Detection and identification of single ribonucleotide monophosphates using a dual in-plane nanopore sensor made in a thermoplastic via replication.

We report the generation of ∼8 nm dual in-plane pores fabricated in a thermoplastic via nanoimprint lithography (NIL). These pores were connected in series with nanochannels, one of which served as a flight tube to allow the identification of single molecules based on their molecular-dependent apparent mobilities (i.e., dual in-plane nanopore sensor). Two different thermoplastics were investigated including poly(methyl methacrylate), PMMA, and cyclic olefin polymer, COP, as the substrate for the sensor both of which were sealed using a low glass transition cover plate (cyclic olefin co-polymer, COC) that could be thermally fusion bonded to the PMMA or COP substrate at a temperature minimizing nanostructure deformation. Unique to these dual in-plane nanopore sensors was two pores flanking each side of the nanometer flight tube (50 × 50 nm, width × depth) that was 10 µm in length. The utility of this dual in-plane nanopore sensor was evaluated to not only detect, but also identify single ribonucleotide monophosphates (rNMPs) by using the travel time (time-of-flight, ToF), the resistive pulse event amplitude, and the dwell time. In spite of the relatively large size of these in-plane pores (∼8 nm effective diameter), we could detect via resistive pulse sensing (RPS) single rNMP molecules at a mass load of 3.9 fg, which was ascribed to the unique structural features of the nanofluidic network and the use of a thermoplastic with low relative dielectric constants, which resulted in a low RMS noise level in the open pore current. Our data indicated that the identification accuracy of individual rNMPs was high, which was ascribed to an improved chromatographic contribution to the nano-electrophoresis apparent mobility. With the ToF data only, the identification accuracy was 98.3%. However, when incorporating the resistive pulse sensing event amplitude and dwell time in conjunction with the ToF and analyzed via principal component analysis (PCA), the identification accuracy reached 100%. These findings pave the way for the realization of a novel chip-based single-molecule RNA sequencing technology.

Quantification of all 12 canonical ribonucleotides by real-time fluorogenic in vitro transcription.

Enzymatic methods to quantify deoxyribonucleoside triphosphates have existed for decades. In contrast, no general enzymatic method to quantify ribonucleoside triphosphates (rNTPs), which drive almost all cellular processes and serve as precursors of RNA, exists to date. ATP can be measured with an enzymatic luminometric method employing firefly luciferase, but the quantification of other ribonucleoside mono-, di-, and triphosphates is still a challenge for a non-specialized laboratory and practically impossible without chromatography equipment. To allow feasible quantification of ribonucleoside phosphates in any laboratory with typical molecular biology and biochemistry tools, we developed a robust microplate assay based on real-time detection of the Broccoli RNA aptamer during in vitro transcription. The assay employs the bacteriophage T7 and SP6 RNA polymerases, two oligonucleotide templates encoding the 49-nucleotide Broccoli aptamer, and a high-affinity fluorogenic aptamer-binding dye to quantify each of the four canonical rNTPs. The inclusion of nucleoside mono- and diphosphate kinases in the assay reactions enabled the quantification of the mono- and diphosphate counterparts. The assay is inherently specific and tolerates concentrated tissue and cell extracts. In summary, we describe the first chromatography-free method to quantify ATP, ADP, AMP, GTP, GDP, GMP, UTP, UDP, UMP, CTP, CDP and CMP in biological samples.

AICAr to SAICAr ratio can serve as additional marker of AICAr use.

