Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

Rolling circle amplification (RCA) of plasmid or genomic DNA using random hexamers and bacteriophage phi29 DNA polymerase has become increasingly popular in the amplification of template DNA in DNA sequencing. We have found that the mutant protein of single-stranded DNA binding protein (SSB) from Thermus thermophilus (Tth) HB8 enhances the efficiency of amplification of DNA templates. In addition, the TthSSB mutant protein increased the specificity of phi29 DNA polymerase. We have overexpressed the native and mutant forms of TthSSB protein in Escherichia coli and purified them to homogeneity. In vitro, these proteins were found to bind specifically to single-stranded DNA. Addition of TthSSB mutant protein to RCA halved the elongation time required for phi29 DNA polymerase to synthesize DNA fragments in RCA. Furthermore, the presence of the TthSSB mutant protein essentially eliminates nonspecific DNA products in RCA reactions.     

Polymorphism and higher order structures of protein nanofibers from crude mixtures of fish lens crystallins: toward useful materials

Protein nanofibers are emerging as useful biological nanomaterials for a number of applications, but to realize these applications requires a cheap and readily available source of fibril-forming protein material. We have identified fish lens crystallins as a feedstock for the production of protein nanofibers and report optimized methods for their production. Altering the conditions of formation leads to individual protein nanofibers assembling into much larger structures. The ability to control the morphology and form higher order structures is a crucial step in bottom up assembly of bionanomaterials. Cell toxicity assays suggest no adverse impact of these structures on mammalian cell proliferation. There are many possible applications for protein nanofibers; here we illustrate their potential as templates for nanowire formation, with a simple gold plating process.     

RCA combined nanoparticle-based optical detection technique for protein microarray: a novel approach

Developing a readily available biosensor with excellent performances is the main focus of many research groups. Recently, major breakthroughs in miniaturization of molecular analysis have produced DNA and protein microarrays. The aim of our group is to develop a sensitive technique for analyzing signals on protein microarray by applying the surface plasmon resonance (SPR) method. This new detection technique for specific molecular binding utilizes rolling circles amplification (RCA) post-signal processing method [Nat. Genet. 19 (1998) 225-232] and optical visualization by nanogold particle-labeled molecules on a micro-structured chip surface. By covalent bonding of the RCA primer to the detection antibody guarantees that the linkage between the analyte and the amplified RCA product is maintained during the assay. Experimental results show that RCA has significantly enhanced sensitivity compared to conventional methods. This combination of an easily detectable signal with chip technology should have the potential to become a successful commercial application.     

Inhibiting protein aggregation with nanomaterials: The underlying mechanisms and impact factors

Protein aggregation is correlated with the onset and progression of protein misfolding diseases (PMDs). Inhibiting the generation of toxic aggregates of misfolded proteins has been proposed as a therapeutic approach for PMDs. Due to their unique properties, nanomaterials have been extensively investigated for their ability to inhibit protein aggregation and have shown great potential in the diagnosis and treatment of PMDs. However, the precise mechanisms by which nanomaterials interact with amyloidogenic proteins and the factors influencing these interactions remain poorly understood. Consequently, developing a rational design strategy for nanomaterials that target specific proteins in PMDs has been challenging. In this review, we elucidate the effects of nanomaterials on protein aggregation and describe the mechanisms through which nanomaterials interfere with protein aggregation. The major factors impacting protein-nanomaterial interaction such as size, charge, concentration, surface modification and morphology that can be rationally addressed to achieve the desired effects of nanomaterials on protein aggregation are summarized. The prospects and challenges to the clinical application of nanomaterials for the treatment of PMDs are also discussed.     

Perspectives of different colour-emissive nanomaterials in fluorescent ink,LEDs, cell imaging,and sensing of various analytes

In the past 2 decades, multicolour light-emissive nanomaterials have gained significant interest in chemical and biological sciences because of their unique optical properties. These materials have drawn much attention due to their unique characteristics towards various application fields. The development of novel nanomaterials has become the pinpoint for different application areas. In this review, the recent progress in the area of multicolour-emissive nanomaterials is summarized. The different emissions (white, orange, green, red, blue, and multicolour) of nanostructure materials (metal nanoclusters, quantum dots, carbon dots, and rare earth-based nanomaterials) are briefly discussed. The potential applications of different colour-emissive nanomaterials in the development of fluorescent inks, light-emitting diodes, cell imaging, and sensing devices are briefly summarized. Finally, the future perspectives of multicolour-emissive nanomaterials are discussed.     

