The cytosine analogues 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) stand out among fluorescent bases due to their unquenched fluorescence emission in double-stranded DNA. Recently, we reported a method for the generation of densely tCo-labeled DNA by polymerase chain reaction (PCR) that relied on the use of the extremely thermostable Deep Vent polymerase. We have now developed a protocol that employs the more commonly used Taq polymerase. Supplementing the PCR with Mn2+ or Co2+ ions dramatically increased the amount of dtCoTP incorporated, and thus enhanced the brightness of the PCR products. The resulting PCR products could be easily detected in gels based on their intrinsic fluorescence. The Mn2+ ions modulate the PCR by improving the bypass of template tCo and the overall catalytic efficiency. In contrast to the lower fidelity during tCo bypass, Mn2+ improved the ability of Taq polymerase to distinguish between dtCoTP and dTTP when copying a template dA. Interestingly, Mn2+ ions hardly affect the fluorescence emission of tC(o), whereas the coordination of Co2+ ions with the phosphate groups of DNA and nucleotides statically quenches tC(o) fluorescence with small reciprocal Stern Vollmer constants of 10 to 300 μM.
polymerase chain reaction; fluorescence; quenching; taq; manganese; cobalt; kinetics
We utilized a series of pyrimidine analogues modified at O2, N-3, and N4/O4 to determine if two B family DNA polymerases, human DNA polymerase α and herpes simplex virus I DNA polymerase, choose whether or not to polymerize pyrimidine dNTPs using the same mechanisms they use for purine dNTPs. Removing O2 of a pyrimidine dNTP vastly decreased incorporation by these enzymes and also compromised fidelity in the case of C analogues, while removing O2 from the templating base had more modest effects. Removing the Watson-Crick hydrogen bonding groups of N-3 and N4/O4 greatly impaired polymerization, both of the resulting dNTP analogues as well as polymerization of natural dNTPs opposite these pyrimidine analogues when present in the template strand. Thus, the Watson-Crick hydrogen bonding groups of a pyrimidine clearly play an important role in enhancing correct dNTP polymerization, but are not essential for preventing misincorporation. These studies also indicate that DNA polymerases recognize bases extremely asymmetrically, both in terms of whether they are a purine or pyrimidine and whether they are in the template or are the incoming dNTP. The mechanistic implications of these results regarding how polymerases discriminate between right and wrong dNTPs are discussed.
Fidelity; misincorporation; kinetics; base-pair; nucleotide
Protein degradation via the ubiquitin-proteasome pathway is important for a diverse number of cellular processes ranging from cell signaling to development. Disruption of the ubiquitin pathway occurs in a variety of human diseases, including several cancers and neurological disorders. Excessive proteolysis of tumor suppressor proteins, such as p27, occurs in numerous aggressive human tumors. To discover small-molecule inhibitors that potentially prevent p27 degradation, we developed a series of screening assays, including a cell-based screen of a small-molecule compound library and two novel nucleotide exchange assays. Several small-molecule inhibitors, including NSC624206, were identified and subsequently verified to prevent p27 ubiquitination in vitro. The mechanism of NSC624206 inhibition of p27 ubiquitination was further unraveled using the nucleotide exchange assays and shown to be due to antagonizing ubiquitin activating enzyme (E1). We determined that NSC624206 and PYR-41, a recently reported inhibitor of ubiquitin E1, specifically block ubiquitin-thioester formation but have no effect on ubiquitin adenylation. These studies reveal a novel E1 inhibitor that targets a specific step of the E1 activation reaction. NSC624206 could, therefore, be potentially useful for the control of excessive ubiquitin-mediated proteolysis in vivo.