AICAr (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, commonly referred to as AICAR) is an adenosine monophosphate-activated protein kinase agonist previously investigated for its therapeutic potential which has been shown to improve exercise performance in laboratory animals. For this reason, the World Anti-Doping Agency prohibits the use of AICAr in sports. AICAr can easily be detected by means of liquid chromatography-mass spectrometry, but being an endogenous metabolite, it cannot be discriminated from AICAr of a non-natural origin. Population-based concentration thresholds have been suggested as a means to identify suspicious samples that would require further analysis by carbon isotope ratio mass spectrometry (CIR); however, it remains at the discretion of the laboratory how to apply them. Here, the urinary ratio of AICAr to SAICA-riboside (SAICAr) that is a closely related purine metabolite was investigated. In an athlete population of 5517 samples, this ratio was relatively narrowly distributed with median values and 99th percentiles of 3.3 and 9.3, and 4.2 and 14 in male and female athletes, respectively. Analysis of urine samples obtained from an AICAr administration study demonstrated that the AICAr/SAICAr ratio can serve in addition to AICAr concentration as a valuable diagnostic trigger for follow-up analysis by CIR. Conceivably, this combination can offer better retrospectivity than AICAr concentration alone by allowing to decrease the AICAr concentration threshold without significantly increasing the number of suspicious samples.

Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples.

Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for understanding diseases caused by defects in dNTP metabolism. The dNTPs are measured by methods based on either HPLC or DNA polymerization. An advantage with the HPLC-based techniques is that the parallel analysis of ribonucleoside triphosphates (rNTPs) can serve as an internal quality control of nucleotide integrity and extraction efficiency. We have developed a Freon-free trichloroacetic acid-based method to extract cellular nucleotides and an isocratic reverse phase HPLC-based technique that is able to separate dNTPs, rNTPs and ADP in a single run. The ability to measure the ADP levels improves the control of nucleotide integrity, and the use of an isocratic elution overcomes the shifting baseline problems in previously developed gradient-based reversed phase protocols for simultaneously measuring dNTPs and rNTPs. An optional DNA-polymerase-dependent step is used for confirmation that the dNTP peaks do not overlap with other components of the extracts, further increasing the reliability of the analysis. The method is compatible with a wide range of biological samples and has a sensitivity better than other UV-based HPLC protocols, closely matching that of mass spectrometry-based detection.

Glutamine Antagonist GA-607 Causes a Dramatic Accumulation of FGAR which can be used to Monitor Target Engagement.

BACKGROUND: Metabolomic analyses from our group and others have shown that tumors treated with glutamine antagonists (GA) exhibit robust accumulation of formylglycinamide ribonucleotide (FGAR), an intermediate in the de novo purine synthesis pathway. The increase in FGAR is attributed to the inhibition of the enzyme FGAR amidotransferase (FGAR-AT) that catalyzes the ATP-dependent amidation of FGAR to formylglycinamidine ribonucleotide (FGAM). While perturbation of this pathway resulting from GA therapy has long been recognized, no study has reported systematic quantitation and analyses of FGAR in plasma and tumors. OBJECTIVE: Herein, we aimed to evaluate the efficacy of our recently discovered tumor-targeted GA prodrug, GA-607 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate), and demonstrate its target engagement by quantification of FGAR in plasma and tumors. METHODS: Efficacy and pharmacokinetics of GA-607 were evaluated in a murine EL4 lymphoma model followed by global tumor metabolomic analysis. Liquid chromatography-mass spectrometry (LC-MS) based methods employing the ion-pair chromatography approach were developed and utilized for quantitative FGAR analyses in plasma and tumors. RESULTS: GA-607 showed preferential tumor distribution and robust single-agent efficacy in a murine EL4 lymphoma model. While several metabolic pathways were perturbed by GA-607 treatment, FGAR showed the highest increase qualitatively. Using our newly developed sensitive and selective LC-MS method, we showed a robust >80- and >10- fold increase in tumor and plasma FGAR levels, respectively, with GA-607 treatment. CONCLUSION: These studies describe the importance of FGAR quantification following GA therapy in cancer and underscore its importance as a valuable pharmacodynamic marker in the preclinical and clinical development of GA therapies.

Evaluation of repair activity by quantification of ribonucleotides in the genome.