Single-stranded DNA binding protein facilitates specific enrichment of circular DNA molecules using rolling circle amplification

Many techniques in molecular biology require the use of pure nucleic acids in general and circular DNA (plasmid or mitochondrial) in particular. We have developed a method to separate these circular molecules from a mixture containing different species of nucleic acids using rolling circle amplification (RCA). RCA of plasmid or genomic DNA using random hexamers and bacteriophage Phi29 DNA polymerase has become increasingly popular for the amplification of template DNA in DNA sequencing protocols. Recently, we reported that the mutant single-stranded DNA binding protein (SSB) from Thermus thermophilus (TthSSB) HB8 eliminates nonspecific DNA products in RCA reactions. We developed this method for separating circular nucleic acids from a mixture having different species of nucleic acids. Use of the mutant TthSSB resulted in an enhancement of plasmid or mitochondrial DNA content in the amplified product by approximately 500×. The use of mutant TthSSB not only promoted the amplification of circular target DNA over the background but also could be used to enhance the amplification of circular targets over linear targets.     

Rolling-circle amplification under topological constraints

We have performed rolling-circle amplification (RCA) reactions on three DNA templates that differ distinctly in their topology: an unlinked DNA circle, a linked DNA circle within a pseudorotaxane-type structure and a linked DNA circle within a catenane. In the linked templates, the single-stranded circle (dubbed earring probe) is threaded, with the aid of two peptide nucleic acid openers, between the two strands of double-stranded DNA (dsDNA). We have found that the RCA efficiency of amplification was essentially unaffected when the linked templates were employed. By showing that the DNA catenane remains intact after RCA reactions, we prove that certain DNA polymerases can carry out the replicative synthesis under topological constraints allowing detection of several hundred copies of a dsDNA marker without DNA denaturation. Our finding may have practical implications in the area of DNA diagnostics.     

Gadolinium-containing carbon nanomaterials for magnetic resonance imaging: Trends and challenges

Gadolinium-containing carbon nanomaterials are a new class of contrast agent for magnetic resonance imaging. They are characterized by a superior proton relaxivity to any current commercial gadolinium contrast agent and offer the possibility to design multifunctional contrasts. Intense efforts have been made to develop these nanomaterials because of their potential for better results than the available gadolinium contrast agents. The aim of the present work is to provide a review of the advances in research on gadolinium-containing carbon nanomaterials and their advantages over conventional gadolinium contrast agents. Due to their enhanced proton relaxivity, they can provide a reliable imaging contrast for cells, tissues or organs with much smaller doses than currently used in clinical practice, thus leading to reduced toxicity (as shown by cytotoxicity and biodistribution studies). Their active targeting capability allows for improved MRI of molecular or cellular targets, overcoming the limited labelling capability of available contrast agents (restricted to physiological irregularities during pathological conditions). Their potential of multifunctionality encompasses multimodal imaging and the combination of imaging and therapy.     

Site-directed chemically-modified magnetic enzymes: fabrication,improvements, biotechnological applications and future prospects

Numerous enzymes of biotechnological importance have been immobilized on magnetic nanoparticles (MNP) via random multipoint attachment, resulting in a heterogeneous protein population with potential reduction in activity due to restriction of substrate access to the active site. Several chemistries are now available, where the modifier can be linked to a single specific amino acid in a protein molecule away from the active-site, thus enabling free access of the substrate. However, rarely these site-selective approaches have been applied to immobilize enzymes on nanoparticles. In this review, for the first time, we illustrate how to adapt site-directed chemical modification (SDCM) methods for immobilizing enzymes on iron-based MNP. These strategies are mainly chemical but may additionally require genetic and enzymatic methods. We critically examine each method and evaluate their scope for simple, quick, efficient, mild and economical immobilization of enzymes on MNP. The improvements in the catalytic properties of few available examples of immobilized enzymes are also discussed. We conclude the review with the applications and future prospects of site-selectively modified magnetic enzymes and potential benefits of this technology in improving enzymes, including cold-adapted homologues, modular enzymes, and CO₂-sequestering, as well as non-iron based nanomaterials.     

