PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing.

Fluorescence-based sequencing is playing an increasingly important role in efforts to identify DNA polymorphisms and mutations of biological and medical interest. The application of this technology in generating the reference sequence of simple and complex genomes is also driving the development of new computer programs to automate base calling (Phred), sequence assembly (Phrap) and sequence assembly editing (Consed) in high throughput settings. In this report we describe a new computer program known as PolyPhred that automatically detects the presence of heterozygous single nucleotide substitutions by fluorescencebased sequencing of PCR products. Its operations are integrated with the use of the Phred, Phrap and Consed programs and together these tools generate a high throughput system for detecting DNA polymorphisms and mutations by large scale fluorescence-based resequencing. Analysis of sequences containing known DNA variants demonstrates that the accuracy of PolyPhred with single pass data is >99% when the sequences are generated with fluorescent dye-labeled primers and approximately 90% for those prepared with dye-labeled terminators.     

Influence of aptamer-targeted antibiofilm agents for treatment of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> biofilms

Biofilms are bacterial communities consisting of numerous extracellular polymeric substances. Infections caused by biofilm-forming bacteria are considered to be a major threat to health security and so novel approaches to control biofilm are of importance. Aptamers are single-strand nucleic acid molecules that have high selectivity to their targets. Single-walled carbon nanotubes (SWNTs) are common nanomaterials and have been shown to be toxic to bacterial biofilms. The aim of this study was to test whether an aptamer could play a role as targeting agents to enhance the efficiency of anti-biofilm agents. Hence, two complexes (aptamer–SWNTs and aptamer–ciprofloxacin–SWNTs) based on an aptamer which targets Pseudomonas aeruginosa and SWNTs were constructed. Both complexes were assessed against P. aeruginosa biofilms. In vitro tests demonstrated that the aptamer–SWNTs could inhibit ~36% more biofilm formation than SWNTs alone. Similarly, the aptamer–ciprofloxacin–SWNTs had a higher anti-biofilm efficiency than either component or simple mixtures of two components. Our study underscores the potential of aptamers as targeting agents for anti-biofilm compounds, as well as providing a new strategy to control biofilms.     

Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-β) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C.     

RNA and DNA aptamers in cytomics analysis.

BACKGROUND: The systematic evolution of ligands by exponential enrichment (SELEX) technique is a combinatorial library approach in which DNA or RNA molecules (aptamers) are selected by their ability to bind their protein targets with high affinity and specificity, comparable to that of monoclonal antibodies. In contrast to antibodies conventionally selected in animals, aptamers are generated by an in vitro selection process, and can be directed against almost every target, including antigens like toxins or nonimmunogenic targets, against which conventional antibodies cannot be raised. METHODS: Aptamers are ideal candidates for cytomics, as they can be attached to fluorescent reporters or nanoparticles in order to study biological function by fluorescence microscopy, by flow cytometry, or to quantify the concentration of their target in biological fluids or cells using ELISA, RIA, and Western blot assays. RESULTS: We demonstrate the in vitro selection of anti-kinin B1 receptor aptamers that could be used to determine B1 receptor expression during inflammation processes. These aptamers specifically recognize their target in a Northern-Western blot assay, and bind to their target protein whenever they are exposed in the membrane. CONCLUSIONS: Currently, aptamers are linked to fluorescent reporters. We discuss here the present status and future directions concerning the use of the SELEX technique in cytomics.     

Aptamer evolution for array-based diagnostics

Closed loop aptameric directed evolution, (CLADE) is a technique enabling simultaneous discovery, evolution, and optimization of aptamers. It was previously demonstrated using a fluorescent protein, and here we extend its applicability with the generation of surface-bound aptamers for targets containing no natural fluorescence. Starting from a random population, in four generations CLADE produced a new aptamer to thrombin with high specificity and affinity. The best aptameric sequence was void of the set of four guanine repeats typifying thrombin aptamers and, thus, highlights the benefits of evolution performed in an environment closely mimicking the final diagnostic application.     

