Simultaneously monitoring DNA binding and helicase-catalyzed DNA unwinding by fluorescence polarization

A new method for helicase-catalyzed DNA unwinding is described. This assay takes advantage of the substantial change in fluorescence polarization (FP) upon helicase binding and DNA unwinding. The low anisotropy value, due to the fast tumbling of the free oligonucleotide in solution, increases abruptly upon binding of helicase to the fluorescein-labeled oligonucleotide. The high anisotropy of the helicase– DNA complex decreases as the fluorescein-labeled oligonucleotide is released from the complex through helicase-catalyzed DNA unwinding. This FP signal can be measured in real time by fluorescent spectroscopy. This assay can simultaneously monitor DNA binding and helicase-catalyzed DNA unwinding. It can also be used to determine the polarity in DNA unwinding mediated by helicase. This FP assay should facilitate the study of the mechanism by which helicase unwinds duplex DNA, and also aid in screening for helicase inhibitors, which are of growing interest as potential anticancer agents.     

Measuring single-molecule DNA hybridization by active control of DNA in a nanopore

We present a novel application of active voltage control of DNA captured in a nanopore to regulate the amount of time the DNA is available to molecules in the bulk phase that bind to the DNA. In this work, the control method is used to measure hybridization between a single molecule of DNA captured in a nanopore and complementary oligonucleotides in the bulk phase. We examine the effect of oligonucleotide length on hybridization, and the effect of DNA length heterogeneity on the measurements. Using a mathematical model, we are able to deduce the binding rate of complementary oligonucleotides, even when DNA samples in experiments are affected by heterogeneity in length. We analyze the lifetime distribution of DNA duplexes that are formed in the bulk phase and then pulled against the pore by reversing the voltage. The lifetime distribution reveals several dissociation modes. It remains to be resolved whether these dissociation modes are due to DNA heterogeneity or correspond to different states of duplex DNA. The control method is unique in its ability to detect single-molecule complex assembly in the bulk phase, free from external force and with a broad (millisecond-to-second) temporal range.     

Thermolytic release of covalently linked DNA oligonucleotides and their conjugates from controlled-pore glass at near neutral pH

The functionalization of long chain alkylamine controlled-pore glass (CPG) with a 3-hydroxypropyl-(2-cyanoethyl)thiophosphoryl linker and its conversion to the support 7 has led to the synthesis of DNA oligonucleotides and their 3'- or (3',5')-conjugates. Indeed, CPG support 7 has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20-mers. Unlike conventional succinylated CPG supports, this distinctively functionalized support allows oligonucleotide deprotection and removal of the deprotection side products to proceed without releasing the oligonucleotide into the aqueous milieu. When freed from deprotection side products, the DNA oligonucleotide is thermolytically released from the support within 2 h under nearly neutral conditions (pH 7.2, 90 degrees C). The quality of these oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated CPG supports in terms of shorter than full length oligonucleotide contaminants and overall yields. The versatility of the thermolytic CPG support 7 is further demonstrated by the synthesis of a DNA oligonucleotide (20-mer) and its conjugation with an azido and alkynyl groups at both 5'-and 3'-termini, respectively. The functionality of the (3',5')-heteroconjugated oligonucleotide 18 is verified by its circularization to the DNA oligonucleotide 19 under "click" chemistry conditions.     

Probing distance and electrical potential within a protein pore with tethered DNA

DNA molecules tethered inside a protein pore can be used as a tool to probe distance and electrical potential. The approach and its limitations were tested with alpha-hemolysin, a pore of known structure. A single oligonucleotide was attached to an engineered cysteine to allow the binding of complementary DNA strands inside the wide internal cavity of the extramembranous domain of the pore. The reversible binding of individual oligonucleotides produced transient current blockades in single channel current recordings. To probe the internal structure of the pore, oligonucleotides with 5' overhangs of deoxyadenosines and deoxythymidines up to nine bases in length were used. The characteristics of the blockades produced by the oligonucleotides indicated that single-stranded overhangs of increasing length first approach and then thread into the transmembrane beta-barrel. The distance from the point at which the DNA was attached and the internal entrance to the barrel is 43 A, consistent with the lengths of the DNA probes and the signals produced by them. In addition, the tethered DNAs were used to probe the electrical potential within the protein pore. Binding events of oligonucleotides with an overhang of five bases or more, which threaded into the beta-barrel, exhibited shorter residence times at higher applied potentials. This finding is consistent with the idea that the main potential drop is across the alpha-hemolysin transmembrane beta-barrel, rather than the entire length of the lumen of the pore. It therefore explains why the kinetics and thermodynamics of formation of short duplexes within the extramembranous cavity of the pore are similar to those measured in solution, and bolsters the idea that a "DNA nanopore" provides a useful means for examining duplex formation at the single molecule level.     