Duality of polynucleotide substrates for Phi29 DNA polymerase: 3'-->5' RNase activity of the enzyme

Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3'-->5' DNA exonucleolytic activities). Here we report that Phi29 DNA polymerase exonucleolyticaly degrades ssRNA. The RNase activity acts in a 3' to 5' polarity. Alanine replacements in conserved exonucleolytic site (D12A/D66A) inactivated RNase activity of the enzyme, suggesting that a single active site is responsible for cleavage of both substrates: DNA and RNA. However, the efficiency of RNA hydrolysis is approximately 10-fold lower than for DNA. Phi29 DNA polymerase is widely used in rolling circle amplification (RCA) experiments. We demonstrate that exoribonuclease activity of the enzyme can be used for the target RNA conversion into a primer for RCA, thus expanding application potential of this multifunctional enzyme and opening new opportunities for RNA detection.     

Artificial site-specific DNA-nicking system based on common restriction enzyme assisted by PNA openers

We report on the peptide nucleic acid (PNA)-directed design of a DNA-nicking system that enables selective and quantitative cleavage of one strand of duplex DNA at a designated site, thus mimicking natural nickases and significantly extending their potential. This system exploits the ability of pyrimidine PNAs to serve as openers for specific DNA sites by invading the DNA duplex and exposing one DNA strand for oligonucleotide hybridization. The resultant secondary duplex can act as a substrate for a restriction enzyme, which ultimately creates a nick in the parent DNA. We demonstrate that several restriction enzymes of different types could be successfully used in the PNA-assisted system we developed. Importantly, the enzyme cleavage efficiency is basically not impaired on such artificially generated substrates, compared with the efficiency on regular DNA duplexes. Our design originates a vast class of semisynthetic rare-cleaving DNA nickases, which are essentially absent at present. In addition, we show that the site-specific PNA-assisted nicking of duplex DNA can be engaged in a rolling-circle DNA amplification (RCA) reaction. This new RCA format demonstrates the practical potential of the novel biomolecular tool we propose for DNA technology and DNA diagnostics.     

Protein detection via direct enzymatic amplification of short DNA aptamers

Aptamers are single-stranded nucleic acids that fold into defined tertiary structures to bind target molecules with high specificities and affinities. DNA aptamers have garnered much interest as recognition elements for biodetection and diagnostic applications due to their small size, ease of discovery and synthesis, and chemical and thermal stability. Here we describe the design and application of a short DNA molecule capable of both protein target binding and amplifiable bioreadout processes. Because both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids can be omitted. The DNA aptamer is designed to be amplified directly by either polymerase chain reaction (PCR) or rolling circle amplification (RCA) processes, taking advantage of real-time amplification monitoring techniques for target detection. A combination of both RCA and PCR provides a wide protein target dynamic range (1 microM to 10 pM).     

碳纳米材料的环境行为及其对环境中污染物迁移归趋的影响

碳纳米材料具有广阔的应用前景,近年来已成为一大研究热点.工程碳纳米材料的大量生产和使用将不可避免地造成这些材料向环境中的释放,可能带来环境和生态风险.一方面,碳纳米材料本身具有环境毒性,另一方面碳纳米材料对环境中有毒有害污染物有较强的吸附性能,因此会影响污染物迁移转化等环境行为.目前,对碳纳米材料生态风险的研究主要集中于碳纳米材料对生物体可能的毒性,而对其自身环境行为以及影响污染物迁移归趋等方面的研究较少.本文简要概述了碳纳米材料的来源、暴露途径、环境行为以及对污染物迁移归趋的影响,阐述了这些研究对于评估碳纳米材料的环境和生态风险所具有的重要意义.     

Homologous recombination in Arabidopsis seeds along the track of energetic carbon ions

Nanomaterials are already used today and offer even greater use and benefits in the future. The progress of nanotechnology must be accompanied by investigations of their potential harmful effects. For airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. While standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. We have investigated two genotoxic endpoints - alkaline Comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow - in a combined study 72 h after a single instillation of 18 μg gold nanoparticles (NP) into the trachea of male adult Wistar rats. The administration of three test materials differing only in their primary particle size (2, 20 and 200 nm) did not lead to relevant DNA damage in the mentioned tests. The measurement of clinical pathology parameters in bronchoalveolar lavage fluid (BALF) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. Minor histopathology findings occurred in the lung of the animals exposed to 20 nm and 200 nm sized nanomaterials. In conclusion, under the conditions of this study the different sized gold NP tested were non-genotoxic and showed no systemic and local adverse effects at the given dose.     

