Primer Extension Assay Research Articles

Preparation of Stable Nitrogen Mustard DNA Interstrand Cross-Link Analogs for Biochemical and Cell Biological Studies.

Nitrogen mustards (NMs) react with two bases on opposite strands of a DNA duplex to form a covalent linkage, yielding adducts called DNA interstrand cross-links (ICLs). This prevents helix unwinding, blocking essential processes such as replication and transcription. Accumulation of ICLs causes cell death in rapidly dividing cells, especially cancer cells, making ICL-forming agents like NMs valuable in chemotherapy. However, the repair of ICLs can contribute to chemoresistance through a number of pathways that remain poorly understood. One of the impediments in studying NM ICL repair mechanisms has been the difficulty of generating site-specific and stable NM ICLs. Here, we describe two methods to synthesize stable NM ICL analogs that make it possible to study DNA ICL repair. As a proof of principle of the suitability of these NM ICLs for biochemical and cell biological studies, we use them in primer extension assays with Klenow polymerase. We show that the NM ICL analogs block the polymerase activity and remain intact under our experimental conditions.

Analysis of Yeast Telomerase by Primer Extension Assays.

Telomeres are specialized nucleoprotein structures located at eukaryotic chromosomal termini, which are required for chromosome stability and are maintained by a reverse transcriptase named telomerase. Budding yeast has served as an extremely useful model system for analyzing telomere maintenance because the organism offers a wide range of genetic and biochemical tools. Several milestones in telomerase research have been reached through investigation of the yeast system. For example, the consequence of telomerase loss was first characterized in the budding yeast Saccharomyces cerevisiae. The catalytic component of telomerase (telomerase reverse transcriptase; TERT) was likewise initially cloned from this organism. Moreover, much of the current understanding of the structure and function of the telomerase complex was derived from yeast studies. In this chapter, we discuss one of the most useful tools for investigating yeast telomerase mechanisms and regulation: the primer extension assay. This assay can be used to examine the overall activity as well as the processivity of telomerase, which represents a unique aspect of telomerase enzymology. It can also be employed to analyze the mechanisms of telomerase regulatory proteins.

Physiological Mg2+ Conditions Significantly Alter the Inhibition of HIV-1 and HIV-2 Reverse Transcriptases by Nucleoside and Non-Nucleoside Inhibitors in Vitro.

Reverse transcriptases (RTs) are typically assayed in vitro with 5-10 mM Mg2+, whereas the free Mg2+ concentration in cells is much lower. Artificially high Mg2+ concentrations used in vitro can misrepresent different properties of human immunodeficiency virus (HIV) RT, including fidelity, catalysis, pausing, and RNase H activity. Here, we analyzed nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in primer extension assays at different concentrations of free Mg2+. At low concentrations of Mg2+, NRTIs and dideoxynucleotides (AZTTP, ddCTP, ddGTP, and 3TCTP) inhibited HIV-1 and HIV-2 RT synthesis less efficiently than they did with large amounts of Mg2+, whereas inhibition by the "translocation-defective RT inhibitor" EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) was unaffected by Mg2+ concentrations. Steady-state kinetic analyses revealed that the reduced level of inhibition at low Mg2+ concentrations resulted from a 3-9-fold (depending on the particular nucleotide and inhibitor) less efficient incorporation (based on kcat/Km) of these NRTIs under this condition compared to incorporation of natural dNTPs. In contrast, EFdATP was incorporated with an efficiency similar to that of its analogue dATP at low Mg2+ concentrations. Unlike NRTIs, NNRTIs (nevirapine, efavirenz, and rilviripine), were approximately 4-fold (based on IC50 values) more effective at low than at high Mg2+ concentrations. Drug-resistant HIV-1 RT mutants also displayed the Mg2+-dependent difference in susceptibility to NRTIs and NNRTIs. In summary, analyzing the efficiency of inhibitors under more physiologically relevant low-Mg2+ conditions yielded results dramatically different from those from measurements using commonly employed high-Mg2+ in vitro conditions. These results also emphasize differences in Mg2+ sensitivity between the translocation inhibitor EFdATP and other NRTIs.

5'-Terminal nucleotide variations in human cytoplasmic tRNAHisGUG and its 5'-halves.