ubiquitin E1; inhibitor; p27kip1; ubiquitin; proteolysis
Protein ubiquitination plays an important role in the regulation of almost every aspect of eukaryotic cellular function; therefore, its destabilization is often observed in most human diseases and cancers. Consequently, developing inhibitors of the ubiquitination system for the treatment of cancer has been a recent area of interest. Currently, only a few classes of compounds have been discovered to inhibit the ubiquitin-activating enzyme (E1) and only one class is relatively selective in E1 inhibition in cells. We now report that Largazole and its ester and ketone analogs selectively inhibit ubiquitin conjugation to p27Kip1 and TRF1 in vitro. The inhibitory activity of these small molecules on ubiquitin conjugation has been traced to their inhibition of the ubiquitin E1 enzyme. To further dissect the mechanism of E1 inhibition, we analyzed the effects of these inhibitors on each of the two steps of E1 activation. We show that Largazole and its derivatives specifically inhibit the adenylation step of the E1 reaction while having no effect on thioester bond formation between ubiquitin and E1. E1 inhibition appears to be specific to human E1 as Largazole ketone fails to inhibit the activation of Uba1p, a homolog of E1 in Schizosaccharomyces pombe. Moreover, Largazole analogs do not significantly inhibit SUMO E1. Thus, Largazole and select analogs are a novel class of ubiquitin E1 inhibitors and valuable tools for studying ubiquitination in vitro. This class of compounds could be further developed and potentially be a useful tool in cells.
The influenza RNA-dependent RNA polymerase (RdRp) both replicates the flu's RNA genome and transcribes its mRNA. Replication occurs de novo; however, initiation of transcription requires a 7-methylguanosine 5’ capped primer that is “snatched” from host mRNA via endonuclease and cap binding functions of the influenza polymerase. A key question is how the virus regulates the relative amounts of transcription and replication. We found that the concentration of a capped cellular mRNA, the concentration of the 5’-end of the viral RNA, and the concentration of RdRp all regulate the relative amounts of replication versus transcription. The host mRNA, from which the RdRp snatches its capped primer, acts to upregulate transcription and repress replication. Elevated concentrations of the RdRp itself switch the influenza polymerase towards replication, likely through an oligomerization of the polymerase. The 5’-end of the vRNA template both activates replication and inhibits transcription of the vRNA template, thereby indicating that RdRp contains an allosteric binding site for the 5’ end of the vRNA template. These data provides insights into the regulation of RdRp throughout the viral life cycle and how it synthesizes the appropriate amounts of viral mRNA and replication products (vRNA and cRNA).
Nucleotide analogues represent a major class of anti-cancer and anti-viral drugs, and provide an extremely powerful tool for dissecting the mechanisms of DNA and RNA polymerases. While the basic assays themselves are relatively straight-forward, a key issue is to appropriately design the studies to answer the mechanistic question of interest. This article addresses the major issues involved in designing these studies, and some of the potential difficulties that arise in interpreting the data. Examples are given both of the type of analogues typically used, the experimental approaches with different polymerases, and issues with data interpretation.
polymerase; nucleotide; DNA; RNA; kinetics
The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of UL5, UL8, and UL52, possesses 5′ to 3′ helicase, single-stranded DNA (ssDNA)-dependent ATPase, primase, and DNA binding activities. In this study we confirm that the UL5-UL8-UL52 complex has higher affinity for forked DNA than for ssDNA and fails to bind to fully annealed double-stranded DNA substrates. In addition, we show that a single-stranded overhang of greater than 6 nucleotides is required for efficient enzyme loading and unwinding. Electrophoretic mobility shift assays and surface plasmon resonance analysis provide additional quantitative information about how the UL5-UL8-UL52 complex associates with the replication fork. Although it has previously been reported that in the absence of DNA and nucleoside triphosphates the UL5-UL8-UL52 complex exists as a monomer in solution, we now present evidence that in the presence of forked DNA and AMP-PNP, higher-order complexes can form. Electrophoretic mobility shift assays reveal two discrete complexes with different mobilities only when helicase-primase is bound to DNA containing a single-stranded region, and surface plasmon resonance analysis confirms larger amounts of the complex bound to forked substrates than to single-overhang substrates. Furthermore, we show that primase activity exhibits a cooperative dependence on protein concentration while ATPase and helicase activities do not. Taken together, these data suggest that the primase activity of the helicase-primase requires formation of a dimer or higher-order structure while ATPase activity does not. Importantly, this provides a simple mechanism for generating a two-polymerase replisome at the replication fork.
The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not coordinated leading and lagging strand synthesis.