A sequence-independent strategy for detection and cloning of circular DNA virus genomes by using multiply primed rolling-circle amplification

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with phi29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 x 10(4)-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.     

Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA

We present a novel Phi29 DNA polymerase application in RCA-based target RNA detection and analysis. The 3′→5′ RNase activity of Phi29 DNA polymerase converts target RNA into a primer and the polymerase uses this newly generated primer for RCA initiation. Therefore, using target RNA-primed RCA, padlock probes may be targeted to inner RNA sequences and their peculiarities can be analyzed directly. We demonstrate that the exoribonucleolytic activity of Phi29 DNA polymerase can be successfully applied in vitro and in situ. These findings expand the potential for detection and analysis of RNA sequences distanced from 3′-end.     

Electrochemical strategy for sensing protein phosphorylation

We herein report a novel electrochemical method in this paper to monitor protein phosphorylation and to assay protein kinase activity based on Zr(4+) mediated signal transition and rolling circle amplification (RCA). First, substrate peptide immobilized on a gold electrode can be phosphorylated by protein kinase A. Then, Zr(4+) links phosphorylated peptide and DNA primer probe by interacting with the phosphate groups. After the introduction of the padlock probe and phi29 DNA polymerase, RCA is achieved on the surface of the electrode. As the RCA product, a very long DNA strand, may absorb a large number of electrochemical speices, [Ru(NH(3))(6)](3+), via the electrostatic interaction, localizing them onto the electrode surface, initiated by protein kinase A, a sensitive electrochemical method to assay the enzyme activity is proposed. The detection limit of the method is as low as 0.5 unit/mL, which might promise this method as a good candidate for monitoring phosphorylation in the future.     

Conditions of activity bubble uniqueness in dynamic neural fields

Dynamic neural fields (DNFs) offer a rich spectrum of dynamic properties like hysteresis, spatiotemporal information integration, and coexistence of multiple attractors. These properties make DNFs more and more popular in implementations of sensorimotor loops for autonomous systems. Applications often imply that DNFs should have only one compact region of firing neurons (activity bubble), whereas the rest of the field should not fire (e.g., if the field represents motor commands). In this article we prove the conditions of activity bubble uniqueness in the case of locally symmetric input bubbles. The qualitative condition on inhomogeneous inputs used in earlier work on DNFs is transfered to a quantitative condition of a balance between the internal dynamics and the input. The mathematical analysis is carried out for the two-dimensional case with methods that can be extended to more than two dimensions. The article concludes with an example of how our theoretical results facilitate the practical use of DNFs.     

Biomedical applications and potential health risks of nanomaterials: molecular mechanisms

Nanotechnologies, defined as techniques aimed to conceive, characterize and produce material at the nanometer scale, represent a fully expanding domain, and one can predict without risk that production and utilization of nanomaterials will increase exponentially in the coming years. Applications of nanotechnologies are numerous, in constant development, and their potential use in the medical field as diagnosis and therapeutics tools is very attractive. The size particularity of these nanomaterials gives them novel properties, allowing them to adopt new comportments because of the laws of quantum physics that exist at this scale. However, worries are expressed regarding the exact properties that make these nanomaterials attractive, and questions are raised regarding their potential toxicity, their long-term secondary effects or their biodegradability, particularly when thinking of their use in the (nano)medical field. These questions are justified by the knowledge of the toxic effects of atmospheric pollution micrometric particles on health, and the fear to get an amplification of these effects because of the size of the materials blamed. In this paper, we first expose the sensed medical applications of nanomaterials, and the physicochemical and molecular determinants potentially responsible for nanomaterials biological effects. Finally, we present a synthesis of the actual knowledge regarding toxicological effects of nanomaterials. It is clear that, in regard to the almost empty field of what is known on the subject, there's an urge to better understand biological effects of nanomaterials, which will allow their safe use, in particular in the nanomedicine field.