Transfer RNAs (tRNAs) are fundamental adapter components of translational machinery. tRNAs can further serve as a source of tRNA-derived noncoding RNAs that play important roles in various biological processes beyond translation. Among all species of tRNAs, tRNAHisGUG has been known to uniquely contain an additional guanosine residue at the −1 position (G−1) of its 5′-end. To analyze this −1 nucleotide in detail, we developed a TaqMan qRT-PCR method that can distinctively quantify human mature cytoplasmic tRNAHisGUG containing G−1, U−1, A−1, or C−1 or lacking the −1 nucleotide (starting from G1). Application of this method to the mature tRNA fraction of BT-474 breast cancer cells revealed the presence of tRNAHisGUG containing U−1 as well as the one containing G−1. Moreover, tRNA lacking the −1 nucleotide was also detected, thus indicating the heterogeneous expression of 5′-tRNAHisGUG variants. A sequence library of sex hormone-induced 5′-tRNA halves (5′-SHOT-RNAs), identified via cP-RNA-seq of a BT-474 small RNA fraction, also demonstrated the expression of 5′-tRNAHisGUG halves containing G−1, U−1, or G1 as 5′-terminal nucleotides. Although the detected 5′-nucleotide species were identical, the relative abundances differed widely between mature tRNA and 5′-half from the same BT-474 cells. The majority of mature tRNAs contained the −1 nucleotide, whereas the majority of 5′-halves lacked this nucleotide, which was biochemically confirmed using a primer extension assay. These results reveal the novel identities of tRNAHisGUG molecules and provide insights into tRNAHisGUG maturation and the regulation of tRNA half production.

Mutagenicity of Ochratoxin A: Role for a Carbon-Linked C8-Deoxyguanosine Adduct?

Ochratoxin A (OTA) is a fungal toxin that is considered to be a potent kidney carcinogen in rodent models. The toxin produces double strand breaks and has a propensity for deletions, single-base substitutions, and insertions. The toxin reacts covalently with DNA to afford a C8-2'-deoxyguanosine carbon-linked adduct (OT-dG) as the major lesion in animal tissues. Incorporation of model C-linked C8-aryl-dG adducts into the G3 site of the NarI sequence demonstrates a tendency to induce base substitutions and deletion mutations in primer extension assays using model polymerases. The degree of misincorporation induced by the C-linked C8-dG adducts correlates with an ability to adopt the promutagenic syn conformation within the NarI duplex as predicted by molecular dynamics (MD) simulations. MD simulations of the OT-dG adduct within the NarI duplex predict an even greater degree of conformational flexibility, suggesting enhanced in vitro mutagenicity compared to the simpler model C-linked C8-dG adducts. Together these findings support the role of OT-dG in promoting OTA-mediated mutagenicity and carcinogenicity in animal studies.

Replicative Bypass of O2-Alkylthymidine Lesions in Vitro.

DNA alkylation represents a major type of DNA damage and is generally unavoidable due to ubiquitous exposure to various exogenous and endogenous sources of alkylating agents. Among the alkylated DNA lesions, O2-alkylthymidines (O2-alkyldT) are known to be persistent and poorly repaired in mammalian systems and have been shown to accumulate in the esophagus, lung, and liver tissue of rats treated with tobacco-specific N-nitrosamines, i.e., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). In this study, we assessed the replicative bypass of a comprehensive set of O2-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, in template DNA by conducting primer extension assays with the use of major translesion synthesis DNA polymerases. The results showed that human Pol η and, to a lesser degree, human Pol κ, but not human polymerase ι or yeast polymerase ζ, were capable of bypassing all O2-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that human Pol η exhibited high frequencies of misincorporation of dCMP opposite those O2-alkyldT lesions bearing a longer straight-chain alkyl group. However, the nucleotide misincorporation opposite branched-chain lesions was not selective, with dCMP, dGMP, and dTMP being inserted at similar efficiencies, though the total frequencies of nucleotide misincorporation opposite the branched-chain lesions differed and followed the order of O2-iPrdT > O2-iBudT > O2-sBudT. Together, the results from the present study provided important knowledge about the effects of the length and structure of the alkyl group in the O2-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.

Suspension Array for Multiplex Detection of Eight Fungicide-Resistance Related Alleles in Botrytis cinerea.