Ribonucleotides incorporated in the genome are a source of endogenous DNA damage and also serve as signals for repair. Although recent advances of ribonucleotide detection by sequencing, the balance between incorporation and repair of ribonucleotides has not been elucidated. Here, we describe a competitive sequencing method, Ribonucleotide Scanning Quantification sequencing (RiSQ-seq), which enables absolute quantification of misincorporated ribonucleotides throughout the genome by background normalization and standard adjustment within a single sample. RiSQ-seq analysis of cells harboring wild-type DNA polymerases revealed that ribonucleotides were incorporated nonuniformly in the genome with a 3'-shifted distribution and preference for GC sequences. Although ribonucleotide profiles in wild-type and repair-deficient mutant strains showed a similar pattern, direct comparison of distinct ribonucleotide levels in the strains by RiSQ-seq enabled evaluation of ribonucleotide excision repair activity at base resolution and revealed the strand bias of repair. The distinct preferences of ribonucleotide incorporation and repair create vulnerable regions associated with indel hotspots, suggesting that repair at sites of ribonucleotide misincorporation serves to maintain genome integrity and that RiSQ-seq can provide an estimate of indel risk.

Electrokinetic identification of ribonucleotide monophosphates (rNMPs) using thermoplastic nanochannels.

With advances in the design and fabrication of nanofluidic devices during the last decade, there have been a few reports on nucleic acid analysis using nanoscale electrophoresis. The attractive nature of nanofluidics is the unique phenomena associated with this length scale that are not observed using microchip electrophoresis. Many of these effects are surface-related and include electrostatics, surface roughness, van der Waals interactions, hydrogen bonding, and the electric double layer. The majority of reports related to nanoscale electrophoresis have utilized glass-based devices, which are not suitable for broad dissemination into the separation community because of the sophisticated, time consuming, and high-cost fabrication methods required to produce the relevant devices. In this study, we report the use of thermoplastic nanochannels (110 nm x 110 nm, depth x width) for the free solution electrokinetic analysis of ribonucleotide monophosphates (rNMPs). Thermoplastic devices with micro- and nanofluidic networks were fabricated using nanoimprint lithography (NIL) with the structures enclosed via thermal fusion bonding of a cover plate to the fluidic substrate. Unique to this report is that we fabricated devices in cyclic olefin copolymer (COC) that was thermally fusion bonded to a COC cover plate. Results using COC/COC devices were compared to poly(methyl methacrylate), PMMA, devices with a COC cover plate. Our results indicated that at pH = 7.9, the electrophoresis in free solution resulted in an average resolution of the rNMPs >4 (COC/COC device range = 1.94 - 8.88; PMMA/COC device range = 1.4 - 7.8) with some of the rNMPs showing field-dependent electrophoretic mobilities. Baseline separation of the rNMPs was not possible using PMMA- or COC-based microchip electrophoresis. We also found that COC/COC devices could be assembled and UV/O3 activated after device assembly with the dose of the UV/O3 affecting the magnitude of the electroosmotic flow, EOF. In addition, the bond strength between the substrate and cover plate of unmodified COC/COC devices was higher compared to PMMA/COC devices. The large differences in the electrophoretic mobilities of the rNMPs afforded by nanoscale electrophoresis will enable a new single-molecule sequencing platform we envision, which uses molecular-dependent electrophoretic mobilities to identify the constituent rNMPs generated from an intact RNA molecule using a processive exonuclease. With optimized nanoscale electrophoresis, the rNMPs could be identified via mobility matching at an accuracy >99% in both COC/COC and PMMA/COC devices.

High-resolution ion mobility spectrometry-mass spectrometry of isomeric/isobaric ribonucleotide variants.