In vitro fluorescent analysis of preprotein import into chloroplasts

Despite recent progress in fluorescence techniques employed to observe protein localization in living cells, the in vitro chloroplastic protein transport assay remains a useful tool for determining the destinations of proteins. Although an in vitro synthesized, radiolabeled precursor protein is frequently used as the transport substrate, we have developed a transport assay system with a non-radiolabeled precursor protein that carries an epitope tag and is overexpressed in Escherichia coli. Thus, a transported protein can be detected by immunoblotting (Inoue et al., Plant Physiol. Biochem., 46, 541-549 (2008)). Here, we propose another in vitro protein transport system that combines fluorescence techniques. We attempted to use two types of precursors: a green fluorescent protein (GFP)-fused precursor and a fluorescent dye-labeled one. Both were successfully imported into chloroplasts. However, the fluorescent dye-labeled precursor was more advantageous than the GFP-fused precursor in the in vitro system.     

Analytical applications of aptamers

So far, several bio-analytical methods have used nucleic acid probes to detect specific sequences in RNA or DNA targets through hybridisation. More recently, specific nucleic acids, aptamers, selected from random sequence pools, have been shown to bind non-nucleic acid targets, such as small molecules or proteins. The development of in vitro selection and amplification techniques has allowed the identification of specific aptamers, which bind to the target molecules with high affinity. Many small organic molecules with molecular weights from 100 to 10,000 Da have been shown to be good targets for selection. Moreover, aptamers can be selected against difficult target haptens, such as toxins or prions. The selected aptamers can bind to their targets with high affinity and even discriminate between closely related targets.

Aptamers can thus be considered as a valid alternative to antibodies or other bio-mimetic receptors, for the development of biosensors and other analytical methods. The production of aptamers is commonly performed by the SELEX (systematic evolution of ligands by exponential enrichment) process, which, starting from large libraries of oligonucleotides, allows the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction.

Aptamers are suitable for applications based on molecular recognition as analytical, diagnostic and therapeutic tools. In this review, the main analytical methods, which have been developed using aptamers, will be discussed together with an overview on the aptamer selection process.     

RNA aptamers that functionally interact with green fluorescent protein and its derivatives

Green Fluorescent Protein (GFP) and related fluorescent proteins (FPs) have been widely used to tag proteins, allowing their expression and subcellular localization to be examined in real time in living cells and animals. Similar fluorescent methods are highly desirable to detect and track RNA and other biological molecules in living cells. For this purpose, we have developed a group of RNA aptamers that bind GFP and related proteins, which we term Fluorescent Protein-Binding Aptamers (FPBA). These aptamers bind GFP, YFP and CFP with low nanomolar affinity and binding decreases GFP fluorescence, whereas slightly augmenting YFP and CFP brightness. Aptamer binding results in an increase in the pKa of EGFP, decreasing the 475 nm excited green fluorescence at a given pH. We report the secondary structure of FPBA and the ability to synthesize functional multivalent dendrimers. FPBA expressed in live cells decreased GFP fluorescence in a valency-dependent manner, indicating that the RNA aptamers function within cells. The development of aptamers that bind fluorescent proteins with high affinity and alter their function, markedly expands their use in the study of biological pathways.     

Selection of DNA aptamers using atomic force microscopy

Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind to their targets with very high affinity. Therefore, we propose the Systematic Evolution of Ligands by an EXponential enrichment (SELEX) method using AFM to obtain aptamers that have a strong affinity for target molecules. In this study, thrombin was chosen as the target molecule, and an ‘AFM-SELEX’ cycle was performed. As a result, selected cycles were completed with only three rounds, and many of the obtained aptamers had a higher affinity to thrombin than the conventional thrombin aptamer. Moreover, one type of obtained aptamer had a high affinity to thrombin as well as the anti-thrombin antibody. AFM-SELEX is, therefore, considered to be an available method for the selection of DNA aptamers that have a high affinity for their target molecules.     