Stimulation of RecA-mediated D-loop formation by oligonucleotide-directed triple-helix formation: guided homologous recombination (GOREC)

Oligonucleotide-directed triple helix formation provides an elegant rational basis for gene-specific DNA targeting and has been widely used to interfere with gene expression ("antigene" strategies) and as a molecular tool for biological studies. Various strategies have been developed to introduce sequence modifications in genomes. However, the low efficiency of the overall process in eucaryotic cells impairs efficient recovery of recombinant genomes. Since one limiting step in homologous recombination is the targeting to the homologous sequence, we have tested the contribution of an oligonucleotide-directed triple helix formation on the RecA-dependent association of an oligonucleotide and its homologous target on duplex DNA (D-loop formation). For this study, the recombinant ssDNA fragment was noncovalently associated to a triple helix-forming oligonucleotide. The physicochemical and biochemical characteristics of the triple helix and D-loop structures formed by the complex molecules in the presence or in the absence of RecA protein were determined. We have demonstrated that the triple helix-forming oligonucleotide increases the efficiency of D-loop formation and the RecA protein speeds up also the triple helix formation. The so-called "GOREC" (for guided homologous recombination) approach can be developed as a novel tool to improve the efficiency of directed mutagenesis and gene alteration in living organisms.     

DNA purification by triple-helix affinity precipitation

Recent advances in DNA-based medicine (gene therapy, genetic vaccination) have intensified the necessity for pharmaceutical-grade plasmid DNA purification at comparatively large scales. In this contribution triple-helix affinity precipitation is introduced for this purpose. A short, single-stranded oligonucleotide sequence (namely (CTT)(7)), which is capable of recognizing a complementary sequence in the double-stranded target (plasmid) DNA, is linked to a thermoresponsive N-isopropylacrylamide oligomer to form a so-called affinity macroligand (AML). At 4 degrees C, i.e., below its critical solution temperature, the AML binds specifically to the target molecule in solution; by raising the temperature to 40 degrees C, i.e., beyond the critical solution temperature of the AML, the complex can be precipitated quantitatively. After redissolution of the complex at lower temperature, the target DNA can be released by a pH shift to slightly alkaline conditions (pH 9.0). Yields of highly pure (plasmid) DNA were routinely between 70% and 90%. Non-specific co- precipitation of either the target molecule by the non-activated AML precursor or of contaminants by the AML were below 7% and presumably due to physical entrapment of these molecules in the wet precipitate. Ligand efficiencies were at least 1 order of magnitude higher than in triple-helix affinity chromatography.     

HIV-1 integrase inhibition by modified oligonucleotides: optimization of the inhibitor structure

Integration of human immunodeficiency virus type 1 DNA into the infected cell genome is one of the key steps of the viral replication cycle. Therefore viral enzyme integrase, which realizes the integration, is of interest as a target for new antiviral drugs. Conjugates of 11-mer single stranded oligonucleotides with hydrophobic molecules are shown to be efficient integrase inhibitors since they induce dissociation of the integrase-viral DNA complex. The effect of the oligonucleotide length and structure as well as the structure of hydrophobic molecules on the conjugate inhibitory activity has been studied. Conjugates with eosin and oleic acid are shown to be the most active. Conjugates of these molecules with 2'-O-methyl-oligonucleotide inhibit integrase at 50-100 nM and have no influence on a number of other DNA-binding enzymes.     

