Toward automated nucleic acid enzyme selection.

Methods for automation of nucleic acid selections are being developed. The selection of aptamers has been successfully automated using a Biomek 2000 workstation. Several binding species with nanomolar affinities were isolated from diverse populations. Automation of a deoxyribozyme ligase selection is in progress. The process requires eleven times more robotic manipulations than an aptamer selection. The random sequence pool contained a 5' iodine residue and the ligation substrate contained a 3' phosphorothioate. Initially, a manual deoxyribozyme ligase selection was performed. Thirteen rounds of selection yielded ligators with a 400-fold increase in activity over the initial pool. Several difficulties were encountered during the automation of DNA catalyst selection, including effectively washing bead-bound DNA, pipetting 50% glycerol solutions, purifying single strand DNA, and monitoring the progress of the selection as it is performed. Nonetheless, automated selection experiments for deoxyribozyme ligases were carried out starting from either a naive pool or round eight of the manually selected pool. In both instances, the first round of selection revealed an increase in ligase activity. However, this activity was lost in subsequent rounds. A possible cause could be mispriming during the unmonitored PCR reactions. Potential solutions include pool redesign, fewer PCR cycles, and integration of a fluorescence microtiter plate reader to allow robotic 'observation' of the selections as they progress.     

核酸探针的原理及应用

近年来核酸探针作为一种基因诊断技术逐渐被人们接受,综述了核酸探针的设计原理、制作方法及其在生物领域中的应用,为中学生物学教学提供参考。     

Application of fluorescence melting curve analysis for dual DNA detection using single peptide nucleic acid probe

Peptide nucleic acid (PNA) is an artificially synthesized polymer. PNA oligomers show greater specificity in binding to complementary DNAs. Using this PNA, fluorescence melting curve analysis (FMCA) for dual detection was established. Genomic DNA of Mycoplasma fermentans and Mycoplasma hyorhinis was used as a template DNA model. By using one PNA probe, M. fermentans and M. hyorhinis could be detected and distinguished simultaneously in a single tube. The developed PNA probe is a dual‐labeled probe with fluorescence and quencher dye. The PNA probe perfectly matches the M. fermentans 16s rRNA gene, with a melting temperature of 72°C. On the other hand, the developed PNA probe resulted in a mismatch with the 16s rRNA gene of M. hyorhinis, with a melting temperature of 44–45°C. The melting temperature of M. hyorhinis was 27–28°C lower than that of M. fermentans. Due to PNA's high specificity, this larger melting temperature gap is easy to create. FMCA using PNA offers an alternative method for specific DNA detection. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:730–735, 2015     

Time-resolved detection probe for homogeneous nucleic acid analyses in one-step format

We report here an extension of homogeneous assays based on fluorescence intensity and lifetime measuring on DNA hybridization. A novel decay probe that allows simple one-step nucleic acid detection with subnanomolar sensitivity, and is suitable for closed-tube applications, is introduced. The decay probe uses fluorescence resonance energy transfer (FRET) between a europium chelate donor and an organic fluorophore acceptor. The substantial change in the acceptor emission decay time on hybridization with the target sequence allows the direct separation of the hybridized and unhybridized probe populations in a time-resolved measurement. No additional sample manipulation or self-hybridization of the probes is required. The wavelength and decay time of a decay probe can be adjusted according to the selection of probe length and acceptor fluorophore, thereby making the probes applicable to multiplexed assays. Here we demonstrate the decay probe principle and decay probe-based, one-step, dual DNA assay using celiac disease-related target oligonucleotides (single-nucleotide polymorphisms [SNPs]) as model analytes. Decay probes showed specific response for their complementary DNA target and allowed good signal deconvolution based on simultaneous optical and temporal filtering. This technique potentially could be used to further increase the number of simultaneously detected DNA targets in a simple one-step homogeneous assay.     

Fluorescence detection of single nucleotide polymorphisms using nucleic acid probe containing tricyclic base-linked acyclonucleoside

A reliable and simple method for detecting nucleobase mutations is very important clinically because sequence variations in human DNA cause genetic diseases and genetically influenced traits. A majority of sequence variations are attributed to single nucleotide polymorphisms (SNPs). Here, we developed a method for SNP detection using DNA probes that contained a fluorescent tricyclic base-linked acyclonucleoside N. The type of nucleobases involved in the SNP sites in an RNA target could be determined using four DNA probes containing N. Further, we found that the SNP in the RNA target could be detected by a visible color. Thus, this system would provide a novel and simple method for detecting SNPs in an RNA target.     