Horseradish peroxidase-driven fluorescent labeling of nanotubes with quantum dots

We describe the first enzyme-driven technique for fluorescent labeling of single-walled carbon nanotubes (SWNTs). The labeling was performed via enzymatic biotinylation of nanotubes in the tyramide-horseradish peroxidase (HRP) reaction. Both direct and indirect fuorescent labeling of SWNTs was achieved using either biotinyl tyramide or fluorescently tagged tyramides. Biotinylated SWNTs later reacted with streptavidin-conjugated fluorophores. Linking semiconductor nanocrystals, quantum dots (Q-dots), to the surface of nanotubes resulted in their fluorescent visualization, whereas conventional fluorophores bound to SWNTs directly or through biotin-streptavidin linkage, were completely quenched. Enzymatic biotinylation permits fluorescent visualization of carbon nanotubes, which could be useful for a number of biomedical applications. In addition, other organic molecules such as proteins, antibodies, or DNA can be conjugated to biotinylated SWNTs using this approach.     

Aptamers in clinical diagnostics

Aptamers are single-stranded DNA or RNA oligonucleotides selected in vitro from combinatorial libraries in a process called SELEX (Systematic Evolution of Ligands by EXponential Enrichment). Aptamers play a role of artificial nucleic acid ligands that can recognize and bind to various organic or inorganic target molecules with high specificity and affinity. They can discriminate even between closely related targets and can be easily chemically modified for radioactive, fluorescent and enzymatic labeling or biostability improvement. Aptamers can thus be considered as universal receptors that rival antibodies in diagnostics as a tool of molecular recognition. To date aptamers have been successively used instead of monoclonal antibodies in flow cytometry, immunochemical sandwich assays and in vivo imaging as well to detect wide range of small or large biomolecules.     

孔雀石绿适配体生物传感器的研究进展

荧光适配体作为一种无需标记的荧光探针,具有许多潜在的优势,并被应用于多种靶物质(如ATP、RNA)的检测,是目前适配体研究领域的热点。孔雀石绿适配体(malachite green aptamer,MGA)属于荧光适配体,其能通过配体诱导折叠形成结合口袋,进而促进孔雀石绿(malachite green,MG)的发光。目前,已经筛选得到的MGA的种类较少,主要介绍了已知的MG RNA适配体及其变构体和MG DNA适配体的特性,以及影响MG-MGA复合物荧光强度的因素。同时,还对主要的MG衍生物和共聚物进行了总结。最后,综述了MGA在生物传感、荧光成像等方面的应用,并对MGA的发展方向进行了展望,以期为MGA在生物检测、生物成像等方面的应用提供指导。     

Developing implantable optical biosensors

Nanobiotechnologists are developing devices that can measure specific enzymes and proteins. These devices are expected to detect single enzyme or protein molecules accurately, providing highly sensitive biosensing applications. A recent study by Strano and co-workers shows that single-walled carbon nanotubes (SWNTs) hold great promise as implantable biosensors. Although most researchers have focused on substrate-oriented biosensors, Strano and colleagues have shown that the inherent fluorescent properties of suspended individual SWNTs can be used for solution-phase beta-D-glucose sensing.     

Visual detection of specific, native interactions between soluble and microbead-tethered alpha-helices from membrane proteins.

Using peptides tethered to polymer microbeads, we have developed a technique for measuring the interactions between the transmembrane alpha-helices of membrane proteins and for screening combinatorial libraries of peptides for members that interact with specific helices from membrane proteins. The method was developed using the well-characterized homodimerization sequence of the membrane-spanning alpha-helix from the erythrocyte membrane protein glycophorin A (GPA). As a control, we also tested a variant with a dimer-disrupting alteration of a critical glycine residue to leucine. To test for detectable, native interactions between detergent-solubilized and microbead-tethered alpha-helices, we incubated fluorescent dye-labeled GPA analogues in sodium dodecyl sulfate solution with microbeads that contained covalently attached GPA analogues. When the dye-labeled peptide in solution and the bead-tethered peptide both contained the native glycophorin A sequence, the microbeads readily accumulated the dye through lateral peptide-peptide interactions and were visibly fluorescent under UV light. When either the peptide in solution or the peptide attached to the beads contained the glycine to leucine change, the beads did not accumulate any dye. The usefulness of this method for screening tethered peptide libraries was tested by incubating dye-labeled, native sequence peptides in detergent solution with a few native sequence beads plus an excess of beads containing the variant glycine to leucine sequence. When the dye-labeled peptide in solution was present at a concentration of > or =2 microM, the few native sequence beads were visually distinguishable from the others because of their bright fluorescence. Using this model system, we have shown that it is possible to visually detect specific, native interactions between alpha-helices from membrane proteins using peptides tethered to polymer microbeads. It will thus be possible to use this method to measure the specific lateral interactions that drive the folding and organization of membrane proteins and to screen combinatorial libraries of peptides for members that interact with them.     

