Sugar-Pucker Force-Induced Transition in Single-Stranded DNA

The accurate knowledge of the elastic properties of single-stranded DNA (ssDNA) is key to characterize the thermodynamics of molecular reactions that are studied by force spectroscopy methods where DNA is mechanically unfolded. Examples range from DNA hybridization, DNA ligand binding, DNA unwinding by helicases, etc. To date, ssDNA elasticity has been studied with different methods in molecules of varying sequence and contour length. A dispersion of results has been reported and the value of the persistence length has been found to be larger for shorter ssDNA molecules. We carried out pulling experiments with optical tweezers to characterize the elastic response of ssDNA over three orders of magnitude in length (60–14 k bases). By fitting the force-extension curves (FECs) to the Worm-Like Chain model we confirmed the above trend:the persistence length nearly doubles for the shortest molecule (60 b) with respect to the longest one (14 kb). We demonstrate that the observed trend is due to the different force regimes fitted for long and short molecules, which translates into two distinct elastic regimes at low and high forces. We interpret this behavior in terms of a force-induced sugar pucker conformational transition (C3′-endo to C2′-endo) upon pulling ssDNA.     

慢性粒细胞白血病ssDNA适配子PCR扩增条件的优化

目的优化慢性粒细胞白血病(Chronic myelogenous leukemia,CML)ssDNA适配子的PCR扩增条件。方法以合成的核苷酸适配子为模板并设计引物,根据引物解链温度(Tm值)设置不同的退火温度(38.0、39.2、41.2、44.2、48.3、51.5、53.6、55.0℃)、循环次数(12、16、20、24、28、30、35、38、40)及非限制性与限制性引物比例(1∶1、30∶1、50∶1、70∶1、80∶1、100∶1),进行PCR扩增,优化CML ssDNA适配子对称PCR及间接不对称PCR扩增条件,并验证优化的PCR条件的重复性。结果对称PCR扩增的最佳退火温度为44.2℃,最佳循环次数为30次;间接不对称PCR的最佳循环次数为38次,非限制性引物与限制性引物浓度比例为50∶1时,可获得理想的ssDNA和较少的dsDNA;优化的PCR条件重复性较好。结论优化了CML ssDNA适配子的PCR扩增条件,为筛选到特异性的ssDNA适配子提供了参考,也为后续试验奠定了基础。     

Covalent attachment of single-stranded DNA to carbon paste electrode modified by activated carboxyl groups

A new method of the electrode modification and DNA immobilization for a biosensor is reported. Outer layer of a conventional carbon paste electrode (CPE) was modified with carboxyl groups by mixing stearic acid with the paste. Single-stranded deoxyribonucleic acid was attached to the modified electrode through a linker - ethylenediamine. Immobilization process was performed in the presence of activators - water soluble 1-ethyl-3(3′-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS). Stearic acid concentration and other experimental parameters of the procedure were optimized. Covalent immobilization of DNA on the electrode surface exhibits some advantages as compared to simple adsorption mainly due to the fact that nucleic acid chains are bound to an electrode surface by one end only and it ensures structural flexibility and increases hybridization without DNA leakage. Modified electrode with immobilized (21-mer) oligonucleotide as a specific probe was successfully applied in preliminary investigations for the detection of bar gene commonly used in genetically modified food.     

Assembly of Single-Stranded DNA Onto HOPG Surface at Different Temperature: Atomic Force Microscopy Study

Assembly of long single‐stranded DNA (ssDNA) and short oligodeoxynucleotides onto bare highly oriented pyrolytic graphite (HOPG) at different temperature has been studied. It was indicated that both long ssDNA and oligodeoxynucleotides can sequentially form network, straight chains, and layer structures when the adsorption temperature was changed from room temperature, 37–55°C. High‐resolution atomic force microscopy (AFM) imaging of the layer structures revealed that they are composed of parallel ssDNA chains with relatively higher height and tend to form patterns with three‐fold symmetry. These new findings are significantly important for understanding assembly characterization of ssDNA. In addition, this assembly method for ssDNA is expected to be used for preparation of DNA structures in biosensing and DNA‐based nanodevices. SCANNING 34: 302–308, 2012. © 2012 Wiley Periodicals, Inc.     

