulty in etching [1]. To address this challenge, we have developed a novel fabrication method utilizing fluorine-based dry etching to controllably fabricate TiO2 nanostructures, and chemically modify their surfaces employing readily accessible surface functionalization methods. In our fabrication protocol (Fig 1), a chromium (Cr) etch mask provides increased resistance to dry etching and allows a wider range of aspect ratios and sidewall angles through its high etch selectivity. The patterning process using electron-beam (e-beam) lithography allows high dimensional uniformity of nanostructures. Additionally, free nanoparticles can be generated from them through mechanical cleaving (Fig 1f, Fig 2). We have optimized TiO2 etching parameters for two types of fluorine-based plasma etching configurations. First, for the trifluoromethane (CHF3)-based plasma etching of TiO2 with a reactive ion etching (RIE) machine, we obtain high etch selectivity (~15:1) as well as controlled sidewall angles. Depending on the amount of added oxygen (O2) gas flow, the angles can be varied from positive to negative, including vertical sidewalls (Fig 3a). Interestingly, with higher O2 gas flow, the fabrication of hourglass-shaped nanocylinders is also possible (Fig 3b). Second, we also optimize the sulfur hexafluoride (SF6)-based plasma etching of TiO2 with an inductively coupled plasma (ICP)-RIE machine. It allows us to achieve higher etch rates of 100-200 nm/min, compared to those of 30-50 nm/min in CHF3-based RIE, possibly due to high ion density and less passivation. Increasing ICP power, hence providing higher ion density, results in more positive sidewall angles (Fig 3c) due to more rapid Cr mask etch rates which reduce etch selectivity. On the other hand, with a reduced ICP power, we can fabricate nanocylinders with nearly vertical sidewalls (Fig 3d), demonstrating sufficient etch selectivity (~12:1). As we use the rutile-phase TiO2 that is the most difficult to etch [2], our method is expected to be directly applicable to the other phases with only minor modifications of a few etching parameters. For surface functionalization of TiO2 surfaces, we utilize widely used epoxysilanes as surface linker molecule (Fig 4a) [3]. The dense, homogeneous surface coating is demonstrated by attaching fluorophores to the epoxysilane-coated surface and imaging their fluorescence emission (Fig 4b). Moreover, the functionalization can be selectively performed only on top of nanocylinders (Fig 4c) with partial PMMA coating (Fig 1e). The introduced fabrication and functionalization methods can be employed in diverse applications that exploit the unique physical and chemical properties of TiO2 at the nanoscale, including biomolecular sensing

Fabrication and surface functionalization of highly birefringent rutile particles for trapping in an optical torque wrench

The optical torque wrench (OTW) allows the direct application and measure- ment of torque on biomolecules, such as DNA or DNA-protein complexes, or rotary motors like the F0F1-ATP-synthase or the bacterial flagellar motor. The applicable torque of the OTW is a function of the size and birefringence of the particle. Quartz has proven a convenient material, but its quite low birefringence limits full investigation of torque-speed relationships of diverse biological systems. In contrast, rutile exhibits a much higher birefringence - exceeding that of quartz by a factor of 30 - but its utilization has been infrequent because of the difficulties in optical trapping and fabrication. To enhance the applicability of the OTW, we have improved both the design and fabrication of cylindrical rutile particles. We have employed finite element method calculations to determine the optimal dimension of stably trappable rutile cylinders. To obtain rutile cylinders with the optimal dimensions, we developed a protocol for full control of size and sidewall angle. In our fabrica- tion protocol, a chromium etch mask provides increased resistance to dry etching and allows the fabrication of structures with both high aspect ratio and anisotropy. Also, the sidewall angle of cylinders can be readily tuned by adjusting a single process parameter, namely the oxygen flow rate during dry etching. The fabricated cylinders were characterized in the OTW setup to reveal their linear and angular trapping properties. The fabrication process is compatible with common chemical functionalization procedures and permits covalent biomolecule attachment. To enhance biomolecule coverage, we used ethanolamine and poly(ethylene glycol) as biomolecular crosslinkers to obtain homogenous and dense coatings. Our recent results, in which we use functionalized, trapped rutile cylinders to study single biomolecules and motor proteins, will be presented