dsDNA Template Research Articles

DNA Amplification via Polymerase Chain Reaction Inside Miniemulsion Droplets with Subsequent Poly(n‐butylcyanoacrylate) Shell Formation and Delivery of Polymeric Capsules into Mammalian Cells

There is a growing interest in the development of stable nanocapsules that could deliver the bioactive compounds within the living organism, and to release them without causing any toxic effects. Here the miniemulsion droplets were first used as "nanoreactors" for the amplification of single-molecule dsDNA template (476 and 790 base pairs) through PCR. Afterwards, each droplet was surrounded with a biodegradable PBCA shell by interfacial anionic polymerization, enabling therefore to deliver the PCR products into the cells. The size of the initial miniemulsion droplets and the final polymeric capsules was in the range of 250 and 320 nm, mainly depending on the type of the continuous phase and presence of dsDNA template molecules. The formation of PCR products was resolved with gel electrophoresis and detected with fluorescence spectroscopy in the presence of DNA specific dye (SYBRGreen). TEM studies were performed to prove the formation of the polymeric shell. The shell thickness was measured to be within 5-15 nm and the average molecular weight of the formed PBCA polymer was around 75000 g · mol(-1) . For the cell uptake experiments, the obtained nanocapsules were transferred from the organic phase into aqueous medium containing a water-soluble surfactant. The effect of the surfactant type (anionic, cationic or non-ionic) on the HeLa cell viability and nanocapsule uptake behavior was studied by CLSM and FACS. Confocal analysis demonstrated that nanocapsules stabilized with cationic (CTMA-Cl) and non-ionic (Lutensol AT50) surfactants show almost the same uptake, whereas capsules redispersed in anionic (SDS) surfactant possess a 30% higher uptake. The release of the encapsulated material within the cell was studied on the example of Cy5-labeled oligonucleotides showing the colocalization with mitochondria of MSCs cells.

Site-Selective Binding of Nanoparticles to Double-Stranded DNA via Peptide Nucleic Acid “Invasion”

We demonstrate a novel method for by-design placement of nano-objects along double-stranded (ds) DNA. A molecular intercalator, designed as a peptide nucleic acid (PNA)-DNA chimera, is able to invade dsDNA at the PNA-side due to the hybridization specificity between PNA and one of the duplex strands. At the same time, the single-stranded (ss) DNA tail of the chimera, allows for anchoring of nano-objects that have been functionalized with complementary ssDNA. The developed method is applied for interparticle attachment and for the fabrication of particle clusters using a dsDNA template. This method significantly broadens the molecular toolbox for constructing nanoscale systems by including the most conventional not yet utilized DNA motif, double helix DNA.

Lambda Exonuclease Activity Measured Using Optical Tweezers with Active Force Clamp Control

Exonucleases are part of many genetic recombination and repair systems. We use high-resolution optical tweezers to study bacteriophage lambda exonuclease. We trap a dumbbell construct (bead-DNA-bead) in an inverted microscope by dividing a CW laser beam into a stationary trap and a steerable trap. The inter-trap distance is actively controlled with 200 kHz update rate by acousto-optical deflectors (AOD) and field programmable gate array -card. In addition, we control the sample temperature by flowing heating fluid through aluminum jacket around the trapping objective. We follow the exonuclease activity through the length change of a 48kb dsDNA template to ssDNA-form. The dsDNA tether is attached from one strand to two streptavidin coated beads, leaving one 5’ phosphorylated end free for exonuclease to start catalytic removal of mononucleotides. At low forces the length of the ssDNA is significantly shorter than the dsDNA. The template is held in a dumbbell construct at constant force with active feedback control. This allows us to follow the enzymatic activity of the lambda exonuclease over ten micrometers while simultaneously applying a constant load between 0 and 50 pN. We apply different loads on the DNA template to measure how the template tension affects the activity of the lambda exonuclease. We also measure the activity under different ambient temperatures.

