Search

Fluorescent RNA is an important analytical tool in medical diagnostics, RNA cytochemistry, and RNA aptamer development. We have synthesized the fluorescent ribonucleotide analogue 1,3-diaza-2-oxophenothiazine-ribose-5'-triphosphate (tCTP) and tested it as substrate for T7 RNA polymerase in transcription reactions, a convenient route for generating RNA in vitro. When transcribing a guanine, T7 RNA polymerase incorporates tCTP with 2-fold higher catalytic efficiency than CTP and efficiently polymerizes additional NTPs onto the tC. Remarkably, T7 RNA polymerase does not incorporate tCTP with the same ambivalence opposite guanine and adenine with which DNA polymerases incorporate the analogous dtCTP. While several DNA polymerases discriminated against a d(tC-A) base pair only by factors < 10, T7 RNA polymerase discriminates against tC-A base pair formation by factors of 40 and 300 when operating in the elongation and initiation mode, respectively. These catalytic properties make T7 RNA polymerase an ideal tool for synthesizing large fluorescent RNA, as we demonstrated by generating a ~800 nucleotide RNA in which every cytosine was replaced with tC. 10.1021/ac902456n

Fluorescent DNA of high molecular weight is an important tool for studying the physical properties of DNA and DNA--protein interactions, and it plays a key role in modern biotechnology for DNA sequencing and detection. While several DNA polymerases can incorporate large numbers of dye-linked nucleotides into primed DNA templates, the amplification of the resulting densely labeled DNA strands by polymerase chain reaction (PCR) is problematic. Here, we report a method for high density labeling of DNA in PCR reactions employing the 5'triphosphate of 1,3-diaza-2-oxo-phenoxazine (tCo) and Deep Vent DNA polymerase, tCo is a fluorescent cytosine analogue that absorbs and emits light at 365 and 460 nm, respectively. We obtained PCR products that were fluorescent enough to directly visualize them in a gel by excitation with long [IV light, thus eliminating the need for staining with ethidium bromide. Reactions with Taq polymerase failed to produce PCR products in the presence of only small amounts of dtCoTP. A comparative kinetic study of Taq and Deep Vent polymerase revealed that Taq polymerase, although it inserts dtCoTP with high efficiency opposite G, is prone to forming mutagenic tCo-A base pairs and does not efficiently extend base pairs containing tCo. These kinetics features explain the poor outcome of the PCR reactions with Taq polymerase. Since tCo substitutes structurally for cytosine, the presented labeling method is believed to be less invasive than labeling with dye-linked nucleotides and, therefore, produces DNA that is ideally suited for biophysical studies. 10.1021/ac9017555

In previous work, a simple diagnostic DNA microarray that targeted only the matrix gene segment of influenza A (MChip) was developed and evaluated with patient samples. In this work, the analytical utility of the MChip for detection and subtyping of an emerging virus was evaluated with a diverse set of A/H5N1 influenza viruses. A total of 43 different highly pathogenic A/H5N1 viral isolates that were collected from diverse geographic locations, including Vietnam, Nigeria, Indonesia, and Kazakhstan, representing human, feline, and a variety of avian infections spanning the time period 2003-2006 were used in this study. A probabilistic artificial neural network was developed for automated microarray image interpretation through pattern recognition. The microarray assay and subsequent subtype assignment by the artificial neural network resulted in correct identification of 24 'unknown' A/H5N1 positive samples with no false positives. Analysis of a data set composed of A/H5N1, A/H3N2, and A/HIN1 positive samples and negative controls resulted in a clinical sensitivity of 97% and a clinical specificity of 100%.

The design and characterization of a low-density micro-array for subtyping influenza A is presented. The micro-array consisted of 15 distinct oligonucleotides designed to target only the matrix gene segment of influenza A. An artificial neural network was utilized to automate micro-array image interpretation. The neural network was trained to recognize fluorescence image patterns for 68 known influenza viruses and subsequently used to identify 53 unknowns in a blind study that included 39 human patient samples and 14 negative control samples. The assay exhibited a clinical sensitivity of 95% and clinical specificity of 92%.

The ability of herpes primase to bind and polymerise a series of NTP analogues containing bases that varied significantly in terms of shape, hydrophobicity, and hydrogen-bonding potential is measured. While enzyme bound all of these analogues with similar affinity, it only efficiently polymerized those analogues that could form Watson-Crick hydrogen bonds with the template base.