In this report, we explored the benefits of cyclic ion mobility (cIM) mass spectrometry in the analysis of isomeric post-transcriptional modifications of RNA. Standard methyl-cytidine samples were initially utilized to test the ability to correctly distinguish different structures sharing the same elemental composition and thus molecular mass. Analyzed individually, the analytes displayed characteristic arrival times (tD ) determined by the different positions of the modifying methyl groups onto the common cytidine scaffold. Analyzed in mixture, the widths of the respective signals resulted in significant overlap that initially prevented their resolution on the tD scale. The separation of the four isomers was achieved by increasing the number of passes through the cIM device, which enabled to fully differentiate the characteristic ion mobility behaviors associated with very subtle structural variations. The placement of the cIM device between the mass-selective quadrupole and the time-of-flight analyzer allowed us to perform gas-phase activation of each of these ion populations, which had been first isolated according to a common mass-to-charge ratio and then separated on the basis of different ion mobility behaviors. The observed fragmentation patterns confirmed the structures of the various isomers thus substantiating the benefits of complementing unique tD information with specific fragmentation data to reach more stringent analyte identification. These capabilities were further tested by analyzing natural mono-nucleotide mixtures obtained by exonuclease digestion of total RNA extracts. In particular, the combination of cIM separation and post-mobility dissociation allowed us to establish the composition of methyl-cytidine and methyl-adenine components present in the entire transcriptome of HeLa cells. For this reason, we expect that this technique will benefit not only epitranscriptomic studies requiring the determination of identity and expression levels of RNA modifications, but also metabolomics investigations involving the analysis of natural extracts that may possibly contain subsets of isomeric/isobaric species.

Diazo Reagent Labeling with Mass Spectrometry Analysis for Sensitive Determination of Ribonucleotides in Living Organisms.

Ribonucleotide analogues and their related phosphorylated metabolites play critical roles in tumor metabolism. However, determination of the endogenous ribonucleotides from the complex biological matrix is still a challenge due to their high structural similarity and high polarity that will lead to the low retention and low detection sensitivities by liquid chromatogram mass spectrometry analysis. In this study, we developed the diazo reagent labeling strategy with mass spectrometry analysis for sensitive determination of ribonucleotides in the living organism. A pair of light and heavy stable isotope labeling reagents, 2-(diazomethyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-(diazomethyl)-N-methyl-N-phenyl-benzamide (d5-2-DMBA), were synthesized to label ribonucleotides. 2-DMBA showed high specificity and high efficiency for the labeling of ribonucleotides. Our results demonstrated that the detection sensitivities of 12 ribonucleotides increased by 17-174-fold upon 2-DMBA labeling. The obtained limits of detection (LODs) of ribonucleotides ranged from 0.07 fmol to 0.41 fmol. Using this method, we achieved the sensitive and accurate detection of ribonucleotides from only a few cells (8 cells). To the best of our knowledge, this is the highest detection sensitivity for ribonucleotides ever reported. In addition, we found that the contents of almost all of these ribonucleotides were significantly increased in human breast carcinoma tissues compared to tumor-adjacent normal tissues, suggesting that endogenous ribonucleotides may play certain functional roles in the regulation of cancer development and formation. This method also can be potentially applied in the analysis of phosphorylated compounds.

Absolute Quantification of RNA or DNA Using Acid Hydrolysis and Mass Spectrometry.

Accurate, traceable quantification of ribonucleotide or deoxyribonucleotide oligomers is achievable using acid hydrolysis and isotope dilution mass spectrometry (ID-MS). In this work, formic acid hydrolysis is demonstrated to generate stoichiometric release of nucleobases from intact oligonucleotides, which then can be measured by ID-MS, facilitating true and precise absolute quantification of RNA, short linearized DNA, or genomic DNA. Surrogate nucleobases are quantified with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow, using multiple reaction monitoring (MRM). Nucleobases were chromatographically resolved using a novel cation-exchange separation, incorporating a pH gradient. Trueness of this quantitative assay is estimated from agreement among the surrogate nucleobases and by comparison to concentrations provided for commercial materials or Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST). Comparable concentration estimates using NanoDrop spectrophotometry or established from droplet-digital polymerase chain reaction (ddPCR) techniques agree well with the results. Acid hydrolysis-ID-LC-MS/MS provides excellent quantitative selectivity and accuracy while enabling traceability to mass unit. Additionally, this approach can be uniquely useful for quantifying modified nucleobases or mixtures.