TEFM (c17orf42) is necessary for transcription of human mtDNA

Here we show that c17orf42, hereafter TEFM (transcription elongation factor of mitochondria), makes a critical contribution to mitochondrial transcription. Inactivation of TEFM in cells by RNA interference results in respiratory incompetence owing to decreased levels of H- and L-strand promoter-distal mitochondrial transcripts. Affinity purification of TEFM from human mitochondria yielded a complex comprising mitochondrial transcripts, mitochondrial RNA polymerase (POLRMT), pentatricopeptide repeat domain 3 protein (PTCD3), and a putative DEAD-box RNA helicase, DHX30. After RNase treatment only POLRMT remained associated with TEFM, and in human cultured cells TEFM formed foci coincident with newly synthesized mitochondrial RNA. Based on deletion mutants, TEFM interacts with the catalytic region of POLRMT, and in vitro TEFM enhanced POLRMT processivity on ss- and dsDNA templates. TEFM contains two HhH motifs and a Ribonuclease H fold, similar to the nuclear transcription elongation regulator Spt6. These findings lead us to propose that TEFM is a mitochondrial transcription elongation factor.

Aptamer-Based Sensitive Detection of Target Molecules via RT-PCR Signal Amplification

In the efforts to explore an aptamer-based approach for target sensing and detection with higher sensitivity and specificity, instead of directly labeling aptamer with fluorophores, we proposed a new strategy by attaching a polymerase chain reaction (PCR) template to an oligonucleotide aptamer selected by systematic evolution of ligands by exponential enrichment (SELEX), so that after aptamer target binding, the template moiety serves as the PCR template in real-time quantitative PCR (RT-PCR), and therefore, the binding event can be reported by the following RT-PCR signals. Using the subtractive SELEX method, the oligonucleotide aptamers specific for the Fc fragment of mouse IgG were selected and subjected to coupling with the PCR dsDNA template by using overlap and the asymmetric extension PCR method. The target binding affinity of the PCR template tethered aptamer has been proven by electrophoretic mobility shift assay (EMSA), and further template tethered aptamer mediated real-time quantitative PCR (A-PCR) was conducted to validate the application for such a template tethered aptamer to be a sensitive probe for IgG detection. The results show that the protocols of A-PCR can detect 10-fold serial dilutions of the target, demonstrating a new mechanism to convert aptamer target binding events to amplified RT-PCR signal, and the feasibility of the PCR template tethered aptamer as a facile, specific, and sensitive target probing and detection is established. This new approach also has potential applications in multiple parallel target detection and analysis in a wide range of research fields.

The DNA/RNA-Dependent RNA Polymerase QDE-1 Generates Aberrant RNA and dsRNA for RNAi in a Process Requiring Replication Protein A and a DNA Helicase

Author SummarySmall RNA molecules (20–30 nucleotides) play important roles in many cellular processes in eukaryotic organisms by silencing gene expression. To generate the many forms of small RNAs, DNA is first transcribed to produce single-stranded RNA (ssRNA), which then is converted to double-stranded RNA (dsRNA) by an RNA-dependent RNA polymerase (RdRP). However, it is not clear how the ssRNA templates are synthesized from DNA and specifically recognized by RdRPs amidst a sea of single-stranded, cellular RNAs. We previously showed that in the filamentous fungus Neurospora the production of one type of small RNA called qiRNA, which is specifically induced after DNA damage, requires the RdRP QDE-1. Here, we investigated the precise contributions of QDE-1 to the synthesis of ssRNA and dsRNA. We show that QDE-1 is surprisingly promiscuous in its template choice in that it is able to synthesize RNA from both ssRNA and single-stranded DNA (ssDNA). These results suggest that QDE-1 first generates ssRNA from a DNA template and then converts the ssRNA into dsRNA; this combination of activities in one protein ensures the specific action by RdRP on aberrant RNA in lieu of other single-stranded cellular RNA. In addition, we identified Replication Protein A, a ssDNA-binding protein that interacts with QDE-1, as an essential factor for small RNA production. Furthermore, we were able to reconstitute synthesis of dsRNA from ssDNA in a test tube using purified QDE-1 and RPA proteins, demonstrating the ability of this relatively simple biosynthetic system to generate the nucleic acid trigger for gene regulation. Together, these results uncover the details of a new and important small RNA production mechanism in cells.