Rapid detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water using peptide nucleic acid probes

A new chemiluminescent in situ hybridization (CISH) method that provides simultaneous detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water within 1 working day has been developed. Individual micro-colonies of P. aeruginosa were detected directly on membrane filters following 5 h of growth by use of soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeted to a species-specific sequence in P. aeruginosa rRNA. Within each micro-colony, reaction of the peroxidase with a chemiluminescent substrate generated light that was subsequently captured by film or with a digital camera system. Each spot of light represented one micro-colony of P. aeruginosa. Sensitivity and specificity for the identification of P. aeruginosa were 100% as determined by testing 28 P. aeruginosa strains and 17 other bacterial species that included closely related Pseudomonas species. Furthermore, the number of micro-colonies of P. aeruginosa represented by light spots correlated with counts of visible colonies following sustained growth. We conclude that PNA CISH speeds up traditional membrane filtration techniques and adds the specificity of PNA probe technology to generate fast and definitive results.     

Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC₅₀ = 1.4 μM), suramin sodium salt (IC₅₀ = 3.6 μM), NF 023 hydrate (IC₅₀ = 6.2 μM) and tyrphostin AG 538 (IC₅₀ = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.     

Nonradioactive detection of nucleic acid by the universal probe system

A convenient and nonradioactive method for DNA hybridization tests termed the "Universal probe system" has been developed. This method is based on the principle of sandwich hybridization. This system consists of two single-stranded DNA probes (a primary probe and a biotin-labeled secondary probe). The primary probe is prepared from a chimeric phage-plasmid vector containing the complementary sequence to a target gene. The secondary probe has a sequence complementary to the vector portion of the primary probe and is labeled with biotin via the transamination reaction. An advantage of this method is that the single-stranded primary probe can be prepared with ease by using the chimeric phage-plasmid vector system, thereby avoiding tedious labeling of individually different probes. As the primary probe is not modified with biotin and other labels, it conserves the sequence to be hybridized with a target. Accordingly, the primary probe containing a relatively short hybridizing region (ca. 50 bp) can efficiently hybridize with the target. In fact, the universal probe is sensitive enough to detect a single-copy human gene on Southern blots.     

Fluorescence energy transfer as a probe for nucleic acid structures and sequences.

The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes.     

Interference-based detection of nucleic acid targets on optically coated silicon

Sequence-specific detection of polynucleotides typically requires modified reporter probes that are labeled with radioactive, fluorescent, or luminescent moieties. Although these detection methods are capable of high sensitivity, they require instrumentation for signal detection. In certain settings, such as clinical point of care, instrumentation might be impractical or unavailable. Here we describe a detection approach in which formation of a nucleic acid hybrid is enzymatically transduced into a molecular thin film that can be visually detected in white light. The system exploits a flat, optically coated silicon-based surface to which capture oligonucleotides are covalently attached. The optimized system is capable of detection of nucleic acid targets present at sub-attomole levels. To supplement visual detection, signals can be quantitated by a charge-coupled device. The design and composition of the optical surface, optimization of immobilization chemistry for attachment of capture probes, and characterization of the efficiency of the hybridization process are presented. We describe the application of this system to detection of a clinically relevant target, the mecA gene present in methicillin-resistant Staphylococcus aureus.     

Survivin mRNA antagonists using locked nucleic acid, potential for molecular cancer therapy

We have investigated the effects of different locked nucleic acid modified antisense mRNA antagonists against Survivin in a prostate cancer model. These mRNA antagonists were found to be potent inhibitors of Survivin expression at low nanomolar concentrations. Additionally there was a pronounced synergistic effect when combining the mRNA antagonists against Survivin with the chemotherapeutic Taxol. This effect was demonstrated at concentrations of antagonists far lower than any previously demonstrated, indicating the high potential of locked nucleic acid for therapeutic use. Further characterisations in vivo are ongoing.     