A simple and high-throughput assay to detect fungicide resistance is required for large-scale monitoring of the emergence of resistant strains of Botrytis cinerea. Using suspension array technology performed on a Bio-Plex 200 System, we developed a single-tube allele-specific primer extension assay that can simultaneously detect eight alleles in one reaction. These eight alleles include E198 and 198A of the β-Tubulin gene (BenA), H272 and 272Y of the Succinate dehydrogenase iron–sulfur subunit gene (SdhB), I365 and 365S of the putative osmosensor histidine kinase gene (BcOS1), and F412 and 412S of the 3-ketoreductase gene (erg27). This assay was first established and optimized with eight plasmid templates containing the DNA sequence variants BenA-E198, BenA-198A, SdhB-H272, SdhB-272Y, BcOS1-I365, BcOS1-365S, erg27-F412, and erg27-412S. Results indicated that none of the probes showed cross-reactivity with one another. The minimum limit of detection for these genotypes was one copy per test. Four mutant plasmids were mixed with 10 ng/μL wild-type genomic DNA in different ratios. Detection sensitivity of mutant loci was 0.45% for BenA-E198A, BcOS1-I365S, and erg27-F412S, and was 4.5% for SdhB-H272Y. A minimum quantity of 0.1 ng of genomic DNA was necessary to obtain reliable results. This is the first reported assay that can simultaneously detect mutations in BenA, SdhB, BcOS1, and erg27.

A deafness-associated tRNAAsp mutation alters the m1G37 modification, aminoacylation and stability of tRNAAsp and mitochondrial function.

In this report, we investigated the pathogenic mechanism underlying the deafness-associated mitochondrial(mt) tRNAAsp 7551A > G mutation. The m.7551A > G mutation is localized at a highly conserved nucleotide(A37), adjacent (3′) to the anticodon, which is important for the fidelity of codon recognition and stabilization in functional tRNAs. It was anticipated that the m.7551A > G mutation altered the structure and function of mt-tRNAAsp. The primer extension assay demonstrated that the m.7551A > G mutation created the m1G37 modification of mt-tRNAAsp. Using cybrid cell lines generated by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mitochondrial DNA(mtDNA)-less (ρo) cells, we demonstrated the significant decreases in the efficiency of aminoacylation and steady-state level of mt-tRNAAsp in mutant cybrids, compared with control cybrids. A failure in metabolism of mt-tRNAAsp caused the variable reductions in mtDNA-encoded polypeptides in mutant cybrids. Impaired mitochondrial translation led to the respiratory phenotype in mutant cybrids. The respiratory deficiency lowed mitochondrial adenosine triphosphate production and increased the production of oxidative reactive species in mutant cybrids. Our data demonstrated that mitochondrial dysfunctions caused by the m.7551A > G mutation are associated with deafness. Our findings may provide new insights into the pathophysiology of maternally transmitted deafness that was manifested by altered nucleotide modification of mitochondrial tRNA.

Plasmid pPCP1-derived sRNA HmsA promotes biofilm formation of Yersinia pestis.

BackgroundThe ability of Yersinia pestis to form a biofilm is an important characteristic in flea transmission of this pathogen. Y. pestis laterally acquired two plasmids (pPCP1and pMT1) and the ability to form biofilms when it evolved from Yersinia pseudotuberculosis. Small regulatory RNAs (sRNAs) are thought to play a crucial role in the processes of biofilm formation and pathogenesis.ResultsA pPCP1-derived sRNA HmsA (also known as sR084) was found to contribute to the enhanced biofilm formation phenotype of Y. pestis. The concentration of c-di-GMP was significantly reduced upon deletion of the hmsA gene in Y. pestis. The abundance of mRNA transcripts determining exopolysaccharide production, crucial for biofilm formation, was measured by primer extension, RT-PCR and lacZ transcriptional fusion assays in the wild-type and hmsA mutant strains. HmsA positively regulated biofilm synthesis-associated genes (hmsHFRS, hmsT and hmsCDE), but had no regulatory effect on the biofilm degradation-associated gene hmsP. Interestingly, the recently identified biofilm activator sRNA, HmsB, was rapidly degraded in the hmsA deletion mutant. Two genes (rovM and rovA) functioning as biofilm regulators were also found to be regulated by HmsA, whose regulatory effects were consistent with the HmsA-mediated biofilm phenotype.ConclusionHmsA potentially functions as an activator of biofilm formation in Y. pestis, implying that sRNAs encoded on the laterally acquired plasmids might be involved in the chromosome-based regulatory networks implicated in Y. pestis-specific physiological processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0793-5) contains supplementary material, which is available to authorized users.

