Rapid, multiplexed, sensitive and specific molecular detection is of great demand in gene profiling, drug screening, clinical diagnostics and environmental analysis. One of the major challenges in ...multiplexed analysis is to identify each specific reaction with a distinct label or 'code'. Two encoding strategies are currently used: positional encoding, in which every potential reaction is preassigned a particular position on a solid-phase support such as a DNA microarray, and reaction encoding, where every possible reaction is uniquely tagged with a code that is most often optical or particle based. The micrometer size, polydispersity, complex fabrication process and nonbiocompatibility of current codes limit their usability. Here we demonstrate the synthesis of dendrimer-like DNA-based, fluorescence-intensity-coded nanobarcodes, which contain a built-in code and a probe for molecular recognition. Their application to multiplexed detection of the DNA of several pathogens is first shown using fluorescence microscopy and dot blotting, and further demonstrated using flow cytometry that resulted in detection that was sensitive (attomole) and rapid.
Thermostable enzymes combine catalytic specificity with the toughness required to withstand industrial reaction conditions. Stabilized enzymes also provide robust starting points for evolutionary ...improvement of other protein properties. We recently created a library of at least 2,300 new active chimeras of the biotechnologically important cytochrome P450 enzymes. Here we show that a chimera's thermostability can be predicted from the additive contributions of its sequence fragments. Based on these predictions, we constructed a family of 44 novel thermostable P450s with half-lives of inactivation at 57 degrees C up to 108 times that of the most stable parent. Although they differ by as many as 99 amino acids from any known P450, the stable sequences are catalytically active. Among the novel functions they exhibit is the ability to produce drug metabolites. This chimeric P450 family provides a unique ensemble for biotechnological applications and for studying sequence-stability-function relationships.
Charge-Reversal Amphiphiles for Gene Delivery Prata, Carla A. H; Zhao, Yuxing; Barthelemy, Philippe ...
Journal of the American Chemical Society,
10/2004, Letnik:
126, Številka:
39
Journal Article
Recenzirano
Delivering a missing gene or a functional substitute of a defective gene has the potential to revolutionize current medical care. Of the two gene delivery approaches, viral and synthetic vectors, ...synthetic cationic vectors possess several practical advantages but suffer from poor transfection efficiency. A new approach to gene delivery using charge-reversal amphiphiles is described. This synthetic vector transforms from a cationic to an anionic amphiphile intracellularly. This amphiphile performs two roles: first, it binds and then releases DNA, and second, as an anionic multicharged amphiphile, it destabilizes lipid bilayers. A charge-reversal amphiphile was synthesized and fully characterized, including the supramolecular complex it forms with DNA. Enhanced gene transfection was observed using these vectors compared to current cationic amphiphiles.
We report a single-molecule fluorescence study on the diffusion of star-branched polymer dendrimer-like DNA (DL-DNA). The DL-DNA molecules were synthesized from ligating Y-shaped DNA to the fourth ...generation. It was found through single particle tracking that the diffusion coefficient of DL-DNA changes in a nonmonotonic fashion with its increased concentration possibly due to arm arrest and arm retraction. The diffusion of DL-DNA in linear lambda DNA solution displayed a monotonic concentration dependence 1 order of magnitude greater than the diffusion of DL-DNA in DL-DNA solution. This difference is attributed to the different conformation of DL-DNA and lambda DNA and the entanglement of lambda DNA with a large radius of gyration. Our diffusion study facilitates DL-DNA transportation for drug delivery.
DNA possesses many desirable chemical/physical properties as a polymeric material. With the myriad of tools available to manipulate DNA, there is great potential for using DNA as a generic instead of ...a genetic material. Although much progress has been made in DNA computing and DNA nanotechnology, the full achievement of DNA-based materials has not yet been realized. As almost all DNA molecules are either linear or circular, to rationally construct DNA materials one must first create additional shapes of DNA as basic building blocks. In addition, these DNA building blocks must be readily incorporated into larger structures in a controlled manner. Here, we show the controlled assembly of dendrimer-like DNA (DL-DNA) from Y-shaped DNA (Y-DNA). The synthesis of Y-DNA and controlled assembly of DL-DNA were robust and efficient; the resulting DL-DNA was stable and almost monodisperse. The multivalent DNA dendrimers can be either isotropic or anisotropic, providing great potential to link other entities.
A major challenge in clinical diagnostics and environmental analysis is the difficulty in rapid and sensitive detection of multiple target molecules simultaneously (i.e., multiplexed detections). Our ...group has designed and synthesized a dendrimer-like DNA (DL-DNA) that is multivalent and anisotropic; using this unique DNA structure, we have developed a fluorescence-tagged nanobarcode system for multiplex detection. This nanobarcode system allows the rapid and sensitive detection of multiple pathogens simultaneously using the ratios of two different fluorescent dyes, green and red, with which different DL-DNAs are labeled. The key step of our nanobarcode model lies in the monodisperse preparation of DL-DNA. Two methods, solution phase and solid phase, are presented here. With slight modifications, this platform technology can also be extended to the multiplexed detection of RNA and proteins. This protocol can be completed in 2-5 d.
Signal-encoded microparticles and nanoparticles have been used to label many reactions simultaneously for target identification in assays, and thus are an indispensable part of multiplex ...technologies. With the increasing demand for multiplexed molecular detection, encoded particles have evolved from pattern encoding to signal-intensity encoding, and also from signal-molecule encapsulation to signal-molecule tagging. The fabrication and utilization of such nano- and microparticles should advance multiplexed analysis. This short review focuses on how these encoded particles work and briefly touches on their applications in multiplexed molecular detection.
Through in-depth analysis on history and current situations of fishery development in Poyang Lake area,this paper presents location,resource and environment advantages in development of ecological ...fishery.According to orientation of leading functions,Poyang Lake can be divided into breeding development,capture operation,enhancement protection and ecological rehabilitation functional zones.In accordance with industrial foundation and ecological advantages of cities and counties in Poyang Lake,it determines the reasonable distribution of 8 leading industries:river crabs,shrimps(Procambarus clarkia),eels,Pengze crucian carp,Siniperca chuatsi,Channa argus,Ietalurus Punetaus,and pearl.On the basis of systematically summing up exploration experience,it comes up with the development path"ecology oriented fishery and industrialization oriented ecological fishery"for ecological fishery in Poyang Lake area.It proposes a series of key technological measures,including reducing fertilizer and drugs,increasing output and benefits,low consumption and emission,high quality and safety,and standardization,to realize a benign cycle of"cost saving,benefit increasing,quality improving and low carbon"and the multi-win objective of coordination between ecology,economy and society.
PDF