Rapid sampling of yeast cells and automated assays of adenylate, citrate, pyruvate and glucose-6-phosphate pools

A sampling technique for yeast cells with a very short interval between harvesting and inactivation of the cells is described. HClO₄ serves as inactivating agent. Optimal extraction is achieved by three freeze-thaw cycles of the acidic solution followed by neutralization with 2 m KHCO₃ to pH 7.5. Methods are presented for the automated fluorometric analyses of ATP, ADP, AMP, glucose-6-phosphate, pyruvate and citrate, as well as for the preparation of crude cell extracts.     

Rapid evaluation of peat-base legume inoculant using immunomagnetic beads for cell retrieval and fluorescent nucleic acid probes for viability analysis

Problems associated with traditional methods of evaluating legume inoculants using the most-probable-number plant nodule grow-out test include technician time, expense, growth chamber space, and the length of time (30 days) required to conduct the tests. Through the use of specific monoclonal antibodies, a biotin-labelled intermediate antibody, and streptavidin-labelled magnetic micro-beads, we were able to rapidly remove and purify rhizobial cells from peat inoculants used as quality control samples in the Canadian Legume Inoculant and Pre-Inoculated Seed Testing Program. Viability of the recovered cells was evaluated with a commercial kit employing differential fluorescent nucleic acid probes allowing viability to be rapidly determined via visual examination with a fluorescence microscope. Total elapsed time for complete evaluation of an inoculant was about 90 minutes.     

Buckyballs conjugated with nucleic acid sequences identifies microorganisms in live cell assays

Background

Rapid identification of bacteria can play an important role at the point of care, evaluating the health of the ecosystem, and discovering spatiotemporal distributions of a bacterial community. We introduce a method for rapid identification of bacteria in live cell assays based on cargo delivery of a nucleic acid sequence and demonstrate how a mixed culture can be differentiated using a simple microfluidic system.

Methods

C60 Buckyballs are functionalized with nucleic acid sequences and a fluorescent reporter to show that a diversity of microorganisms can be detected and identified in live cell assays. The nucleic acid complexes include an RNA detector, targeting a species-specific sequence in the 16S rRNA, and a complementary DNA with an attached fluorescent reporter. As a result, each bacterium can be detected and visualized at a specific emission frequency through fluorescence microscopy.

Results

The C60 probe complexes can detect and identify a diversity of microorganisms that include gram-position and negative bacteria, yeast, and fungi. More specifically, nucleic-acid probes are designed to identify mixed cultures of Bacillus subtilis and Streptococcus sanguinis, or Bacillus subtilis and Pseudomonas aeruginosa. The efficiency, cross talk, and accuracy for the C60 probe complexes are reported. Finally, to demonstrate that mixed cultures can be separated, a microfluidic system is designed that connects a single source-well to multiple sinks wells, where chemo-attractants are placed in the sink wells. The microfluidic system allows for differentiating a mixed culture.

Conclusions

The technology allows profiling of bacteria composition, at a very low cost, for field studies and point of care.

     

Bioluminescence regenerative cycle (BRC) system: theoretical considerations for nucleic acid quantification assays

A novel application of bioluminescence for nucleic acid quantification, the bioluminescence regenerative cycle (BRC), is described in theoretical terms and supported by preliminary experimental data. In the BRC system, pyrophosphate (PPi) molecules are released during biopolymerization and are counted and correlated to DNA copy number. The enzymes ATP-sulfurylase and firefly luciferase are employed to generate photons quantitatively from PPi. Enzymatic unity-gain positive feedback is implemented to amplify photon generation and to compensate for decay in light intensity by self-regulation. The cumulative total of photons can be orders of magnitude higher than in typical chemiluminescent processes. A system level theoretical model is developed, taking into account the kinetics of the regenerative cycle, contamination, and detector noise. Data and simulations show that the photon generation process achieves steady state for the time range of experimental measurements. Based on chain reaction theory, computations show that BRC is very sensitive to variations in the efficiencies of the chemical reactions involved and less sensitive to variations in the quantum yield of the process. We show that BRC can detect attomolar quantities of DNA (10(-18) mol), and that the useful dynamic range is five orders of magnitude. Sensitivity is not constrained by detector performance but rather by background bioluminescence caused by contamination by either PPi or ATP (adenosine triphosphate).