Novel DNA Binding Properties of the Mcm10 Protein from Saccharomyces cerevisiae

The Mcm10 protein is essential for chromosomal DNA replication in eukaryotic cells. We purified the Saccharomyces cerevisiae Mcm10 (ScMcm10) and characterized its DNA binding properties. Electrophoretic mobility shift assays and surface plasmon resonance analysis showed that ScMcm10 binds stably to both double strand (ds) DNA and single strand (ss) DNA. On short DNA templates of 25 or 50 bp, surface plasmon resonance analysis showed a approximately 1:1 stoichiometry of ScMcm10 to dsDNA. On longer dsDNA templates, however, multiple copies of ScMcm10 cooperated in the rapid assembly of a large, stable nucleoprotein complex. The amount of protein bound was directly proportional to the length of the DNA, with an average occupancy spacing of 21-24 bp. This tight spacing is consistent with a nucleoprotein structure in which ScMcm10 is aligned along the helical axis of the dsDNA. In contrast, the stoichiometry of ScMcm10 bound to ssDNA of 20-50 nucleotides was approximately 3:1 suggesting that interaction with ssDNA induces the assembly of a multisubunit ScMcm10 complex composed of at least three subunits. The tight packing of ScMcm10 on dsDNA and the assembly of a multisubunit complex on ssDNA suggests that, in addition to protein-DNA, protein-protein interactions may be involved in forming the nucleoprotein complex. We propose that these DNA binding properties have an important role in (i) initiation of DNA replication and (ii) formation and maintenance of a stable replication fork during the elongation phase of chromosomal DNA replication.

A comprehensive assay for targeted multiplex amplification of human DNA sequences

We developed a robust and reproducible methodology to amplify human sequences in parallel for use in downstream multiplexed sequence analyses. We call the methodology SMART (Spacer Multiplex Amplification Reaction), and it is based, in part, on padlock probe technology. As a proof of principle, we used SMART technology to simultaneously amplify 485 human exons ranging from 100 to 500 bp from human genomic DNA. In multiple repetitions, >90% of the targets were successfully amplified with a high degree of uniformity, with 70% of targets falling within a 10-fold range and all products falling within a 100-fold range of each other in abundance. We used long padlock probes (LPPs) >300 bases in length for the assay, and the increased length of these probes allowed for the capture of human sequences up to 500 bp in length, which is optimal for capturing most human exons. To engineer the LPPs, we developed a method that generates ssDNA molecules with precise ends, using an appropriately designed dsDNA template. The template has appropriate restriction sites engineered into it that can be digested to generate nucleotide overhangs that are suitable for lambda exonuclease digestion, producing a single-stranded probe from dsDNA. The SMART technology is flexible and can be easily adapted to multiplex tens of thousands of target sequences in a single reaction.

Expedient placement of two fluorescent dyes for investigating dynamic DNA protein interactions in real time.

Many questions in molecular and cellular biology can be reduced to questions about 'who talks to whom, when and how frequently'. Here, we review approaches we have used with single-pair fluorescence resonance energy transfer (spFRET) to follow the motions between two well-placed fluorescent probes to ask similar questions. We describe two systems. We have used a nucleosomal system in which the naked DNA molecule has the acceptor and donor dyes too far apart for FRET to occur whereas the dyes are close enough in the reconstituted nucleosome for FRET. As these individual nucleosomes were tethered on a surface, we could follow dynamics in the repositioning of these two dyes, inferring that nucleosomes stochastically and reversibly open and close. These results imply that most of the DNA on the nucleosome can be sporadically accessible to regulatory proteins and proteins that track the DNA double helix. In the case of following the binding of recombination protein RecA to double-stranded DNA (dsDNA) and the RecA filament displacement by DNA helicase motor PcrA, the dsDNA template is prepared with the two dyes close enough to each other to generate high FRET. Binding of the RecA molecules to form a filament lengthens the dsDNA molecule 1.5-fold and reduces the FRET accordingly. Once added, DNA motor protein helicase PcrA can displace the RecA filament with concomitant return of the DNA molecule to its original B-form and high FRET state. Thus, appropriately placed fluorescent dyes can be used to monitor conformational changes occurring in DNA and or proteins and provide increased sensitivity for investigating dynamic DNA-protein interactions in real time.