Method for Quantification of Ribonucleotides and Deoxyribonucleotides in Human Cells Using (Trimethylsilyl)diazomethane Derivatization Followed by Liquid Chromatography-Tandem Mass Spectrometry.

Investigation into intracellular ribonucleotides (RNs) and deoxyribonucleotides (dRNs) is important for studies of the mechanism of many biological processes, such as RNA and DNA synthesis and DNA repair, as well as metabolic and therapeutic efficacy of nucleoside analogues. However, current methods are still unsatisfactory for determination of nucleotides in complex matrixes. Here we describe a novel method for the determination of RN and dRN pools in cells based on fast derivatization with (trimethylsilyl)diazomethane (TMSD) followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Derivatization was accomplished in 3 min, and each derivatized nucleotide not only had a sufficient retention on reversed-phase column by introduction of methyl groups but also exhibited a unique ion transition which consequently eliminated mutual interference in LC-MS/MS. Chromatographic separation was performed on a C18 column with a simple acetonitrile-water gradient elution system, which avoided contamination and ion suppression caused by ion-pairing reagents. The developed method was fully validated and applied to the analysis of RNs and dRNs in cell samples. Moreover, results demonstrated that the applicability of this method could be extended to nucleoside analogues and their metabolites and could facilitate many applications in future studies.

Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.

Information about the intracellular concentration of dNTPs and NTPs is important for studies of the mechanisms of DNA replication and repair, but the low concentration of dNTPs and their chemical similarity to NTPs present a challenge for their measurement. Here, we describe a new rapid and sensitive method utilizing hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the simultaneous determination of dNTPs and NTPs in biological samples. The developed method showed linearity (R2 > 0.99) in wide concentration ranges and could accurately quantify dNTPs and NTPs at low pmol levels. The intra-day and inter-day precision were below 13%, and the relative recovery was between 92% and 108%. In comparison with other chromatographic methods, the current method has shorter analysis times and simpler sample pre-treatment steps, and it utilizes an ion-pair-free mobile phase that enhances mass-spectrometric detection. Using this method, we determined dNTP and NTP concentrations in actively dividing and quiescent mouse fibroblasts.

A possible mechanism for lincomycin induction of secondary metabolism in Streptomyces coelicolor A3(2).

Lincomycin forms cross-links within the peptidyl transferase loop region of the 23S ribosomal RNA (rRNA) of the 50S subunit of the bacterial ribosome, which is the site of peptide bond formation, thereby inhibiting protein synthesis. We have previously reported that lincomycin at concentrations below the minimum inhibitory concentration potentiates the production of secondary metabolites in actinomycete strains, suggesting that activation of these strains by utilizing the dose-dependent response of lincomycin could be used to effectively induce the production of cryptic secondary metabolites. Here, we aimed to elucidate the fundamental mechanisms underlying lincomycin induction of secondary metabolism in actinomycetes. In the present study, the dose-dependent response of lincomycin on gene expression of the model actinomycete Streptomyces coelicolor A3(2) and possible relationships to secondary metabolism were investigated. RNA sequencing analysis indicated that lincomycin produced enormous changes in gene expression profiles. Moreover, reverse transcription PCR and/or comparative proteome analysis revealed that in S. coelicolor A3(2), lincomycin, which was used at concentrations for markedly increased blue-pigmented antibiotic actinorhodin production, rapidly enhanced expression of the gene encoding the lincomycin-efflux ABC transporter, the 23S rRNA methyltransferase, and the ribosome-splitting factor to boost the intrinsic lincomycin resistance mechanisms and to reconstruct the probably stalled 70S ribosomes with lincomycin; and in contrast temporarily but dramatically reduced mRNA levels of housekeeping genes, such as those encoding FoF1 ATP synthase, RNA polymerase, ribosomal proteins, and transcription and translation factors, with an increase in intracellular NTPs. A possible mechanism for lincomycin induction of secondary metabolism in S. coelicolor A3(2) is discussed on the basis of these results.