The human DNA primase synthesizes short RNA primers that DNA polymerase alpha further elongates. The data indicates that human primase requires the formation of Watson-Crick hydrogen bonds in order to polymerize a NTP, a situation very different than what is observed with some DNA polymerases.

Research shows that while the NTPase activity of the herpes simplex virus-1 helicase-primase is unaffected by template sequence of the double-stranded DNA templates, the primase activity is markedly influenced by the template sequence. The primer synthes is dependent on the presence of a 3'-G-pyr-pyr triplet in the template and its flanking sequences.

Research has been conducted on DNA primase which synthesizes short RNA oligonucleotides. The development of the potent primase inhibitors carried out via the reaction of 2'-modified sugars with bases incapable of Watson-Crick hydrogen bonding is described.

Results suggest that the 'pol beta-like' region of p58 subunit of human DNA primase participates in the initiation of primer synthesis, its elongation and translocation, and counting. Mutations in the p58 region affects both initiation and elongation of primers.

Results reveal that the CMP-sialic acid transporter mediates transport of a variety of nucleotide monophosphates. For sugar transport, the protein requires a complete ribose ring for binding. Addition of a methyl group to N3 markedly reduces binding, suggesting that the binding inhibition is due to stearic hindrance.

The kinetics of primer synthesis were examined in relation to the regulation of the p49-p58 primase complex and the interaction of the primase subunits with DNA. Results suggested a model of primer synthesis regulation in which the newly synthesized primer-template species binds to p58 and regulates further primer synthesis. In this model, it is also theorized that p58 mediates the transfer of primers from the primase active site to DNA polymerase alpha.

A series of synthetic 43-mer templates was examined to investigate the interaction of the DNA polymerase alpha complex with abasic sites. Results showed that abasic lesions did not lead to strong chain termination during primase-coupled DNA polymerase alpha activity. Moreover, it was demonstrated that the mechanism of bypass was likely to involve DNA polymerase activity since abasic lesions were strong inhibitors of primase. Biological implications of these findings are also provided.

A study was conducted to examine in detail the effects of Mn2+ on eukaryotic DNA primase both in the absence and presence of 5 mM Mg2+. Results showed that low concentrations of Mn2+ dramatically increase the rate of both elongation and initiation in a template dependent manner and decrease the sensitivity of primase to inhibition by anions. It was also observed that Mn2+ alters the sequence specificity for primer synthesis.

Synthetic templates of defined sequence [d(TC)30] and wild-type DNA sequences were utilized to determine the influence of nucleoside triphosphates (NTP) concentration in the initiation of primer synthesis. Measures of primase-coupled alpha polymerase activity on different NTP concentrations revealed that at higher concentrations, NTP allowed primase to initiate synthesis at all regions of the DNA. Furthermore, it also determines which nucleotide is found as the second nucleotide of the primer.

The study of polymerization of NTPs and arbinofuranosyladenosine triphosphate (araATP) at DNA polymerase alpha catalyzed elongation of primase-synthesized primers reveals that the initiation of new strands of DNA by pol alpha-primase may be a means for inhibiting replication. Polymerization of araATP does not lead to chain termination but the next correct nucleotide is added in a way that araATP is simply an alternate substrate.

A comparative study of the inhibition of DNA polymerase (pol) alpha, delta and epsilon by penciclovir triphosphate (PCVTP), ganciclovir triphosphate (GCVTP) and acyclovir triphosphate (ACVTP) reveals that GCVTP selectively inhibits pol delta. ACVTP and PCVTP exhibit decreased selectivity toward pol delta. Inhibition of DNA primase by all the three compounds is weak. ACVTP inhibits the elongation of primase-synthesized primers, catalyzed by pol alpha.

Defined-sequence templates and steady-state kinetic studies of primer synthesis and primer-template movement reveal that primase-generated primer-template negatively mediates primase activity. Primase and pol alpha activities are independent before primer generation and coordinated after primer-template synthesis. The coordination requires that the primase generate primer, a unit-length primer be bound to template of proper length and pol alpha's active site interact with primer-template.

Polymerase and exonuclease activities use short, artificial oligonucleotides as substrates for calf thymus DNA polymerase-epsilon. Aphidicolin was studied as a means of repression in the substrates. A pol epsilon.DNA complex is bound to aphidicolin by competing with four dNTPs. The identity of the next template base which will be duplicated does not affect the binding ability of aphidicolin. Exonuclease activity on single-stranded DNA is not repressed by aphidicolin, although it is slightly repressed on double-stranded DNA. A significant activity of the exonuclease is seen even when the polymerase site is totally deterred by a template primer.

