Chemical and enzymatic incorporation of N2-(p-n-butylphenyl)-2''-deoxyguanosine into an oligodeoxyribonucleotide.

An 18mer oligodeoxyribonucleotide containing a N2-(p-n-butylphenyl)-2'-deoxyguanosine (BuPdG) residue at the 3' end has been synthesized by both chemical and enzymatic methods. Chemical synthesis involved attachment of 5'-DMT-BuPdG as the 3'-H-phosphonate to uridine-controlled pore glass (CPG), followed by extension via H-phosphonate chemistry. After oxidation of the backbone, deprotection of bases, and removal from CPG, the uridine residue was removed by periodate cleavage and beta-elimination. The resulting oligomer 3'-phosphate was digested with alkaline phosphatase to give the free BuPdG-18mer. E.coli DNA polymerase I (Klenow) incorporated BuPdGTP at the 3' end of the corresponding 17mer primer annealed to a complementary 29mer template, and the properties of this product were identical to those of chemically synthesized BuPdG-18mer. E.coli DNA polymerase I (Klenow) was unable to extend the BuPdG-18mer, and the 3' to 5' exonuclease activity of the enzyme was unable to remove the modified nucleotide.     

Using 2-aminopurine fluorescence to detect base unstacking in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase

The fluorescence of the base analogue 2-aminopurine (2AP) was used to detect physical changes in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase. Fluorescent enzyme-DNA complexes were formed with 2AP placed in the template strand opposite the primer terminus (the n position) and placed one template position 5' to the primer terminus (the n + 1 position). The fluorescence enhancement for 2AP at the n position was shown to be due to formation of the editing complex, which indicates that the 2AP-T terminal base pair is recognized primarily as a mismatch. 2AP fluorescence at the n + 1 position, however, was a reporter for DNA interactions in the polymerase active center that induce intrastrand base unstacking. T4 DNA polymerase produced base unstacking at the n + 1 position following formation of the phosphodiester bond. Thus, the increase in fluorescence intensity for 2AP at the n + 1 position could be used to measure the nucleotide incorporation rate in primer extension reactions in which 2AP was placed initially at the n + 2 position. Primer extension occurred at the rate of about 314 s(-1). The amount of base unstacking at the template n + 1 position was sensitive to the local DNA sequence. More base unstacking was detected for DNA substrates with an A-T base pair at the primer terminus compared to C-G or G-C base pairs. Since proofreading is also increased by A-T base pairs compared to G-C base pairs at the primer terminus, we propose that base unstacking may provide an opportunity for the DNA polymerase to reexamine the primer terminus.     

Mismatching base-pair dependence of the kinetics of DNA-DNA hybridization studied by surface plasmon fluorescence spectroscopy

Two single-stranded DNAs consisting of complementary base pairs except for one mismatching base pair (MM1) can form double-stranded DNA by molecular recognition. This type of duplex is not as stable as that formed by MM0. In order to add to a better understanding of the physical mechanism of the hybridization and dissociation processes at sensor (chip) surfaces, we studied the kinetics of the MM1 hybridization by surface plasmon fluorescence spectroscopy. Target DNA strands labelled with a fluorescent molecule Cy5 at the 5′ end and hybridizing with the surface-attached probe DNA can be excited by the strong optical field of a surface plasmon resonance mode. The emitted fluorescence can be detected with high sensitivity. The affinity of a duplex was found to depend on the chemical nature, i.e. G–G, G–T etc., and on the position of the mismatching base pair along the 15mer duplex.     

Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides

Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide primers was mixed with biotin-ddNTPs, DNA polymerase and the DNA templates containing the SNPs in a single tube. The nucleotide at the 3′-end of each CFET-labeled oligonucleotide primer was complementary to a particular SNP in the template. Only the CFET-labeled primer that is fully complementary to the DNA template was extended by DNA polymerase with a biotin-ddNTP. We isolated the DNA extension fragments that carry a biotin at the 3′-end by capture with streptavidin-coated magnetic beads, while the unextended primers were eliminated. The biotinylated fluorescent DNA fragments were subsequently analyzed in a multicolor fluorescence electrophoresis system. The distinct fluorescence signature and electrophoretic mobility of each DNA extension product in the electropherogram coded the SNPs without the use of a sizing standard. We simultaneously distinguished six nucleotide variations in synthetic DNA templates and a PCR product from the retinoblastoma tumor suppressor gene. The use of CFET-labeled primers and biotin-ddNTPs coupled with the specificity of DNA polymerase in SBE offered a multiplex method for detecting SNPs.     