This first report of a photoinitiator-nucleotide conjugate demonstrates a novel approach for sensitive, rapid and visual detection of DNA hybridization events. This approach holds potential for various DNA labeling schemes and for applications benefiting from selective DNA-based polymerization initiators. Here, we demonstrate covalent, enzymatic incorporation of an eosin-photoinitiator 2′-deoxyuridine-5′-triphosphate (EITC-dUTP) conjugate into surface-immobilized DNA hybrids. Subsequent radical chain photoinitiation from these sites using an acrylamide/bis-acrylamide formulation yields a dynamic detection range between 500pM and 50nM of DNA target. Increasing EITC-nucleotide surface densities leads to an increase in surface-based polymer film heights until achieving a film height plateau of 280nm ±20nm at 610 ±70 EITC-nucleotides/μm2. Film heights of 10–20 nm were obtained from eosin surface densities of approximately 20 EITC-nucleotides/μm2 while below the detection limit of ~10 EITC-nucleotides/μm2, no detectable films were formed. This unique threshold behavior is utilized for instrument-free, visual quantification of target DNA concentration ranges.
Labeled nucleotides; Photoinitiator; DNA quantification; Microarrays; Signal amplification; Photopolymerization; Surface-Initiated Polymerization
Human DNA primase synthesizes short RNA primers that DNA polymerase α then elongates during the initiation of all new DNA strands. Even though primase misincorporates NTPs at a relatively high frequency, this likely does not impact the final DNA product since the RNA primer is replaced with DNA. We used an extensive series of purine and pyrimidine analogues to provide further insights into the mechanism by which primase chooses whether or not to polymerize a NTP. Primase readily polymerized a size-expanded cytosine analogue, 1, 3-diaza-2-oxo-phenothiazine-NTP, across from a templating G but not across from A. The enzyme did not efficiently polymerize NTPs incapable of forming two Watson-Crick hydrogen bonds with the templating base with the exception of UTP opposite purine deoxyribonucleoside. Likewise, primase did not generate base-pairs between two nucleotides with altered Watson-Crick hydrogen bonding patterns. Examining the mechanism of NTP polymerization revealed that human primase can misincorporate NTPs via both template misreading and a primer-template slippage mechanism. Together, these data demonstrate that human primase strongly depends on Watson-Crick hydrogen bonds for efficient nucleotide polymerization, much more so than the mechanistically related herpes primase, and provide insights into the potential roles of primer-template stability and base tautomerization during misincorporation.
Fidelity; misincorporation; hydrogen bonds; Watson-Crick; DNA polymerase; kinetics
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.
in vitro transcription; kinetics; mutagenesis; minor tautomeric forms; base analogue
Herpes simplex virus-1 primase misincorporates the natural NTPs at frequencies around 1 error per 30 NTPs polymerized, making it one of the least accurate polymerases known. We used a series of nucleotide analogues to further test the hypothesis that primase requires Watson-Crick hydrogen bond formation in order to efficiently polymerize a NTP. Primase could not generate base pairs containing a complete set hydrogen bonds in an altered arrangement (iso-guanine:iso-cytosine), and did not efficiently polymerize dNTPs completely incapable of forming Watson-Crick hydrogen bonds opposite templating bases incapable of forming Watson-Crick hydrogen bonds. Similarly, primase did not incorporate most NTPs containing hydrophobic bases incapable of Watson-Crick hydrogen bonding opposite natural template bases. However, 2-pyridone NTP and 4-methyl-2-pyridone NTP provided striking exceptions to this rule. The effects of removing single Watson-Crick hydrogen bonding groups from either the NTP or templating bases varied from almost no effect to completely blocking polymerization depending both on the parental base pair (G:C vs. A:T/U) and which base pair of the growing primer (second, third or fourth) was examined. Thus, primase does not absolutely need to form Watson-Crick hydrogen bonds in order to efficiently polymerize a NTP. Additionally, we found that herpes primase can misincorporate nucleotides both by misreading the template and by a primer-template slippage mechanism. The mechanistic and biological implications of these results are discussed.