Pre-steady-state and steady-state techniques were used to examine the mechanism by which calf thymus DNA primase synthesizes RNA primers. A central feature of the approach is the use of mixed-base templates of defined sequences. Critical steps in the initiation, polymerization and termination of primer synthesis were identified. In addition, it was found that the role of primase is to synthesize primers that polymerase alpha can elongate.

Inhibition of DNA primase and polymerase alpha (pol alpha) by arabinofuranosylnucleoside triphosphates and related nucleotides were examined. The 2' and 3'-hydroxyls of the incoming nucleotide appear relatively not important for binding to primase. However, the 3'-hydroxyl is important while the 2'-hydroxyl is not essential for polymerization. Moreover, it was shown that RNA primers modulate pol alpha inhibition by nucleotide analogues relative to DNA primers.

A study was conducted to gain insight into the role of the influenza RNA-dependent RNA polymerase (RdRp) virus in regulation of the relative amounts of mRNA transcription and replication of flu RNA genome. The data obtained provides insight into the regulation of RdRp throughout the viral life cycle and its role in synthesizing the appropriate concentration amounts of viral mRNA and replication products (vRNA and cRNA).

An extensive series of purine and pyrimidine analogues were utilized to gain further insights into the mechanisms by which human DNA primase choose to polymerize nucleoside triphosphates (NTPs). The data results provided insight into the strong dependence of human primase on Watson-Crick hydrogen bonds for efficient nucleotide polymerization and the potential roles of primer-template stability and base tautomerization during misincorporation.

Several studies are conducted to explain the properties, as well as the applications of the herpes simplex virus-1 DNA primase, which is shown to be one of the least accurate primnases. The mechanisms involved in the misincorporation of different nucleotides by the primase are also discussed.

A series of kinetic and photo-cross-linking studies were conducted to gain insight into the roles of each subunit in DNA binding and primer synthesis for the helicase-primase complex from herpes simplex virus-1 contains three subunits, UL5, UL52, and UL8. Photo-cross-linking of a RNA primer-template, the product of primer synthesis, containing 5-iodouracil in the template were shown to generate the same apparent cross-linked species.

A study was conducted to analyze human DNA polymerase [alpha], a B family enzyme, and DNA polymerase from Bacillus stearothermophilus (BF). Results indicate that different polymerases interact very differently with base lesions.

The studies related to the incorporation of the fluorescent cytidine analogues 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) by human DNA polymerase [alpha] and Klenow fragment of DNA polymerase I (Escherichia coli) are described. Both enzymes showed the aptitude to readily extend the formed tC(o)-G and tC(o)-A base pairs and are able to incorporate at least four consecutive nucleotide analogues.

A series of dATP and dGTP analogues were used to determine the kinetics of DNA polymerase I from Bacillus stearothermophilus (BF), a prototypical A family polymerase which used N-1, [N.sup.2], N-3, and [N.sup.6] of purine dNTPs to differentiate between right and wrong nucleotide incorporation. The results indicated that BF used N-1, [N.sup.2], N-3, and [N.sup.6] of purine dNTPs both as negative selectors to prevent misincorporation and as positive selectors to enhance correct incorporation.

Several studies are conducted for the identification of the chemical features of the purine 2'-deoxy-5'-triphosphates (dNTPs), which are necessary for an efficient DNA replication by the Herpes Simplex Virus I (HSV-I) DNA polymerase. The structure of the base pair is shown to highly affect the Herpes polymerase.

The interaction of nucleoside triphosphates (NTPs) containing modified sugars is analyzed in order to understand how DNA primase from herpes simplex virus I has catalyzed primer synthesis. The incorporation of a nucleotide whose sugar contains a 2'-OH in the ribo configuration has allowed nucleotide polymerization, whereas the incorporation of a nucleotide containing a 2'-OH in the area configuring has resulted in very strong chain termination.

A study was conducted to identiy the chemical features of purine dNTPs that human pol [alpha] uses to discriminate between right and wrong dNTPs. The findings suggest that during dNTP polymerization pol [alpha] uses a combination of negative (N-1 and N-3) and positive (N-1 and N-6) selectivity to differentiate between right and wrong dNTPs while the shape of the base pair is essentially irrelevant.

