Room temperature ionic liquid doped DNA network immobilized horseradish peroxidase biosensor for amperometric determination of hydrogen peroxide

A novel electrochemical H2O2 biosensor was constructed by embedding horseradish peroxide (HRP) in a 1-butyl-3-methylimidazolium tetrafluoroborate doped DNA network casting on a gold electrode. The HRP entrapped in the composite system displayed good electrocatalytic response to the reduction of H2O2. The composite system could provide both a biocompatible microenvironment for enzymes to keep their good bioactivity and an effective pathway of electron transfer between the redox center of enzymes, H2O2 and the electrode surface. Voltammetric and time-based amperometric techniques were applied to characterize the properties of the biosensor. The effects of pH and potential on the amperometric response to H2O2 were studied. The biosensor can achieve 95% of the steady-state current within 2 s response to H2O2. The detection limit of the biosensor was 3.5 mu M, and linear range was from 0.01 to 7.4 mM. Moreover, the biosensor exhibited good sensitivity and stability. The film can also be readily used as an immobilization matrix to entrap other enzymes to prepare other similar biosensors.

Large-scale, uniform DNA network on 11-mercaptoundecanoic acid modified gold (111) surface: Atomic force microscopy study

Large-scale, uniform plasmid deoxyribonucleic acid (DNA) network has been successfully constructed on 11-mercaptoundecanoic acid modified gold (111) surface using a self-assembly technique. The effect of DNA concentration on the characteristics of the DNA network was investigated by atomic force microscopy. It was found that the size of meshes and the height of fibers in the DNA network could be controlled by varying the concentration of DNA with a constant time of assembly of 24 h.

DNA network structures on various solid substrates investigated by atomic force microscopy

We have fabricated DNA network structures on glass and sapphire substrates. As a comparison, we also formed the network structure on mica substrate. For titanate strontium substrate, however, DNA network can not be obtained even if it is wet-treated by Na2HPO4 solution to make it hydrophilic. We also discuss the factors that affect the DNA networks formed on various substrates.

Construction and control of plasmid DNA network

The influences of different cations on plasmid DNA network structures on a mica substrate were investigated by atomic force microscopy (AFM). Interactions between the DNA strands and mica substrate, and between the DNA strands themselves were more strongly influenced by the complex cations (Fe(phen)(3)(2+), Ni(phen)(3)(2+), and Co(phen)(3)(3+)) than by the simple cations (Mg2+, Mn2+, Ni2+, Ca2+, Co3+). The mesh height of the plasmid DNA network was higher when the complex cations were added to DNA samples. The mesh size decreased with increasing DNA concentration and increased with decreasing DNA concentration in the same cation solution sample. Hence, plasmid DNA network height can be controlled by selecting different cations, and the mesh size can be controlled by adjusting plasmid DNA concentration.

Electrostatic-assembly metallized nanoparticles network by DNA template

Eighteen-nanometer gold and 3.5-nm silver colloidal particles closely packed by cetyltrimethylammonium bromide (CTAB) to form its positively charged shell. The DNA network was formed on a mica Substrate firstly. Later, CTAB-capped gold or silver colloidal solutions were cast onto DNA network surface. It was found that the gold or silver nanoparticles metallized networks were formed owing to the electrostatic-driven template assembling of positive charge of CTAB-capped gold and silver particles on the negatively charged phosphate groups of DNA Molecules by the characterizations of AFM, XPS and UV-vis. This method may provide a novel and simple way to studying nanoparticles assembly conjugating DNA molecules and offer some potential promising applications in nanocatalysis, nanoelectronics, and nanosensor on the basis of the fabricated metal nanoparticles network.