In situ synthesis of amylose/single-walled carbon nanotubes supramolecular assembly

A supramolecular assembly of amylose and single-walled carbon nanotubes (SWNTs) was synthesized in situ through vine-twining polymerization. Raman analysis indicated that the amylose-SWNTs supramolecular assembly was formed after the polymerization and SEM images displayed the twisted ribbons in the SWNTs wrapped by amylose. The dispersion stability of the SWNTs in aqueous solutions was improved by the wrapping of short-chain amylose molecules around the SWNTs.     

Application of microchip electrophoresis in the analysis of RNA aptamer-protein interactions

DNA and RNA can be separated by microchip electrophoresis (ME) and detected using an intercalating fluorescent dye. The advantages of this method are short sensing times (<3 min), avoidance of a radioisotope labeling detection system, relatively low costs, and reduced labor intensity. In the present study, RNA aptamer-protein or -peptide interactions were analyzed using ME and the regression of free aptamers corresponding to unbound RNA was detected as the target protein or peptide increased in a dose-dependent manner. Our results demonstrate the applicability of this method to simple, rapid ligand screening in the interactions between oligonucleotides and their targets.     

Molecular dynamics study of thrombin capture by aptamers TBA26 and TBA29 coupled to a DNA origami

The fabrication of DNA origamis is one of the very few possibilities to create nanostructures with precise atomistic-tailored geometries in a variety of shapes. In addition, these origamis can be functionalized or be impregnated with specialised aptamers in order to convert them into nanosensors or to tune them with pre-specified properties simply by impregnating single-stranded DNA or RNA chains (aptamers) via the precise features of DNA pairing. We performed molecular dynamics simulations to determine the relative energetics associated with the capture of thrombin by two aptamers TBA26 and TBA29 attached to a rectangular DNA origami. The molecular simulations provided detailed structural information of aptamer–enzyme interactions which are crucial for the efficient design of aptamer-based biosensors. In addition, the simulations showed a remarkable selectivity of the biosensor assembly for thrombin. The detection, capture, and sensing of enzymes is of great significance in biomedicine. In particular, the detection of thrombin is a major task in cardiovascular diagnostics and therapeutics. On the other hand, our simulations can be extended to detect biotoxins or any other chemical or biological agent by simply choosing proper aptamers. Finally, the problems due to the large number of atoms involved as well as the quality of the approximations are also discussed.     

Application of Microchip Electrophoresis in the Analysis of RNA Aptamer-Protein Interactions

DNA and RNA can be separated by microchip electrophoresis (ME) and detected using an intercalating fluorescent dye. The advantages of this method are short sensing times (< 3 min), avoidance of a radioisotope labeling detection system, relatively low costs, and reduced labor intensity. In the present study, RNA aptamer-protein or -peptide interactions were analyzed using ME and the regression of free aptamers corresponding to unbound RNA was detected as the target protein or peptide increased in a dose-dependent manner. Our results demonstrate the applicability of this method to simple, rapid ligand screening in the interactions between oligonucleotides and their targets.     

Aptamer Selection Express: A Novel Method for Rapid Single-Step Selection and Sensing of Aptamers

Here we describe a new DNA capture element (DCE) sensing system, based on the quenching and dequenching of a double-stranded aptamer. This system shows very good sensitivity and thermal stability. While quenching, dequenching, and separating the DCE systems made from different aptamers (all selected by SELEX), an alternative method to rapidly select aptamers was developed—the Aptamer Selection Express (ASExp). This process has been used to select aptamers against different types of targets (Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2 bacteriophage, ovalbumin, and botulinum neurotoxin). The DCE systems made from botulinum neurotoxin aptamers selected by ASExp have been investigated. The results of this investigation indicate that ASExp can be used to rapidly select aptamers for the DCE sensing system.