Implementation of AICAR analysis by GC-C-IRMS for anti-doping purposes.

AICAR (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside), is a naturally occurring substance which is part to the World Anti-Doping Agency (WADA) Prohibited List. It is claimed to improve physical performance when administered as a supplement. As for other endogenous compounds such as steroids, the gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis remains an efficient tool to differentiate endogenous substances from exogenous ones. A protocol was described in the literature for the analysis of AICAR by GC-C-IRMS. The aim of the present study was to implement this protocol in our laboratory and to propose solutions to avoid the difficulties encountered. The first point discussed in this study is the derivatization step. Due to the structure of the AICAR molecule, conventional derivatization for GC-C-IRMS such as acetylation could not be applied and silylation was preferred. The improvement of the derivatives stability was achieved thanks to several derivatization conditions tested. This adjustment led to a reproducible derivatization pattern with the 3-TMS form as major derivative product. The second point discussed in this study is the diminution of extracts' background noise. Indeed, the implementation of the published protocol was not easy due to high performance liquid chromatography (HPLC) problems encountered when concentrated urine was injected into our system. Also, too many interferences in the endogenous reference compound fractions were observed. The addition of both a wash step before the HPLC purification and a HPLC purification step for the endogenous reference compound (ERC) fraction allowed us to increase the robustness of the method. This study presents the modified protocol compared to the original protocol as well as the evaluation of the whole method performances. Copyright © 2017 John Wiley & Sons, Ltd.

Doping control study of AICAR in post-race urine and plasma samples from horses.

Acadesine, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, commonly known as AICAR, is a naturally occurring adenosine monophosphate-activated protein kinase (AMPK) activator in many mammals, including humans and horses. AICAR has attracted considerable attention recently in the field of doping control because of a study showing the enhancement of endurance performance in unexercised or untrained mice, resulting in the term 'exercise pill'. Its use has been classified as gene doping by the World Anti-Doping Agency (WADA), and since it is endogenous, it may only be possible to control deliberate administration of AICAR to racehorses after establishment of an appropriate threshold. Herein we report our studies of AICAR in post-race equine urine and plasma samples including statistical studies of AICAR concentrations determined from 1,470 urine samples collected from thoroughbreds and standardbreds and analyzed in Australia, France, and Hong Kong. Quantification methods in equine urine and plasma using liquid chromatography-mass spectrometry were developed by the laboratories in each country. An exchange of spiked urine and plasma samples between the three countries was conducted, confirming no significant differences in the methods. However, the concentration of AICAR in plasma was found to increase upon haemolysis of whole blood samples, impeding the establishment of a suitable threshold in equine plasma. A possible urine screening cut-off at 600 ng/mL for the control of AICAR in racehorses could be considered for adoption. Application of the proposed screening cut-off to urine samples collected after intravenous administration of a small dose (2 g) of AICAR to a mare yielded a short detection time of approximately 4.5 h. Copyright © 2017 John Wiley & Sons, Ltd.

Exploring the Antitumor Mechanism of High-Dose Cytarabine through the Metabolic Perturbations of Ribonucleotide and Deoxyribonucleotide in Human Promyelocytic Leukemia HL-60 Cells.

Despite the apparent clinical benefits of high-dose cytarabine (Ara-C) over lower dose Ara-C in acute myeloid leukemia (AML) therapy, the mechanism behind high-dose Ara-C therapy remains uncertain. In this study, a LC-MS-based method was carried out to investigate the metabolic alteration of ribonucleotide and deoxyribonucleotide in human promyelocytic leukemia cells (HL-60) after treatment with Ara-C to reveal its antitumor mechanism. The metabolic results revealed that four nucleotides (ATP, ADP, CDP, and dCTP) could be used as potential biomarkers indicating the benefit of high-dose Ara-C over lower dose Ara-C treatment. Combining metabolic perturbation and cell cycle analysis, we conjectured that, apart from the acknowledged mechanism of Ara-C on tumor inhibition, high-dose Ara-C could present a specific action pathway. It was suggested that the pronounced rise in AMP/ATP ratio induced by high-dose Ara-C can trigger AMP-activated protein kinase (AMPK) and subsequently Forkhead Box, class O (FoxO), to promote cell cycle arrest. Moreover, the significant decrease in CDP pool induced by high-dose Ara-C might further accelerate the reduction of dCTP, which then aggravates DNA synthesis disturbance. As a result, all of these alterations led to heightened tumor inhibition. This study provides new insight in the investigation of potential mechanisms in the clinical benefits of high-dose Ara-C in therapy for AML.