全细胞SELEX法筛选单增李斯特菌的ssDNA适配体

以单增李斯特菌活细胞为靶标,采用指数富集配基的系统进化技术(Systematic Evolution of Ligands by Exponential Enrichment,SELEX)从含40个随机碱基序列的单链DNA(single-stranded DNA,ss DNA)文库中筛选与之特异性结合的适配体。酶联配体吸附法(ELASA)测定筛选过程中次级文库与单增李斯特菌结合力的变化。对筛选得到的适配体进行序列测定,RNA structure软件预测二级结构,ELASA测定适配体与靶标的结合能力和特异性。结果显示,每轮筛选后次级文库与靶标的亲和力增强,特异性适配体逐步得到富集。测序获得23条适配体,其序列同源性不高,但预测的二级结构有些相似,根据二级结构将其分为3个群,分析结果表明二级结构与适配体的亲和力有关。挑选亲和力较强的21号(Lma-21)和35号(Lma-35)适配体能特异性地识别单增李斯特菌。本研究为开发食源性病原菌单增李斯特菌的新型检测试剂提供了基础材料。     

基于酶联适配体的四环素检测方法研究

通过Mannich法偶联辣根过氧化物酶(HRP)和四环素(tetracycline,TC)分子制备酶标记物,采用棋盘滴定法确定链霉亲和素(streptavidin,SA)的包被浓度为8μg/mL,适配体稀释浓度为20 nmol/L。通过单因素实验优化了检测条件,碳酸盐缓冲液(CB,0.05 mol/L,pH 9.6)为包被液,适配体稀释液选择含有5 mmol/L Mg2+的磷酸缓冲液PBS,TC-HRP稀释度为1:50,标准品稀释液为PBS溶液,适配体孵育1 h,最终建立了四环素酶联适配体检测(enzyme-linked aptamer assay,ELAA)方法。该法半抑制浓度(IC50)为0.705μg/mL,检测限(LOD)为2.5 ng/mL,与其结构类似物(多西环素、土霉素、金霉素)测试中,发现除与金霉素稍有交叉反应(25.9%)外,与其他药物基本没有明显交叉反应。本研究所建立的直接竞争酶联适配体检测方法特异性强、灵敏度高,能够满足四环素定量分析要求,适用于食品中四环素的快速检测。     

Chirality‐Selective Photoluminescence Enhancement of ssDNA‐Wrapped Single‐Walled Carbon Nanotubes Modified with Gold Nanoparticles

In this work, a convenient method to enhance the photoluminescence (PL) of single‐walled carbon nanotubes (SWNTs) in aqueous solutions is provided. Dispersing by single‐stranded DNA (ssDNA) and modifying with gold nanoparticles (AuNPs), about tenfold PL enhancement of the SWNTs is observed. More importantly, the selective PL enhancement is achieved for some particular chiralities of interest over all other chiralities, by using certain specific ssDNA sequences that are reported to recognize these particular chiralities. By forming AuNP–DNA–SWNT nanohybrids, ssDNA serves as superior molecular spacers that on one hand protect SWNT from direct contacting with AuNP and causing PL quench, and on the other hand attract the AuNP in close proximity to the SWNT to enhance its PL. This PL enhancement method can be utilized for the PL analysis of SWNTs in aqueous solutions, for biomedical imaging, and may serve as a prescreening method for the recognition and separation of single chirality SWNTs by ssDNA.     