Templates of defined sequence to investigate the mechanism of NTP misincorporation by DNA primase from herpes simplex virus-I are examined. During the primer synthesis, herpes generated a series of mismatches.

Templates of defined sequence were utilized to investigate the mechanism of primer synthesis by herpes simplex virus 1 helicase-primase. Under steady-state conditions, the rate of primer synthesis and the size distribution of products remained constant with time, suggesting that the rate-limiting steps of primer synthesis occur during primer initiation.

Research shows that human DNA polymerase alpha and Escherichia coli DNA polymerase I Klenow fragment can incorporate nucleotide analogues opposite the canonical bases. Data indicate that this incorporation is up to 4000-fold more efficient than that mediated by an incorrect naturl dNTP. The analogue incorporation by human DNA polymerase alpha is opposite pyrimidine bases, while that of Klenow fragment is opposite purine bases.

Eukaryotic DNA primase initiates the synthesis of all new DNA strands by synthesizing short RNA oligomers on single-stranded DNA. Additionally, primase helps couple replication and repair and is critical for telomere maintenance and, therefore, chromosome stability. In light of the many aspects of DNA metabolism in which primase is involved, understanding the unique features of the mechanism of this enzyme and how it interacts with other proteins will greatly advance our knowledge of DNA replication and repair.

The chemical modification of only a single nucleotide in the human genome can affect an individual's fitness, although the chemical bonds in DNA are exceptionally stable generally. The knowledge of the various types of DNA repair pathways and the study of the roles of a broad family of mechanically distinct DNA polymerases help evolve elaborate mechanisms for repairing damaged DNA, which restore the original chemical information encoded in the genome.

We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on the exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E-DNA complex or polymerization of acyclovir onto the DNA also had a minimal effect on exonuclease activity. The inability of the exonuclease to excise acyclovir from the primer 3'-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the exonuclease, or transfer of the DNA from the polymerase to the exonuclease. Removing the 3'-hydroxyl or the 2'-carbon from the nucleotide at the 3'-terminus of the primer strongly inhibited exonuclease activity, although addition of a 2'-hydroxyl did not affect exonuclease activity. The biological consequences of these results are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization and exonuclease-mediated destruction of herpes DNA. Second, the ability of the exonuclease to rapidly remove a ribonucleotide at the primer 3'-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA. [ABSTRACT FROM AUTHOR]

The herpes polymerase-processivity factor complex consists of the catalytic UL30 subunit containing both polymerase and proofreading exonuclease activities and the UL42 subunit that acts as a processivity factor. Curiously, the highly active exonuclease has minimal impact on the accumulation of mismatches generated by the polymerase activity. We utilized a series of oligonucleotides of defined sequence to define the interactions between the polymerase and exonuclease active sites. Exonuclease activity requires unwinding of two nucleotides of the duplex primer-template. Surprisingly, even though the exonuclease rate is much higher than the rate of DNA dissociation, the exonuclease degrades both single- and double-stranded DNA in a nonprocessive manner. Efficient proofreading of incorrect nucleotides incorporated by the polymerase would seem to require efficient translocation of DNA between the exonuclease and polymerase active sites. However, we found that translocation of DNA from the exonuclease to polymerase active site is remarkably inefficient. Consistent with inefficient translocation, the DNA binding sites for the exonuclease and polymerase active sites appear to be largely independent, such that the two activities appear noncoordinated. Finally, the presence or absence of UL42 did not impact the coordination of the polymerase and exonuclease activities. In addition to providing fundamental insights into how the polymerase and exonuclease function together, these activities provide a rationale for understanding why the exonuclease minimally impacts accumulation of mismatches by the purified polymerase and raise the question of how these two activities function together in vivo. [ABSTRACT FROM AUTHOR]

Tricyclic cytosines (tC and tCO frameworks) have emerged as a unique class of fluorescent nucleobase analogues that minimally perturb the structure of B-form DNA and that are not quenched in duplex nucleic acids. Systematic derivatization of these frameworks is a likely approach to improve on and diversify photophysical properties, but has not so far been examined. Synthetic methods were refined to improve on tolerance for electron-donating and electron-withdrawing groups, resulting in a series of eight new, fluorescent cytidine analogues. Photophysical studies show that substitution of the framework results in a pattern of effects largely consistent across tC and tCO and provides nucleoside fluorophores that are brighter than either parent. Moreover, a range of solvent sensitivities is observed, offering promise that this family of probes can be extended to new applications that require reporting on the local environment. [ABSTRACT FROM AUTHOR]