Sequencing of production-scale synthetic oligonucleotides by enriching for coupling failures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A technique for sequencing oligonucleotides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is described. The series of coupling failure species are extracted from the dimethoxytrityl-on, full-length oligonucleotide in crude synthetic material using C18 stationary-phase cartridges. These concentrated failure species can be easily detected by MALDI-TOF, which determines the mass difference between spectral ions to identify a particular base. The solid-phase extraction step greatly enhances ion signals and mass resolution, and sequencing information is generally obtained from the 5' end up to the first three to four nucleotides at the 3' end. Complete sequence can be generated in conjunction with snake venom phosphodiesterase digestion of purified material. This method eliminates difficulties associated with other mass spectrometric sequencing techniques involving oligonucleotide length; structure; and sugar, base, and backbone modifications. Examples of sequencing a 17-mer composed primarily of 2'-O-methylribonucleotides and a single nonnucleosidic linker and a mixed sugar backbone 51-mer with 2'-O-methylribonucleotides and a homopolymer tail are reported in this study.     

Characterization of degradable polyelectrolyte multilayers fabricated using DNA and a fluorescently-labeled poly(β-amino ester): shedding light on the role of the cationic polymer in promoting surface-mediated gene delivery

Polyelectrolyte multilayers (PEMs) fabricated from cationic polymers and DNA have been investigated broadly as materials for surface-mediated DNA delivery. One attractive aspect of this "multilayered" approach is the potential to exploit the presence of cationic polymer "layers" in these films to deliver DNA to cells more effectively. Past studies demonstrate that these films can promote transgene expression in vitro and in vivo, but significant questions remain regarding roles that the cationic polymers could play in promoting the internalization and processing of DNA. Here, we report physicochemical and in vitro cell-based characterization of DNA-containing PEMs fabricated using fluorescently end-labeled derivatives of a degradable polycation (polymer 1) used in past studies of surface-mediated transfection. This approach permitted simultaneous characterization of polymer and DNA in solution and in cells using fluorescence-based techniques, and provided information about the locations and behaviors of polymer 1 that could not be obtained using other methods. LSCM and flow cytometry experiments revealed that polymer 1 and DNA released from film-coated objects were both internalized extensively by cells and that they were colocalized to a significant extent inside cells (e.g., ~58% of DNA was colocalized with polymer). Fluorescence anisotropy measurements of solutions containing partially eroded films were also consistent with the presence of aggregates of polymer 1 and DNA in solution (e.g., after release from surfaces, but prior to internalization by cells). Our results support the view that polymer 1, which is incorporated into these materials as "layers" rather than as part of optimized, preformed "polyplexes", can act to promote or enhance surface-mediated DNA delivery. More broadly, our results suggest opportunities to improve the delivery properties of DNA-containing PEMs by incorporation of additional "layers" of other conventional cationic polymers designed to address specific intracellular barriers to transfection, such as endosomal escape, more effectively.     

Gel purification of radiolabeled nucleic acids via phosphorimaging: Dip-N-Dot

RNA and DNA oligonucleotides radiolabeled with ³²P or ³³P often require gel electrophoresis to remove undesired side and/or degradation products. Common ways to visualize these molecules after electrophoresis are by ultraviolet (UV) shadowing, which necessarily reduces the specific activity of the oligonucleotide, and by autoradiography using film, which is cumbersome and increases the cost of generating the radiolabeled molecule. A more cost-effective method is to physically inject the gel with a “Dip-N-Dot” solution of dye and radionuclide after electrophoresis but prior to phosphorimaging. The gel can be overlaid on its computer-generated image, allowing the labeled molecules to be visualized quickly.     