Polymerase; fidelity; misincorporation; Watson-Crick; hydrogen bond; kinetics; mechanism
The helicase-primase complex from herpes simplex virus-1 contains three subunits, UL5, UL52, and UL8. We generated each of the potential two subunit complexes, UL5/UL52, UL5/UL8, and UL52/UL8, and used a series of kinetic and photocrosslinking studies to provide further insights into the roles of each subunit in DNA binding and primer synthesis. UL8 increases the rate of primer synthesis by UL5/UL52 by increasing the rate of primer initiation (2 NTPs → pppNpN), the rate-limiting step in primer synthesis. The UL5/UL8 complex lacked any detectable catalytic activity (DNA dependent ATPase, primase, or RNA polymerase using a RNA primer:template and NTPs as substrates), but could still bind DNA, indicating that UL52 must provide some key amino acids needed for helicase function. The UL52/UL8 complex lacked detectable DNA dependent ATPase activity and could not synthesize primers on single-stranded DNA. However, it exhibited robust RNA polymerase activity using a RNA primer:template and NTPs as substrate. Thus, UL52 must contain the entire primase active site needed for phosphodiester bond formation, while UL5 minimally contributes amino acids needed for initiation of primer synthesis. Photocrosslinking experiments using single-stranded templates containing 5-iodouracil either before, in, or after the canonical 3′-GPyPy (Py = T or C) initiation site for primer synthesis showed that only UL5 crosslinked to the DNA. This occurred for both the UL5/UL52 and UL5/UL52/UL8 complexes, and whether or not the reactions contained NTPs. Photocrosslinking of a RNA primer:template, the product of primer synthesis, containing 5-iodouracil in the template generated the same apparent crosslinked species.
Replication; kinetics; NTPs; polymerase; photocrosslinking
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 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 UV 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.
Taq; Deep Vent; DNA polymerase; kinetics; dNTP
To better understand how DNA polymerases interact with mutagenic bases, we examined how human DNA polymerase α (pol α), a B family enzyme, and DNA polymerase from Bacillus stearothermophilus (BF), an A family enzyme, generate adenine:hypoxanthine and adenine:8-oxo-7,8-dihydroguanine (8-oxoG) base pairs. Pol α strongly discriminated against polymerizing dATP opposite 8-oxoG, and removing N1, N6, or N7 further inhibited incorporation, whereas removing N3 from dATP dramatically increased incorporation (32-fold). Eliminating N6 from 3-deaza-dATP now greatly reduced incorporation, suggesting that incorporation of dATP (analogues) opposite 8-oxoguanine proceeds via a Hoogsteen base-pair and that pol α uses N3 of a purine dNTP to block this incorporation. Pol α also polymerized 8-oxo-dGTP across from a templating A, and removing N6 from the template adenine inhibited incorporation of 8-oxoG. The effects of N1, N6, and N7 demonstrated a strong interdependence during formation of adenine:hypoxanthine base-pairs by pol α and N3 of dATP again helps prevent polymerization opposite a templating hypoxanthine. BF very efficiently polymerized 8-oxo-dGTP opposite adenine, and N1 and N7 of adenine appear to play important roles. BF incorporates dATP opposite 8-oxoG less efficiently, and modifying N1, N6, or N7 greatly inhibits incorporation. N6, and to a lesser extent N1, help drive hypoxanthine:adenine base pair formation by BF. The mechanistic implications of these results showing that different polymerases interact very differently with base lesions are discussed.
Fidelity; misincorporation; kinetics; mechanism; base-pair; Hoogsteen
We studied the incorporation of the fluorescent cytidine analogues 1, 3-diaza-2-oxo-phenothiazine (tC) and 1, 3-diaza-2-oxo-phenoxazine (tCo) by human DNA polymerase α and Klenow fragment of DNA polymerase I (E. coli). These tricyclic nucleobases possess the regular hydrogen bonding interface of cytosine but are significantly size expanded toward the major groove. Despite the size alteration both DNA polymerases insert dtCTP and dtCoTP with remarkable catalytic efficiency. Polymerization opposite guanine is comparable to the insertion of dCTP, while the insertion opposite adenine is only ∼4-11 times less efficient than the formation of a T-A base pair. Both enzymes readily extend the formed tC(o)-G and tC(o)-A base pairs, and can incorporate at least 4 consecutive nucleotide analogues. Consistent with these results, both DNA polymerases efficiently polymerize dGTP and dATP when tC and tCo are in the template strand. KF inserts dGTP with a 4- to 9-fold higher probability than dATP, while pol α favors dGTP over dATP by a factor of 30-65. Overall, the properties of tC(o) as templating base and as incoming nucleotide are surprisingly symmetrical and may be universal for A and B family DNA polymerases. This finding suggests that the aptitude for ambivalent base pairing is a consequence of the electronic properties of tC(o).