A novel hydrogen peroxide sensor based on horseradish peroxidase immobilized in DNA films on a gold electrode

A novel third-generation hydrogen peroxide (H2O2) biosensor was developed by immobilizing horseradish peroxidase (HRP) on a biocompatible gold electrode modified with a well-ordered, self-assembled DNA film. Cysteamine was first self-assembled on a gold electrode to provide an interface for the assembly of DNA molecules. Then DNA was chemisorbed onto the self-assembled monolayers (SAMs) of cysteamine to form a network by controlling DNA concentration. The DNA-network film obtained provided a biocompatible microenvironment for enzyme molecules, greatly amplified the coverage of HRP molecules on the electrode surface, and most importantly could act as a charge carrier which facilitated the electron transfer between HRP and the electrode. Finally, HRP was adsorbed on the DNA-network film. The process of the biosensor construction was followed by atomic force microscopy (AFM). Voltammetric and time-based amperometric techniques were employed to characterize the properties of the biosensor derived. The enzyme electrode achieved 95% of the steady-state current within 2 s and had a 0.5 mu mol l(-1) detection limit of H2O2. Furthermore, the biosensor showed high sensitivity, good reproducibility, and excellent long-term stability.

栽培牡丹起源研究

牡丹在中国被称作“花中之王”。我国不仅是全部野生牡丹的原产地，也是栽培牡丹最早的驯化地。野生牡丹共有8个种，分布于云南、四川、湖北、甘肃、陕西、山西、安徽、河南和西藏等9省区，因其具有很大的观赏和药用价值，而在中国和世界温带地区广泛栽培。本研究利用形态特征和4个核基因片段（三个Adh 基因和GPAT基因片断）的核苷酸序列变异对牡丹组的种间系统发育关系进行了分析，并对我国栽培牡丹四个品种群的101个代表品种的可能祖先进行了形态学鉴定和分子诊断标记研究。在此基础上，利用核编码叶绿体表达的GPAT基因的（大内含子）部分序列和叶绿体基因组的trnS – trnG 和 rpS16 – trnQ两个基因间隔区的DNA序列变异重建了栽培牡丹37个代表品种和26个野生居群间的谱系关系。结果表明：（1）GPAT基因树是迄今得到的分辨率最好，并具有很高自展值支持的牡丹组种间系统发育关系树;（2）GPAT基因树和形态学证据一致支持银屏牡丹（P. suffruticosa ssp. yinpingmudan）, 凤丹（P. ostii）, 紫斑牡丹（P. rockii）, 卵叶牡丹（P. qiui）, 和矮牡丹 （P. jishanensis） 参与了栽培牡丹的起源;（3）叶绿体DNA单倍型网络树（network）进一步证实上述5个祖先类群的4个（矮牡丹除外）可能参与了栽培牡丹的母系起源。37个品种的GPAT基因谱系和叶绿体DNA单倍型网络树一致表明银屏牡丹是栽培牡丹最主要的祖先，其次是紫斑牡丹、凤丹、和卵叶牡丹;（4）我们的分子证据不支持形态学证据关于矮牡丹是栽培牡丹最主要的野生祖先的推测;（5）形态学和分子诊断标记证据表明，101个品种中有65.35 % 的品种具有两个以上野生种的特征，18.81 % 品种同时具有 Eco R I (+) 和 InDel51(+)物种特异分子标记。对37个品种的GPAT基因谱系和叶绿体DNA谱系比较发现，其中35个可能是杂种起源。另外，对7个古代牡丹品种（据文献记载）的GPAT基因的不同克隆类型进行测序和谱系分析，结果表明其中4 个为杂种起源。上述证据充分表明杂交和（或）渗入杂交在牡栽培牡丹的起源和进化中发挥了重要作用。根据本研究的结果，结合现有的形态学数据、考古记录，以及有关牡丹栽培和驯化历史的记载，我们对栽培牡丹的起源和驯化历史总结如下。牡丹的栽培迄今有1,600 – 2,000年，栽培牡丹最迟起源于1，500年前。最初通过驯化和对突变的选择获得原始品种。由于牡丹品种可以通过无性和（或）有性方式进行繁殖，其后新的品种通过如下方式产生：（1）对突变的选择，（2）对栽培类型和野生种之间或栽培类型之间杂交和（或）渗入杂交产生的实生苗的选择。由于绝大部分（如果不是全部）早期的原始品种已绝灭，现有栽培牡丹是起源于各种人工和自然进化力共同作用的结果，其中包括多次驯化、人工选择、突变、杂交和渗入杂交等。据作者所知，栽培牡丹的这种 ‘compilospecies’ 起源和驯化模式是目前已研究过的主要栽培作物中未见报道的。 因此，本研究不仅为栽培牡丹的多系起源和驯化历史提供了可信的分子证据，同时也为利用单拷贝基因的内含子序列构建栽培作物及其近缘野生祖先间的种系发生关系提供了成功的例子。另外，本研究也为同时利用核和叶绿体基因组的非编码DNA序列研究杂交在栽培作物的起源和进化的中作用提供了成功的例子。