In Vitro Lesion Bypass Studies of O(4)-Alkylthymidines with Human DNA Polymerase η.

Environmental exposure and endogenous metabolism can give rise to DNA alkylation. Among alkylated nucleosides, O(4)-alkylthymidine (O(4)-alkyldT) lesions are poorly repaired in mammalian systems and may compromise the efficiency and fidelity of cellular DNA replication. To cope with replication-stalling DNA lesions, cells are equipped with translesion synthesis DNA polymerases that are capable of bypassing various DNA lesions. In this study, we assessed human DNA polymerase η (Pol η)-mediated bypass of various O(4)-alkyldT lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, (R)-sBu, or (S)-sBu, in template DNA by conducting primer extension and steady-state kinetic assays. Our primer extension assay results revealed that human Pol η, but not human polymerases κ and ι or yeast polymerase ζ, was capable of bypassing all O(4)-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η preferentially misincorporated dGMP opposite O(4)-alkyldT lesions with a straight-chain alkyl group. The nucleotide misincorporation opposite most lesions with a branched-chain alkyl group was, however, not selective, where dCMP, dGMP, and dTMP were inserted at similar efficiencies opposite O(4)-iPrdT, O(4)-iBudT, and O(4)-(R)-sBudT. These results provide important knowledge about the effects of the length and structure of the alkyl group in O(4)-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.

The Master Quorum-Sensing Regulator OpaR is Activated Indirectly by H-NS in Vibrio parahaemolyticus.

AphA and OpaR are the master regulators of quorum sensing in Vibrio parahaemolyticus and there is reciprocal negative regulation between AphA and OpaR. The histone-like nucleoid structure (H-NS) protein is a global transcriptional repressor of horizontally transferred genes, but a few prokaryotic genes, for example flagella genes, are stimulated by H-NS. The regulation of opaR by H-NS was investigated using the following methods: primer extension, LacZ fusion, quantitative RT-PCR, and electrophoretic mobility shift assay. One transcription start site located at 74bp upstream of opaR was detected and its activity was induced by H-NS. Therefore, a single H-NS-dependent promoter was transcribed for opaR in V. parahaemolyticus. Because the H-NS protein could not bind to the upstream region of opaR and H-NS does not regulate aphA, indirect activation of the transcription of opaR by H-NS cannot be mediated by the AphA repressor.

Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys.

The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.

Bacillus subtilis 6S-2 RNA serves as a template for short transcripts in vivo

The global transcriptional regulator 6S RNA is abundant in a broad range of bacteria. The RNA competes with DNA promoters for binding to the housekeeping RNA polymerase (RNAP) holoenzyme. When bound to RNAP, 6S RNA serves as a transcription template for RNAP in an RNA-dependent RNA polymerization reaction. The resulting short RNA transcripts (so-called product RNAs = pRNAs) can induce a stable structural rearrangement of 6S RNA when reaching a certain length. This rearrangement leads to the release of RNAP and thus the recovery of transcription at DNA promoters. While most bacteria express a single 6S RNA, some harbor a second 6S RNA homolog (termed 6S-2 RNA in Bacillus subtilis). Bacillus subtilis 6S-2 RNA was recently shown to exhibit essentially all hallmark features of a bona fide 6S RNA in vitro, but evidence for the synthesis of 6S-2 RNA-derived pRNAs in vivo has been lacking so far. This raised the question of whether the block of RNAP by 6S-2 RNA might be lifted by a mechanism other than pRNA synthesis. However, here we demonstrate that 6S-2 RNA is able to serve as a template for pRNA synthesis in vivo. We verify this finding by using three independent approaches including a novel primer extension assay. Thus, we demonstrate the first example of an organism that expresses two distinct 6S RNAs that both exhibit all mechanistic features defined for this type of regulatory RNA.

Tracking of the origin of recurrent mutations of the BRCA1 and BRCA2 genes in the North-East of Italy and improved mutation analysis strategy.