DNA-templated preparation of palladium nanoparticles and their application

Abstract A versatile method, based on the metallization of DNA strands, is developed to prepare palladium nanoparticles on indium tin oxide (ITO) coated glass surface. Resulted palladium nanoparticles are distributed along the DNA strands and their size is controllable in the range of 10–100 nm. This is contrast to the branched palladium aggregates formed on bare ITO surface. When used as an electrochemical sensor, the nanoparticles show strong catalytic activity for the reduction of hydrogen peroxide and the oxidation of ascorbic acid, respectively. Higher catalytic activity is observed with the nanoparticles formed on double-stranded DNA (dsDNA) templates than those on single-stranded DNA (ssDNA) templates.

Controlled DNA-Templated Metal Deposition: Towards Ultra-Thin Nanowires

In this paper, we report the metallization of a dsDNA template using a novel photography-derived two-step strategy in which dsDNA is first complexed with Ag(I) ions and then irradiated with UV light at 254 nm. The nucleobases act as light harvesters and sensitizers, triggering the photoreduction of the complexed silver ions. This process yields a silver nanoparticles blueprint along the DNA strand. The silver latent image is then developed by depositing metallic nickel through an electroless plating process. This photography-derived procedure generates very homogeneous and evenly distributed strings of silver-core/nickel-shell nanoparticles. Although still discontinuous, we believe that such chains can serve as the base for obtaining continuous metal nanowires. Furthermore, this process can most likely be extended to other plating metals, resulting in a broadly general procedure for metallizing DNA with a variety of different materials. Because of the intrinsic simplicity in using light as the key step, this methodology might be amenable to large-scale development, eventually leading to a very efficient molecular-photolithography process.

Inhibition of Human Carcinoma and Neuroblastoma Cell Proliferation by Anti-c-myc siRNA

Suppression of c-myc proto-oncogene expression by small interfering RNA (siRNA) in human epidermoid carcinoma KB-3-1 and neuroblastoma SK-N-MC cell lines was investigated. The siRNA duplex targeted to the exon 3 of c-myc mRNA (siRNA-I) was prepared by in vitro transcription using T7 RNA polymerase and short double-stranded DNA (dsDNA) templates. siRNA-I was shown to efficiently decrease c-myc mRNA expression in both tumor cell lines and to arrest their proliferation. Incubation of KB-3-1 cells with 150 nM siRNA-I results in a 92% decrease in the c-myc mRNA level and an 83% decrease in the protein level. In SK-N-MC cells, 150 nM siRNA-I causes a 60% decrease in the c-myc mRNA level and a 55% decrease in the protein level. The reduction of the c-myc mRNA level correlates with the inhibition of cell proliferation; 150 nM siRNA-I causes a 2.5-fold reduction in the SK-N-MC proliferation rate and a 15-fold decrease in the proliferation rate and complete arrest of cell division in KB-3-1 cells. siRNA-I has little effect on proliferation of the IMR-32 cells that overexpress the N-myc but not the c-myc gene, demonstrating that siRNA-I antiproliferation activity is mediated by specific block of c-myc expression.