Solid phase capturable dideoxynucleotides for multiplex genotyping using mass spectrometry

We report an approach using solid phase capturable biotinylated dideoxynucleotides (biotin-ddNTPs) in single base extension for multiplex genotyping by mass spectrometry (MS). In this method, oligonucleotide primers that have different molecular weights and that are specific to the polymorphic sites in the DNA template are extended with biotin-ddNTPs by DNA polymerase to generate 3′-biotinylated DNA products. These products are then captured by streptavidin-coated solid phase magnetic beads, while the unextended primers and other components in the reaction are washed away. The pure extension DNA products are subsequently released from the solid phase and analyzed by matrix-assisted laser desorption/ionization time-of-flight MS. The mass of the extension products is determined using a stable oligonucleotide as a common internal mass standard. Since only the pure extension DNA products are introduced to the MS for analysis, the resulting mass spectrum is free of non-extended primer peaks and their associated dimers, which increases the accuracy and scope of multiplexing in single nucleotide polymorphism (SNP) analysis. The solid phase purification approach also facilitates desalting of the captured oligonucleotides, which is essential for accurate mass measurement by MS. We selected four biotin-ddNTPs with distinct molecular weights to generate extension products that have a 2-fold increase in mass difference compared to that with conventional ddNTPs. This increase in mass difference provides improved resolution and accuracy in detecting heterozygotes in the mass spectrum. Using this method, we simultaneously distinguished six nucleotide variations on synthetic DNA templates mimicking mutations in the p53 gene and two disease-associated SNPs in the human hereditary hemochromatosis gene.     

Affinity thermoprecipitation and recovery of biotinylated biomolecules via a mutant streptavidin-smart polymer conjugate

A system has been developed for reversibly binding and thermoprecipitating biotinylated macromolecules. A high off-rate Ser45Ala (S45A) streptavidin mutant has been covalently conjugated to poly(N-isopropylacrylamide) (PNIPAAm), a temperature-responsive polymer. The resulting conjugate is shown to coprecipitate biotinylated immunoglobulin G (IgG) and a biotinylated oligonucleotide in response to a thermal stimulus. Thermally precipitated biotinylated macromolecules can be released from the S45A-PNIPAAm conjugate by simple treatment with excess free biotin. This release step has been shown to be unique to the mutant streptavidin conjugate-a conjugate of wild type (WT) streptavidin and PNIPAAm does not release bound biotinylated molecules upon treatment with excess free biotin. The capture efficiency (fraction of target molecule precipitated from solution) of the S45A-PNIPAAm conjugate is similar to that of the WT-PNIPAAm conjugate for the biotinylated IgG target molecule (near 100%), but significantly smaller for the biotinylated oligonucleotide target (approximately 60% for the S45A-PNIPAAm conjugate compared to 80% for the WT-PNIPAAm conjugate). The release efficiency (fraction of originally precipitated target molecule released after treatment with free biotin) of the S45A-PNIPAAm conjugate is 70-80% for the biotinylated IgG target and nears 100% for the biotinylated oligonucleotide target. This system demonstrates the use of a high off-rate streptavidin mutant to add reversibility to a system based on smart-polymer-streptavidin conjugates.     

Magnetic DNA affinity purification of ecdysteroid receptor.

A new method for rapid purification to near homogeneity of the ecdysteroid receptor (EcdR) from Drosophila melanogaster nuclear extract is presented. In the first step of the purification procedure the EcdR molecules were radiolabelled with [3H]ponasterone A and the [3H]ponasterone A-EcdR complexes were chromatographed under very mild conditions on Fractogel EMD TMAE(s) ion-exchanger. A 23-fold purified receptor was obtained which can be stored in liquid N2 without loss of activity. The second step involved the use of a magnetic DNA affinity technique where the double stranded hsp 27 oligonucleotide containing EcdR binding sequence was biotin 5'-end labelled and bound to monodisperse superparamagnetic particles coated with streptavidin (Dynabeads M-280 Streptavidin) giving magnetic DNA affinity beads. The chromatographed EcdR-ponasterone A complexes were bound to the magnetic DNA affinity beads and by magnetic separation, wash and elution, a 29,000-fold enriched EcdR preparation was obtained within 1.5 h. This procedure can be applied for other EcdR sources with minor modifications.     