A nanophosphor-based method for selective DNA recovery in Synthosomes

A nanocompartment system composed of an ABA triblock copolymer, where A is poly(dimethylsiloxane) and B is poly(2-methyloxazoline), has been developed for selective recovery and detection of DNA. Translocation of TAMRA-labeled complementary primers into the nanocompartment system has been achieved through two deletion mutants (FhuA Delta1-129; FhuA Delta1-160) of the channel protein FhuA. Translocation was monitored by fluorescence resonance energy transfer through hybridization of the TAMRA-labeled primer to the complementary sequence of a nanophosphor-DNA-conjugate, which reduces its half-life (FhuA Delta1-129, 16.0% reduced; FhuA Delta1-160, 39.0% reduced).     

Quantitative analysis of polymerase chain reaction using anisotropy ratio and relative hydrodynamic volume of fluorescence polarization method.

The method based on the combination of polymerase chain reaction (PCR) and fluorescence polarization is presented. A targeted DNA was amplified with a 5'-fluorescein labeled primer, using a 256 bp DNA fragment of stx2 gene in Escherichia coli O157:H7 (188-443 bp) as a template. The fluorescence anisotropy of the 5'-fluorescein labeled primer increased upon the polymerization through Taq polymerase. The conversion of primer to PCR product was quantitatively monitored by anisotropy ratio and relative hydrodynamic volume. This system was also applied to the determination of E.coli O157:H7.     

Fluorescence of 2-aminopurine reveals rapid conformational changes in the RB69 DNA polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates

We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/template (ddP/T) with 2AP as the templating base (n position) is used. No detectable fluorescence changes occur when a ddP/T with 2AP positioned 5′ to the templating base (n + 1 position) is used. With a deoxy-primer/template (dP/T) with 2AP in the n position, a rapid fluorescence quenching occurs within 2 ms, followed by a second, slower fluorescence quenching with a rate constant similar to base incorporation as determined by chemical quench. With a dP/T having 2AP in the n + 1 position, there is a [dNTP]-dependent fluorescence enhancement that occurs at a rate comparable to dNMP incorporation. Collectively, the results favor a minimal kinetic scheme in which population of two distinct biochemical states of the ternary EDN complex precedes the nucleotidyl transfer reaction. Observed differences between dP/T and ddP/T ternary complexes indicate that the 3′ hydroxyl group of the primer plays a critical role in determining the rate constants of transitions that lead to strong deoxynucleoside triphosphate binding prior to chemistry.     

Determination of the reopening temperature of a DNA hairpin structure in vitro.

A novel method, based upon primer extension, has been developed for measuring the reopening temperature of a single type of DNA hairpin structure. Two DNA oligonucleotides have been utilized and designated as primers 1 and 2. Primer 1, with its 5- and 3'-termini fully complementary to the hairpin flanking sequences, was used to evaluate primer extension conditions, and primer 2, with its 3'-end competing with the DNA hairpin stem, was used to detect the DNA hairpin reopening temperature. A single DNA hairpin structure was formed on the DNA template by thermal denaturation and renaturation, and this hairpin structure was predicted to prevent the annealing of the 3'-end of primer 2 with the template DNA, which leads to no primer extension. By incubating at different temperatures, the DNA hairpin structure can be reopened at a particular temperature where the primer extension can be carried out. This resulted in the appearance of double-stranded DNA that was detected on an agarose gel. This temperature is defined here as the hairpin reopening temperature.     

Probing DNA polymerase-DNA interactions: examining the template strand in exonuclease complexes using 2-aminopurine fluorescence and acrylamide quenching

The bacteriophage T4 DNA polymerase forms fluorescent complexes with DNA substrates labeled with 2-aminopurine (2AP) in the template strand; the fluorescence intensity depends on the position of 2AP. When preexonuclease complexes are formed, complexes at the crossroads between polymerase and exonuclease complexes, 2AP in the +1 position in the template strand is fully free of contacts with the adjacent bases as indicated by high fluorescence intensity and a long fluorescence lifetime of about 10.9 ns. Fluorescence intensity decreases for 2AP in the template strand when the primer end is transferred to the exonuclease active center to form exonuclease complexes, which indicates a change in DNA conformation; 2AP can now interact with adjacent bases, which quenches fluorescence emission. Some polymerase-induced base unstacking for 2AP in the template strand in exonuclease complexes is observed but is restricted primarily to the n and +1 positions, which indicates that the DNA polymerase holds the template strand in a way that forces base unstacking only in a small region near the primer terminus. A hold on the template strand will help to maintain the correct alignment of the template and primer strands during proofreading. Acrylamide quenches 2AP fluorescence in preexonuclease and in exonuclease complexes formed with DNA labeled with 2AP in the template strand, which indicates that the template strand remains accessible to solvent in both complexes. These studies provide new information about the conformation of the template strand in exonuclease complexes that is not available from structural studies.     