Type I collagen-mediated synthesis and assembly of UV-photoreduced gold nanoparticles and their application in surface-enhanced Raman scattering

We reported a simple method to synthesize gold nanoparticles (NPs) by photoreducing HAuCl4 in acetic acid solution in the presence of type I collagen. It was found that the collagen takes an important role in the formation of gold NPs. The introduction of collagen made the shape of the synthesized gold nanocrystals change from triangular and hexangular gold nanoplates to size-uniform NPs. On the other hand, thanks to the special characters of collagen molecules, such as its linear nanostructure, are positively charged when the pH < 7, and the excellent self-assembly ability, photoreduced gold NPs were assembled onto the collagen chains and formed gold NPs films and networks. A typical probe molecule, 4-aminothiophenol, was used to test the surface-enhanced Raman scattering activity of these gold NPs films and networks and the results indicated good Raman activity on these substrates.

A relocated technique of atomic force microscopy (AFM) samples and its application in molecular biology

A kind of simple atomic force microscopy (AFM) relocated technique, which takes advantage of homemade sample locator system, is used for investigating repeatedly imaging of some specific species on the whole substrate (over 1 x 1 cm(2)) with resolution about 400 nm. As applications of this sample locator system, single extended DNA molecules and plasmid DNA network are shown in different AFM operational modes: tapping mode and contact mode with different tips after the substrates have been moved.

RNA editing in kinetoplastids

RNA editing in kinetoplastid protozoa is a post-transcriptional process of uridine insertion or deletion in mitochondrial mRNAs. The process involves two RNA species, the pre-edited mRNA and in most cases a trans-acting guide RNA (gRNA). Sequences within gRNAs define the position and extend of mRNA editing. Both mRNAs and gRNAs are encoded by mitochondrial genes in the kinetoplast DNA (kDNA), which consists of thousands of small circular DNA molecules, called minicircles, encoding thousands of gRNAs, catenated together and with a few mRNA encoding larger circles, the maxicircles, to form a huge DNA network. Editing has been shown to result in translatable mRNAs of bona fide mitochondrial genes as well as novel alternatively edited transcripts that are involved in the maintenance of the kDNA itself. RNA editing occurs within large protein-RNA complexes, editosomes, containing gRNA, preedited and partially edited mRNAs and also structural and catalytically active proteins. Editosomes are diverse in both RNA and protein composition and undergoe structural remodeling during the maturation. The compositional and structural diversity of editosomes further underscores the complexity of the RNA editing process.