Site-directed Mutations of T4 Helicase Loading Protein (gp59) Reveal Multiple Modes of DNA Polymerase Inhibition and the Mechanism of Unlocking by gp41 Helicase

The T4 helicase loading protein (gp59) interacts with a multitude of DNA replication proteins. In an effort to determine the functional consequences of these protein-protein interactions, point mutations were introduced into the gp59 protein. Mutations were chosen based on the available crystal structure and focused on hydrophobic residues with a high degree of solvent accessibility. Characterization of the mutant proteins revealed a single mutation, Y122A, which is defective in polymerase binding and has weakened affinity for the helicase. The interaction between single-stranded DNA-binding protein and Y122A is unaffected, as is the affinity of Y122A for DNA substrates. When standard concentrations of helicase are employed, Y122A is unable to productively load the helicase onto forked DNA substrates. As a result of the loss of polymerase binding, Y122A cannot inhibit the polymerase during nucleotide idling or prevent it from removing the primer strand of a D-loop. However, Y122A is capable of inhibiting strand displacement synthesis by polymerase. The retention of strand displacement inhibition by Y122A, even in the absence of a gp59-polymerase interaction, indicates that there are two modes of polymerase inhibition by gp59. Inhibition of the polymerase activity only requires gp59 to bind to the replication fork, whereas inhibition of the exonuclease activity requires an interaction between the polymerase and gp59. The inability of Y122A to interact with both the polymerase and the helicase suggests a mechanism for polymerase unlocking by the helicase based on a direct competition between the helicase and polymerase for an overlapping binding site on gp59.

Gene Correction in Human Cells Is Promoted by the Bacteriophage Protein Redβ.

Correction of a disease gene mutation by homologous recombination (HR) with a transfected DNA repair template represents an ideal form of gene therapy for many inherited hematological disorders. Therapeutic gene correction via HR in hematopoietic stem cells is unlikely to succeed, however, without major improvements in its efficiency. One promising approach is to use an engineered zinc finger endonuclease to specifically cleave the target gene and so promote HR between a dsDNA template and its chromosomal target (Urnov et al (2005) Nature 435:646-51). We are exploring an alternative approach based on the expression of Redβ, a single stranded (ss) DNA annealing protein from bacteriophage λ. In bacteria, Redβ promotes HR-mediated gene modification using templates with short regions of homology (e.g. 30–100nt). Such HR is independent of RecA-mediated host cell HR. For double stranded (ds) DNA templates, co-expression of λ exonuclease (the red α gene product) is required, but ssDNA templated modifications are particularly efficient and require only Redβ expression. Such properties could be useful for therapeutic gene correction if reproduced in human cells. Preliminary experiments in mouse embryonic stem cells suggested that Redβ can function in mammalian cells (Zhang Y. et al, (2003) BMC Mol. Biol. 4). To explore this further, we have generated a human cell line in which large amounts of Redβ can be expressed inducibly in the nucleus. In contrast to the over-expression of endogenous HR proteins Rad51 (RecA homologue) or Rad52 (ssDNA annealing protein), sustained Redβ over-expression is well tolerated, with no impairment of cell proliferation or viability. We find that Redβ does indeed promote HR in human cells. Surprisingly, dsDNA and ssDNA templated gene modification are stimulated to similar extents by Redβ. Stimulations are modest, but similar to those achieved by human Rad51 over-expression. The effects of co-expressing Red α are under investigation. We have also shown that ssDNA-templated gene correction is stimulated by transient, RNAi-mediated, depletion of the mismatch repair protein MSH2, and that such stimulation is additive with Redβ-stimulated gene correction.

DNA Elongation by the Human DNA Polymerase λ Polymerase and Terminal Transferase Activities Are Differentially Coordinated by Proliferating Cell Nuclear Antigen and Replication Protein A

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9-12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol lambda.

Electrical contacting of glucose oxidase by DNA-templated polyaniline wires on surfaces

Poly(aniline–aniline boronic acid) wires are generated on ds-DNA templates, and the resulting wires exhibit redox functions at neutral pH aqueous solutions. The association of flavin adenine dinucleotide (FAD) to the boronic acid ligand followed by the reconstitution of apo-glucose oxidase on the cofactor units yield an integrated enzyme-electrode where the biocatalyst reveals direct electrical contact with the electrode.