BackgroundAbout 20 % of hereditary breast cancers are caused by mutations in BRCA1 and BRCA2 genes. Since BRCA1 and BRCA2 mutations may be spread throughout the gene, genetic testing is usually performed by direct sequencing of entire coding regions. In some populations, especially if relatively isolated, a few number of recurrent mutations is reported, sometimes caused by founder effect.MethodsBRCA1 and BRCA2 screening for mutations was carried out on 1114 breast and/or ovarian cancer patients complying with the eligibility criteria for BRCA testing. Haplotype analysis was performed on the probands carrying recurrent mutations and their relatives, using two sets of microsatellite markers covering the BRCA1 (D17S588, D17S806, D17S902, D17S1325, D17S855, D17S1328, D17S800, and D17S250) and BRCA2 (D13S220, D13S267, D13S171, D13S1701, D13S1698, D13S260, D13S290, D13S1246) loci. The DMLE + 2.2 software was used to estimate the age of BRCA1 c.676delT and BRCA2 c.7806-2A > G. A multiplex PCR and two different primer extension assays were optimized and used for genotyping the recurrent mutations of the two genes.ResultsIn the time frame of almost 20 years of genetic testing, we have found that five BRCA1 and three BRCA2 mutations are recurrent in a substantial subset of carriers from North-East Italy and neighboring Istria, where they represent more than 50 % of all mutations. Microsatellite analyses identified a common haplotype of different length for each mutation. Age estimation of BRCA1 c.676delT and BRCA2 c.7806-2A > G mutations revealed that they arose in the Friuli Venezia Giulia area about 86 and 94 generations ago, respectively. Suggestion of an association between BRCA2 c.7806-2A > G and risk of breast cancer in males has emerged. Finally, we developed a simple and efficient pre-screeening test, performing an in-house primer extension SNaPshot® assay for the rapid identification of the eight recurrent mutations.ConclusionsProofs of common ancestry has been obtained for the eight recurrent mutations. The observed genotype-phenotype correlation and the proposed rapid mutation detection strategy could improve the clinical management of breast and ovarian patients in North-East of Italy and neighboring geographic areas.

Single Base Primer Extension Assay (SNaPshot) for Rapid Detection of Human Immunodeficiency Virus - 1 Drug Resistance Mutations

The 3' primer extension assay can be employed to interrogate multiple known single nucleotide polymorphisms (SNPs) simultaneously using a multiplex approach. Over 200 Human Immunodeficiency Virus-1 (HIV-1) mutations are currently associated with drug resistance. Identifying these mutations assist in taking appropriate therapeutic decisions such that superior drug regimens can be designed for keeping the viral load below pathogenic level. Presently, reverse transcription of HIV-1 RNA followed by amplification of the pol gene and subsequent nucleotide sequencing is a popular method of HIV-1 drug resistance genotyping. However, it is still a time consuming and laborious protocol and require significant bioinformatics analysis in order to decipher the significance of mutations encountered during analysis. In this study we analyzed 75 HIV-1 positive clinical samples for 2 representative mutations, viz., M41L and M184V using a 3' primer extension protocol popularly known as SNaPshot assay. Twenty four and 36% of the patients were found to harbor the critical M41L and M184V mutations respectively while 20% were found to contain both the mutations together. The results were 100% concordant with DNA sequencing assay which was used as a gold standard. The results substantiate the cost, speed and economy-related advantages of this technology platform for interrogating known mutations of significance within the HIV-1 genome.

Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases.

Cfr and RlmN methyltransferases both modify adenine 2503 in 23S rRNA (Escherichia coli numbering). RlmN methylates position C2 of adenine while Cfr methylates position C8, and to a lesser extent C2, conferring antibiotic resistance to peptidyl transferase inhibitors. Cfr and RlmN show high sequence homology and may be evolutionarily linked to a common ancestor. To explore their individual specificity and similarity we performed two sets of experiments. We created a homology model of Cfr and explored the C2/C8 specificity using docking and binding energy calculations on the Cfr homology model and an X-ray structure of RlmN. We used a trinucleotide as target sequence and assessed its positioning at the active site for methylation. The calculations are in accordance with different poses of the trinucleotide in the two enzymes indicating major evolutionary changes to shift the C2/C8 specificities. To explore interchangeability between Cfr and RlmN we constructed various combinations of their genes. The function of the mixed genes was investigated by RNA primer extension analysis to reveal methylation at 23S rRNA position A2503 and by MIC analysis to reveal antibiotic resistance. The catalytic site is expected to be responsible for the C2/C8 specificity and most of the combinations involve interchanging segments at this site. Almost all replacements showed no function in the primer extension assay, apart from a few that had a weak effect. Thus Cfr and RlmN appear to be much less similar than expected from their sequence similarity and common target.