In vitro selection of molecular beacons

While molecular beacons are primarily known as biosensors for the detection of nucleic acids, it has proven possible to adapt other nucleic acid binding species (aptamers) to function in a manner similar to molecular beacons, yielding fluorescent signals only in the presence of a cognate ligand. Unfortunately, engineering aptamer beacons requires a detailed knowledge of aptamer sequence and structure. In order to develop a general method for the direct selection of aptamer beacons we have first developed a selection method for molecular beacons. A pool of random sequence DNA molecules were immobilized via a capture oligonucleotide on an affinity column, and those variants that could be released from the column by a target oligonucleotide were amplified. After nine rounds of selection and amplification the elution characteristics of the population were greatly improved. A fluorescent reporter in the selected beacons was located adjacent to a DABCYL moiety in the capture oligonucleotide; addition of the target oligonucleotide led to release of the capture oligonucleotide and up to a 17-fold increase in fluorescence. Signaling was specific for the target oligonucleotide, and occurred via a novel mechanism, relative to designed molecular beacons. When the target oligonucleotide is bound it can form a stacked helical junction with an intramolecular hairpin in the selected beacon; formation of the intramolecular hairpin in turn leads to release of the capture oligonucleotide. The ability to select molecular beacons may prove useful for identifying available sites on complex targets, such as mRNAs, while the method for selection can be easily generalized to other, non-nucleic acid target classes.     

Ring closure probabilities for DNA fragments by Monte Carlo simulation

The rate of ligation of DNA molecules into circular forms depends on the ring closure probability, commonly called the j-factor, which is a sensitive measure of the extent to which thermal fluctuations contribute to bending and twisting of DNA molecules in solution. We present a theoretical treatment of the cyclization equilibria of DNA that employs a special Monte Carlo method for generating large ensembles of model DNA chains. Using this method, the chain length dependence of the j-factor was calculated for molecules. in the size range 250 to 2000 base-pairs. The Monte Carlo results are compared with recent analytical theory and experimental data. We show that a value of 475 A for the persistence length of DNA, close to values measured by a number of other methods, is in excellent agreement with the cyclization results. Preliminary applications of the Monte Carlo method to the problem of systematically bent DNA molecules are presented. The calculated j-factor is shown to be very sensitive to the amount of bending in these fragments. This fact suggests that ligase closure measurements of systematically bent DNA molecules should be a useful method for studying sequence-directed bending in DNA.     

The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction

The polymerase chain reaction (PCR) is most effectively performed using a thermostable DNA polymerase such as that isolated from Thermus aquaticus. Since temperature and oligonucleotide length are known to control the specificity of oligonucleotide hybridization, we have investigated the effect of oligonucleotide length, base composition, and the annealing temperature on the specificity and efficiency of amplification by the PCR. Generally, the specificity of PCR is controlled by the length of the oligonucleotide and/or the temperature of annealing of the primer to the template. An empirical relationship between oligonucleotide length and ability to support amplification was determined. This relationship allows for the design of specific oligonucleotide primers. A model is proposed which helps explain the observed dependence of PCR on annealing temperature and length of the primer.     

Multiple labeling of antibodies with dye/DNA conjugate for sensitivity improvement in fluorescence immunoassay

Fluorescence immunoassays are widely used in life science research, medical diagnostics, and environmental monitoring due to the intrinsically high specificity, simplicity, and versatility of immunoassays, as well as the availability of a large variety of fluorescent labeling molecules. However, the sensitivity needs to be improved to meet the ever-increasing demand in the new proteomics era. Here, we report a simple method of attaching multiple fluorescent labels on an antibody with a dye/DNA conjugate to increase the immunoassay sensitivity. In the work, mouse IgG adsorbed on the surface of a 96-well plate was detected by its immunoreaction with biotinylated goat anti-mouse antibody. A 30 base pair double-stranded oligonucleotide terminated with biotin was attached to the antibody through the biotin/streptavidin/biotin interaction. Multiple labeling of the antibody was achieved after a fluorescent DNA probe was added into the solution and bound to the oligonucleotide at high ratios. By comparison with fluorescein-labeled streptavidin, the assay with the dye/DNA label produced up to 10-fold increase in fluorescence intensity, and consequently about 10-fold lower detection limit. The multiple labeling method uses readily available reagents, and is simple to implement. Further sensitivity improvement can be obtained by using longer DNAs for antibody labeling, which can incorporate more fluorescent dyes on each DNA.