Sensitive immobilization-free electrochemical DNA sensor based on isothermal circular strand displacement polymerization reaction

A highly sensitive electrochemical DNA sensor that requires no probe immobilization has been developed based on a target recycling mechanism utilizing a DNA polymerase with a strand displacement activity. The electrochemical detection is realized by taking advantage of the difference in diffusivity between a free ferrocene-labeled peptide nucleic acid (Fc-PNA) and a Fc-PNA hybridized with a complementary DNA, while the DNA polymerase-assisted target recycling leads to signal generation and amplification. The hybridization of the target DNA opens up a stem-loop template DNA with the Fc-PNA hybridized to its extruded 5' end and allows a DNA primer to anneal and be extended by the DNA polymerase, which results in sequential displacement of the target DNA and the Fc-PNA from the template DNA. The displaced target DNA will hybridize with another template DNA, triggering another round of primer extension and strand displacement. The released Fc-PNA, due to its neutral backbone, has much higher diffusivity towards a negatively charged electrode, compared to that when it is hybridized with a negatively charged DNA. Therefore, a significantly enhanced signal of Fc can be observed. The outstanding sensitivity and simplicity make this approach a promising candidate for next-generation electrochemical DNA sensing technologies.     

Fidelity of DNA ligation: a novel experimental approach based on the polymerisation of libraries of oligonucleotides.

Complete libraries of oligonucleotides were used as substrates for Thermus thermophilus DNA ligase, on a M13mp18 ssDNA template. A 17mer primer was used to start a polymerisation process. Ladders of ligation products were analysed by gel electrophoresis. Octa-, nona- and decanucleotide libraries were compared. Nonanucleotides were optimum for polymerisation and up to 15 monomers were ligated. The fidelity of incorporation was studied by sequencing 28 clones (2268 bases) of nonanucleotide polymers, 12 monomers in length. Of the ligated monomers, 79% were the correct complementary sequence. In a total of 57 (2.5%) mispaired bases, there was a strong bias to G.T, G.A, G.G and A.G mismatches. Of the mismatches, 86% were found to be purines on the incoming oligonucleotide, of which 71% were G. There is evidence for clustering of mismatches within specific 9mers and at specific positions within these 9mers. The most frequent mismatches were at the 5'-terminus of the oligonucleotide, followed by the central position. We suggest that sequence selection was imposed by the ligase and not just by base pairing interactions. The ligase directs polymerisation in the 3' to 5' direction which we propose is linked to its role in lagging strand DNA replication.     

An efficient and site-specific gene trimming method

Trimming a DNA strand into a precisely determined fragment can be carried out efficiently by an improved method involving a site-specific trim-primer and a single-stranded DNA template which is generated from a multifunctional vector, pTZ18R, and linearized by using an Eco RI-pTZ18R splinter. A complementary DNA strand is synthesized by DNA polymerase I (Klenow), and the 3'-end of the template upstream from the annealed primer is trimmed by subsequent T4 DNA polymerase reaction. An ATG translation initiator codon or a termination codon can be incorporated into the trim-primer, providing versatility to this single-stranded DNA-initiated gene trimming method that can be applied to subcloning and expression of any DNA fragment with known terminal sequences.     

Detection of activity of telomerase in tumor cells using fiber optical biosensors

Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80-90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48 degrees C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.     

Primer-mediated inhibition of the hydrolysis of template DNA by T5-induced DNA polymerase.

Bacteriophage T5-induced DNA polymerase has an associated 3′→5′ exonuclease activity for which both single-stranded and duplex DNA serve as substrate (1). In this report, we demonstrate that hydrolysis of single-stranded DNA homopolymers (template) is inhibited in the presence of complementary (Watson-Crick sense) oligonucleotides (primer). Almost complete inhibition is observed at a primer/template ratio of ? 0.1. Formation of “H-bonded” primer-template complex seems to be necessary for the inhibition of template hydrolysis because (a) similar amounts of noncomplementary oligonucleotides have no detectable effect on the rate of template hydrolysis, and (b) complementary oligonucleotides lose their inhibitory potential at temperatures where the H-bonded primer-template complex is expected to be unstable. From our data, it appears that the inhibition of template hydrolysis in the presence of primer molecules is due to the preferential binding of the enzyme at the 3′-OH terminus of the primer in the primer-template complex.     

Optical study of DNA surface hybridization reveals DNA surface density as a key parameter for microarray hybridization kinetics

We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22 mer strand (bound-DNA) is immobilized via phenylene-diisothiocyanate linker molecule on the substrate, the dye-labeled (Cy3) complementary strand (free-DNA) is in solution in a reaction chamber. We use total internal reflection fluorescence for surface detection of hybridization. As a new feature we perform a simultaneous real-time measurement of the change of free-DNA concentration in bulk parallel to the total internal reflection fluorescence measurement. We observe that the free-DNA concentration decreases considerably during hybridization. We show how the standard Langmuir kinetics needs to be extended to take into account the change in bulk concentration and explain our experimental results. Connecting both measurements we can estimate the surface density of accessible, immobilized bound-DNA. We discuss the implications with respect to DNA microarray detection.