Fluorogenic Labeling of 5-Formylpyrimidine Nucleotides in DNA and RNA.

5-Formylcytosine (5fC) and 5-formyluracil (5fU) are natural nucleobase modifications that are generated by oxidative modification of 5-methylcytosine and thymine (or 5-methyluracil). Herein, we describe chemoselective labeling of 5-formylpyrimidine nucleotides in DNA and RNA by fluorogenic aldol-type condensation reactions with 2,3,3-trimethylindole derivatives. Mild and specific reaction conditions were developed for 5fU and 5fC to produce hemicyanine-like chromophores with distinct photophysical properties. Residue-specific detection was established by fluorescence readout as well as primer-extension assays. The reactions were optimized on DNA oligonucleotides and were equally suitable for the modification of 5fU- and 5fC-modified RNA. This direct labeling approach of 5-formylpyrimidines is expected to help in elucidating the occurrence, enzymatic transformations, and functional roles of these epigenetic/epitranscriptomic nucleobase modifications in DNA and RNA.

Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors.

Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semi-quantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of ∼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/quantification of specific RNA is required.

Synthesis of C8-N-Arylamine-Modified 2'-Deoxyguanosine-5'-Triphosphates and Their Effects on Primer Extension by DNA Polymerases.

C8-N-arylamine adducts of 2'-deoxyguanosine (2'-dG) play an important role in the induction of the chemical carcinogenesis caused by aromatic amines. C8-N-acetyl-N-arylamine dG adducts that differ in their substitution pattern in the aniline moiety were converted by cycloSal technology into the corresponding C8-N-acetyl-N-arylamine-2'-deoxyguanosine-5'-triphosphates and C8-NH-arylamine-2'-deoxyguanosine-5'-triphosphates. Their conformation preference has been investigated by NOE spectroscopy and DFT calculations. The substrate properties of the C8-dG adducts were studied in primer-extension assays by using Klenow fragment exo(-) of Escherichia coli DNA polymerase I and human DNA polymerase β. It was shown that the incorporation was independent of the substitution pattern in the aryl moiety and the N-acetyl group. Although the triphosphates were poor substrates for the human polymerases, they were incorporated twice before the termination of the elongation process occurred; this might demonstrate the importance of C8-N-arylamine-2'-deoxyguanosine-5'-triphosphates in chemical carcinogenesis.

Efficient detection of frequent eIF2B mutations for the rapid molecular diagnosis of CACH/VWM syndrome

ObjectivesThe aim of this study was to develop a reliable, rapid and cost-effective molecular diagnostic assay allowing widespread routine investigation of eIF2B-related disorders (CACH/VWM syndrome). This heterogeneous disease is caused by autosomal recessive mutations in the genes encoding the five subunits of the translation-initiation factor eIF2B. Such a diagnostic method would be particularly adapted to the apparently acute presentation of the disease. Design and methodsWe developed a multiplex PCR amplification method for 7 genomic regions of the eIF2B genes in a single run. This method targeted the 8 most frequent mutations representing 61.4% of all mutations identified to date in our laboratory. These mutations affected eIF2B2 exon 5, eIF2B3 exon 2, eIF2B4 exons 8 and 11 and eIF2B5 exons 5, 7 and 8. PCR products were then pooled and subjected to a primer-extension assay validated using previously genotyped samples. ResultsThe results were compared to screening and/or direct sequencing methods: 100% agreement between methods confirmed equivalent sensitivity and specificity. The new assay was highly superior in terms of cost, time to results and robustness despite sample heterogeneity. ConclusionsThis genotyping strategy allows the detection of all eIF2B mutations targeted. A second multiplex primer-extension assay is in development to detect the 11 next-most frequent mutations, thus raising the global detection rate to 76.8%. Our approach is widely applicable as it involves standard techniques and equipment. Moreover, it can easily be further adapted to the clinical and genetic heterogeneity of eIF2B-related disorders by including or excluding mutations.