Fluorescent Base Analogue; DNA labeling; Kinetics; Nucleotide polymerization; DNA Replication
We used a series of dATP and dGTP analogues to determine how DNA polymerase I from Bacillus stearothermophilus (BF), a prototypical A family polymerase, uses N-1, N2, N-3, and N6 of purine dNTPs to differentiate between right and wrong nucleotide incorporation. Altering any of these nitrogens had two effects. First, it decreased the efficiency of correct incorporation of the resulting dNTP analogue, with the loss of N-1 and N-3 having the most severe effects. Second, it dramatically increased misincorporation of the resulting dNTP analogues, with alterations in either N-1 or N6 having the most severe impacts. Adding N2 to dNTPs containing the bases adenine and purine increased polymerization opposite T, but also tremendously increased misincorporation opposite A, C, and G. Thus, BF uses N-1, N2. N-3, and N6 of purine dNTPs both as negative selectors to prevent misincorporation and as positive selectors to enhance correct incorporation. Comparing how BF discriminates between right and wrong dNTPs with both B family polymerases and low fidelity polymerases indicates that BF has chosen a unique solution vis-à-vis these other enzymes, and, therefore, that nature has evolved at least three mechanistically distinct solutions.
Fidelity; Kinetics; Polymerization; Nucleotide; Replication
In order to accurately replicate its viral genome, the Herpes Simplex Virus 1 (HSV-1) DNA polymerase usually polymerizes the correct dNTP opposite the template base being replicated. We employed a series of purine-dNTP analogues to determine the chemical features of the base necessary for the herpes polymerase to avoid polymerizing incorrect dNTPs. The enzyme uses N-3 to prevent misincorporation of purine dNTPs, but does not require N-3 for correct polymerization. A free pair of electrons on N-1 also helps prevent misincorporation opposite A, C, and G, and strongly drives polymerization opposite T. N6 contributes a small amount both for preventing misincorporation and for correct polymerization. Within the context of guanine in either the incoming dNTP or the template base being replicated, N2 prevents misincorporation opposite adenine but plays at most a minor role for incorporation opposite C. In contrast, adding N2 to dNTPs of either adenine, purine, 6-chloropurine or 1-deazapurine greatly enhances incorporation opposite C, likely via the formation of a hydrogen bond between N2 of the purine and O2 of the pyrimidine. Herpes polymerase is very sensitive to the structure of the base-pair at the primer 3′-terminus since eliminating N-1, N-3, or N6 from a purine nucleotide at the primer 3′-terminus interfered with polymerization of the next two dNTPs. The biological and evolutionary implications of these data are discussed.
Fidelity; Misincorporation; Selectivity; Kinetics; Nucleotide
We used a series of dNTP analogues in conjunction with templates containing modified bases to elucidate the role that N2 of a purine plays during dNTP polymerization by human DNA polymerase α. Removing N2 from dGTP had small effects during correct incorporation opposite C, but specifically increased misincorporation opposite A. Adding N2 to dATP and related analogues had small and variable effects on the efficiency of polymerization opposite T. However, the presence of N2 greatly enhanced polymerization of these dATP analogues opposite a template C. The ability of N2 to enhance polymerization opposite C likely results from formation of a hydrogen bond between the purine N2 and pyrimidine O2. Even in those cases where formation of a wobble base-pair, tautomerization, and/or protonation of the base-pair between the incoming dNTP and template base cannot occur (Eg., 2-pyridone:purine (or purine analogue) base-pairs), N2 enhanced formation of the base-pair. Importantly, N2 had similar effects on dNTP polymerization both when added to the incoming purine dNTP and the template base being replicated. The mechanistic implications of these results regarding how pol α discriminates between right and wrong dNTPs are discussed.