A conserved mitochondrial outer membrane protein mediates kDNA maintenace in Trypanosoma brucei

Kinetoplastids are defined by the unique organization of their mitochondrial DNA (kDNA). It forms a highly concatenated DNA network that is linked to the basal body of the flagellum by the tripartite attachment complex (TAC). The TAC encompasses intra and extramitochondrial filaments and a highly differentiated region of the two mitochondrial membranes. Here we identify and characterize a mitochondrial outer membrane protein of Trypanosoma brucei that is predominantly localized in the TAC. The protein is essential for growth in both life cycle stages. Immunofluorescence shows that ablation of the protein does not affect kDNA replication but abolishes the segregation of the replicated kDNA network causing rapid loss of kDNA. Besides its role in kDNA maintenance in vivo and in vitro experiments show that the protein is involved in mitochondrial protein import and that it interacts with a recently discovered protein import factor. RNAi experiments in a T. brucei cell line in which the kDNA is dispensable suggest that the essential function is linked to kDNA maintenance. Bioinformatic analysis shows that the studied outer membrane protein has beta-barrel topology and that it belongs to the mitochondrial porin family comprising VDAC, Tom40 and Mdm10. Interestingly, Mdm10 has sofar only been found in yeast. Ist function in protein import and mitochondrial DNA maintenance suggests that the protein in our study is the functional homologue of Mdm10. Thus, the TAC – a defining structure of Kinetoplastids – contains a conserved protein which in yeast and trypanosomes performs the same function. Our study therefore provides an example that trypanosomal biology, rather than being unique, often simply represents a more extreme manifestation of a conserved biological concept.

A conserved mitochondrial outer membrane protein mediates kDNA maintenace in Trypanosoma brucei

Kinetoplastids are defined by the unique organization of their mitochondrial DNA (kDNA). It forms a highly concatenated DNA network that is linked to the basal body of the flagellum by the tripartite attachment complex (TAC). The TAC encompasses intra and extramitochondrial filaments and a highly differentiated region of the two mitochondrial membranes. Here we identify and characterize a mitochondrial outer membrane protein of Trypanosoma brucei that is predominantly localized in the TAC. The protein is essential for growth in both life cycle stages. Immunofluorescence shows that ablation of the protein does not affect kDNA replication but abolishes the segregation of the replicated kDNA network causing rapid loss of kDNA. Besides its role in kDNA maintenance in vivo and in vitro experiments show that the protein is involved in mitochondrial protein import and that it interacts with a recently discovered protein import factor. RNAi experiments in a T. brucei cell line in which the kDNA is dispensable suggest that the essential function is linked to kDNA maintenance. Bioinformatic analysis shows that the studied outer membrane protein has beta-barrel topology and that it belongs to the mitochondrial porin family comprising VDAC, Tom40 and Mdm10. Interestingly, Mdm10 has so far only been found in yeast. Its function in protein import and mitochondrial DNA maintenance suggests that the protein in our study is the functional homologue of Mdm10. Thus, the TAC – a defining structure of Kinetoplastids – contains a conserved protein which in yeast and trypanosomes performs the same function. Our study therefore provides an example that trypanosomal biology, rather than being unique, often simply represents a more extreme manifestation of a conserved biological concept.

Evolution of RNA editing in trypanosome mitochondria

Two different RNA editing systems have been described in the kinetoplast-mitochondrion of trypanosomatid protists. The first involves the precise insertion and deletion of U residues mostly within the coding regions of maxicircle-encoded mRNAs to produce open reading frames. This editing is mediated by short overlapping complementary guide RNAs encoded in both the maxicircle and the minicircle molecules and involves a series of enzymatic cleavage-ligation steps. The second editing system is a C34 to U34 modification in the anticodon of the imported tRNATrp, thereby permitting the decoding of the UGA stop codon as tryptophan. U-insertion editing probably originated in an ancestor of the kinetoplastid lineage and appears to have evolved in some cases by the replacement of the original pan-edited cryptogene with a partially edited cDNA. The driving force for the evolutionary fixation of these retroposition events was postulated to be the stochastic loss of entire minicircle sequence classes and their encoded guide RNAs upon segregation of the single kinetoplast DNA network into daughter cells at cell division. A large plasticity in the relative abundance of minicircle sequence classes has been observed during cell culture in the laboratory. Computer simulations provide theoretical evidence for this plasticity if a random distribution and segregation model of minicircles is assumed. The possible evolutionary relationship of the C to U and U-insertion editing systems is discussed.