RNA Amplification Strategies for cDNA Microarray Experiments

The biological materials available for cDNA microarray studies are often limiting. Thus, protocols have been developed to amplify RNAs isolated from limited amounts of tissues or cells. RNA amplification by in vitro transcription is the most widely used among the available amplification protocols. Two means of generating a dsDNA template for the RNA polymerase are a combination of reverse transcription with conventional second-strand cDNA synthesis and a combination of the switch mechanism at the 5' end of RNA templates (SMART) with reverse transcription, followed by PCR. To date, there has been no systematic comparison of the efficiency of the two amplification strategies. In this study, we performed and analyzed a set of six microarray experiments involving the use of a "regular" (unamplified) microarray experimental protocol and two different RNA amplification protocols. Based on their ability to identify differentially expressed genes and assuming that the results from the regular protocol are correct, our analyses demonstrated that both amplification protocols achieved reproducible and reliable results. From the same amount of starting material, our results also indicated that more amplified RNA can be obtained using conventional second-strand cDNA synthesis than from the combination of SMART and PCR. When the critical issue is the amount of starting RNA, we recommend the conventional second-strand cDNA synthesis as the preferred amplification method.

Assay of Intrinsic Transcript Termination by E. Coli RNA Polymerase on Single-Stranded and Double-Stranded DNA Templates

This chapter describes the principal techniques used for biochemical characterization of transcript termination. The chapter focuses on intrinsic termination by Escherichia coli (E. coli) RNA polymerase on double-stranded (dsDNA) and single-stranded (ssDNA) DNA templates. These methods can be easily adapted to study transcript termination of other RNA polymerases and factor-dependent termination, or extended to study aspects of transcript elongation. To study intrinsic termination of E. coli RNA polymerase, 5ʹ biotinylated linear dsDNA templates generated by polymerase chain reaction (PCR) using one 5ʹ biotinylated oligonucleotide and one unmodified oligonucleotide. The template contains a promoter and an intrinsic termination site. Two solid-phase transcription methods are developed to prepare transcript elongation complexes halted at a distinct position along the DNA template. Use of biotinylated templates provides an attractive alternative to using T4 polynucleotide kinase and [γ 32 P] ATP to radiolabel one of two oligonucleotides required to amplify a linear DNA template by PCR.

Rapid preparation of denatured double-stranded DNA templates for sequencing.

This article proposes protocol for rapid preparation (ds) DNA templates for sequencing based on double-stranded DNA denaturation and its recovery by extraction with Wizard DNA purification resin (Promega). This method is an alternative to commonly used procedure employing denatured-DNA recovery by ethanol precipitation.

Genomic DNA sequencing by SPEL-6 primer walking using hexamer ligation1Published in conjunction with A Wisconsin Gathering Honoring Waclaw Szybalski on the occasion of his 75th year and 20years of Editorship-in-Chief of Gene, 10–11 August 1997, University of Wisconsin, Madison, WI, USA.1

DNA sequencing by SPEL-6 (Sequential Primer Elongation by Ligation of 6-mers) primer walking is based on the rapid assembly of true primers by ligation of several (three to 10) contiguous hexamers complementary to a DNA template saturated with Escherichia coli single-stranded DNA-binding protein. To prove the usefulness and to check the reliability of this method, a 3-kb DNA fragment carrying the genes encoding the EcoVIII restriction-modification (RM) system was sequenced with low redundancy (2.8). The use of both single-stranded (ss) and double-stranded (ds) DNA templates was compared. For this project, 27 primers were assembled by hexamer ligation to form 18-30-nt strings of three to five hexamers. Each primer was designed based on nucleotide sequence determined in a previous run, and was produced in a matter of minutes. The overall length of the easily readable sequencing ladders was about 300-450nt. We found that strong secondary structures in the ss DNA tend to interfere with its template function for the primer assembly by hexamer ligation, especially when they overlap the 3'-end of such a primer. This was easily overcome either by avoiding such hairpin regions or by using longer strings of hexamers, since we show that their ligation is highly cooperative, and ligation efficiency increases with the length of the string (). Some general rules for successful primer assembly and prospects for using the SPEL-6 method for large-scale, fully automated fluorescent sequencing of large genomes are discussed.