Fidelity; misincorporation; base-pair; nucleotide; dITP
We analyzed the interaction of NTPs containing modified sugars to develop a better understanding of how DNA primase from herpes simplex virus I catalyzes primer synthesis. During the NTP binding reaction, primase tolerated a large number of modifications to the sugar ring. Altering the 2′ and 3′ carbons, and even converting the furanose sugar into an acyclic sugar, did not prevent binding. Whether or not the base on the NTP could form a correct base-pair with the template base being replicated also had minimal effects on the binding reaction, indicating that primase does not use this process to discriminate between right and wrong NTPs. Rather, the key feature that primase recognizes to bind a NTP is the 5′-γ-phosphate since converting a NTP into a NDP greatly compromised binding. During the polymerization reaction, primase tolerated substantial modification of the 2′-carbon, including the presence of either an ara or ribo hydroxyl, two hydrogens, or two fluorines. However, polymerization absolutely required that the NTP contain a 3′-hydroxyl and an intact sugar ring. Modifications at the 2′-carbon of the nucleotide at the primer 3′-terminus significantly impaired further polymerization events. Compared to a ribonucleotide, incorporation of a 2-deoxyribo- or 2′,2′-difluoro-2′-deoxyribonucleotide resulted in strong chain termination, while incorporation of an aranucleotide resulted in very strong chain termination. The implications of these data with respect to the mechanism of primase and the relationship between human and herpes primase are discussed.
Influenza A has the ability to rapidly mutate and become resistant to the commonly prescribed influenza therapeutics, thereby complicating treatment decisions.
To design a cost-effective low-density microarray for use in detection of influenza resistance to the adamantanes.
We have taken advantage of functional genomics and microarray technology to design a DNA microarray that can detect the two most common mutations in the M2 protein associated with adamantane resistance, V27A and S31N.
In a blind study of 22 influenza isolates, the Antiviral Resistance-Chip (AVR-Chip) had a success rate of 95% for detecting these mutations. Microarray data from a larger set of samples were further analyzed using an artificial neural network and resulted in a correct identification rate of 94% for influenza virus samples that had V27A and S31N mutations.
The AVR-Chip provided a method for rapidly screening influenza viruses for adamantane sensitivity, and the general approach could be easily extended to detect resistance to other chemotherapeutics.
The importance of global influenza surveillance using simple and rapid diagnostics has been frequently highlighted. For influenza type B, the need exists for discrimination between the two currently circulating major lineages, represented by virus strains B/Victoria/2/87 and B/Yamagata/16/88, as only one of these lineages is represented in seasonal influenza vaccines. Here, the development and characterization of a low-density DNA microarray (designated BChip) designed to detect and identify the two influenza B lineages is presented. The assay involved multiplex nucleic acid amplification and microarray hybridization of viral RNA. Detection and lineage identification was achieved in less than 8 hours. In a study of 62 influenza B virus samples from 19 countries, dating from 1945 to 2005, as well as 5 negative control samples, the assay exhibited 97% sensitivity and 100% specificity. Furthermore, application of a trained artificial neural network to the pattern of relative fluorescence signals resulted in correct lineage assignment for 94% of 50 applicable influenza B viruses, with no false assignments.
influenza B; DNA microarray; influenza diagnostic; virus lineage; Yamagata lineage; Victoria lineage
A novel assay was developed for real-time monitoring of RNA synthesis using a hybridization-based method. In this work, a “broken beacon” in which the fluor and quencher were located on separate but complementary oligonucleotides was used to quantify the amount of RNA production by T7 polymerase. The relative lengths of the fluor-oligo and quencher-oligo, as well as their relative concentrations, were optimized. The experimentally determined limit-of-detection was ~45 nM. The new assay was compared to the “gold-standard” radiolabel ([32P]NTP incorporation) assay for RNA quantification. While the broken beacon assay exhibited a higher limit of detection, it provided an accurate measure of RNA production rates. However, the broken beacon assay provided the significant analytical advantages of i) a real-time and continuous measurement, ii) no requirement for the use of radiolabels or gel-based analysis, and iii) substantial time and labor savings.
RNA AMPLIFICATION; REAL-TIME; RNA POLYMERASE; FLUORESCENCE; DISPLACEMENT PROBES; DETECTION
The robustness of a recently developed diagnostic microarray for influenza, the MChip, was evaluated with 16 historic subtype H1N1 influenza A viruses (A/H1N1), including A/Brevig Mission/1/1918. The matrix gene segments from all 16 viruses were successfully detected on the array. An artificial neural network trained with temporally related A/H1N1 viruses identified A/Brevig Mission/1/1918 as influenza virus A/H1N1 with 94% probability.