Electrochemical investigation of interactions of bilayer lipid membranes (BLMs) with incorporated resorcin[4]arene receptor with ephedrine for the development of a stabilized lipid film biosensor for ephedrine

This work explores the electrochemical investigation of interactions of ephedrine with bilayer lipid membranes (BLMs) in which a resorcin[4]arene receptor or ssDNA were incorporated that can be used for the rapid electrochemical detection of ephedrine. BLMs were composed of egg phosphatidylcholine (PC) and 35% (w/w) dipalmitoyl phosphatidic acid in which the receptor was incorporated or by PC and ssDNA in BLMs without the receptor. The interactions of ephedrine with self-assembled lipid membranes were investigated. These interactions have provided current increases or ion channel events depending on the composition of the lipid film. A comparison of signals between those obtained with self-assembled lipid films with incorporated resorcin[4]arene receptor and DNA was made herein. The use resorcin[4]arene receptor has provided ion current increases which were more sensitive than those in its absence or using BLMs with incorporated DNA. The linear also concentration range was increased in the presence of the resorcin[4]arene receptor. The response times in the presence of the resorcin[4]arene receptor was found to be smaller than those of BLMs without receptor or in the presence of DNA. Freely suspended BLMs with incorporated receptor were used to develop a rapid detection biosensing technique for ephedrine. The interactions of this compound with freely suspended lipid membranes were found to be electrochemically transduced in the form of a transient current signal with duration of seconds, which reproducibly appeared within 20 s after exposure of the membranes to ephedrine. The magnitude of the transient current signal was related to the concentration of the stimulating agent in bulk solution in the micromolar range. No interferences from ascorbic acid were noticed, because of the use of the negatively charged lipids in membranes. A large number of other compounds were also investigated as interferences. Stabilized after storage in air lipid membranes (i.e. the lipid film is formed by polymerisation on supports of glass fiber microfilters) were investigated as practical chemical biosensors for the rapid detection of ephedrine, which will allow further commercialization of these devices. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2′-azobis-(2-methylpropionitrile) was the initiator, whereas the lipid and the receptor were enclosed within the polymer. The interactions of these polymerized lipid films with ephedrine were also found to provide transient current signals with duration of seconds, which reproducibly appeared after about 1.5 min after exposure of the films to ephedrine. The magnitude of the transient current signal was also related to the concentration of the stimulating agent in bulk solution in the micromolar range and these stabilized lipid films can used again after storage in air. The present technique can be used as a practical sensor for the rapid detection of ephedrine and keep prospects for its determination in biofluids of athletes.     

Graphene-DNA hybrid materials: Assembly, applications, and prospects

Among the carbon-based nanomaterials such as carbon nanotubes, fullerenes, graphene and nanodiamonds, graphene received recently widespread attention owing to its exceptional structural, electronic and mechanical properties and potential applications in various domains. However, all currently known forms of graphene materials are not well dispersible or soluble in most common solvents. This limitation deters to explore the chemistry of graphene at the molecular level and its nanobio device applications. One well known solution to this problem is the use of dispersing agents such as polymers, biopolymers, or surfactants in conjunction with the appropriate experimental conditions. Among the various biomolecules, deoxyribonucleic acid (DNA) has emerged as an appealing biomacromolecule for functional materials due to its biocompatibility and renewability in addition to its very interesting double helix structure, which guarantees a range of unique properties that are difficult to detect in other molecules and polymers. Hence, the combination of graphene (a carbon-based nanomaterial), showing exceptional electronic properties, and DNA (a nanostructured biomolecule), having extraordinary recognition properties, demonstrates a new type of nanobio hybrid material. This, in turn, leads to a successful incorporation of the properties of the two different components in new hybrid materials that present important features for potential applications that range from advanced biomedical systems by means of very sensitive electrochemical sensors and biosensors to highly efficient electronics- and optics-based biochips. This article will focus on the recent advancement of the methods available for the chemical functionalization of graphene using DNA by different interactions (covalent or non-covalent and insertion of DNA through graphene nanopore or nanogap), various types of assemblies, and future prospects. Furthermore, the various potential applications of the resulting new nanobio hybrid materials are also highlighted.