Determination of inosine 5'-monophosphate dehydrogenase activity in red blood cells of thiopurine-treated patients using HPLC.

Thiopurine drugs are commonly used in immune diseases and to a lesser extent, in transplant rejection prophylaxis: however interindividual variability in drug response and in the occurrence of adverse events is observed. Genetic variation in thiopurine S-methyltransferase (TPMT) doesn't completely explain the occurrence of all adverse events and drug response variability. The potential implication of other enzymes involved in thiopurine metabolism, such as ITPA, has been investigated over the last decade but little data is available on inosine 5'-monophosphate dehydrogenase (IMPDH) in patients treated with thiopurine drugs. The authors reported a HPLC method to determine IMPDH activity in the red blood cells (RBCs) of thiopurine-treated patients. IMPDH activity was evaluated by enzymatic conversion of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP). The XMP formed was analyzed on a Luna® NH2 stationary phase, a weak anion exchange phase that exhibits both ionic and hydrophobic properties. XMP was eluted below 15min. Intra-assay and inter-assay precisions were below 9% for RBCs supplemented with 2, 40 and 80µmol/L of XMP. IMPDH activity was measured in adults without thiopurine treatment as well as in adult and paediatric patients treated with thiopurines. A wide interindividual variability in IMPDH activity in RBCs was observed. No difference in IMPDH activity was found between untreated subjects and adult and paediatric patients on thiopurine therapy (median value 11.8, 7.9 and 7.7nmol XPM/g Hb/h respectively). The method described is useful in the determination of IMPDH phenotype from patients on thiopurine therapy and in the investigation of the potential relationship between IMPDH activity in RBCs and the occurrence of adverse events and drug response variability.

Effect of the Concentration Difference between Magnesium Ions and Total Ribonucleotide Triphosphates in Governing the Specificity of T7 RNA Polymerase-Based Rolling Circle Transcription for Quantitative Detection.

T7 RNA polymerase-based rolling circle transcription (RCT) is a more powerful tool than universal runoff transcription and traditional DNA polymerase-based rolling circle amplification (RCA). However, RCT is rarely employed in quantitative detection due to its poor specificity for small single-stranded DNA (ssDNA), which can be transcribed efficiently by T7 RNA polymerase even without a promoter. Herein we show that the concentration difference between Mg(2+) and total ribonucleotide triphosphates (rNTPs) radically governs the specificity of T7 RNA polymerase. Only when the total rNTP concentration is 9 mM greater than the Mg(2+) concentration can T7 RNA polymerase transcribe ssDNA specifically and efficiently. This knowledge improves our traditional understanding of T7 RNA polymerase and makes convenient application of RCT in quantitative detection possible. Subsequently, an RCT-based label-free chemiluminescence method for microRNA detection was designed to test the capability of this sensing platform. Using this simple method, microRNA as low as 20 amol could be quantitatively detected. The results reveal that the developed sensing platform holds great potential for further applications in the quantitative detection of a variety of targets.

Global Epitranscriptomics Profiling of RNA Post-Transcriptional Modifications as an Effective Tool for Investigating the Epitranscriptomics of Stress Response.

The simultaneous detection of all the post-transcriptional modifications (PTMs) that decorate cellular RNA can provide comprehensive information on the effects of changing environmental conditions on the entire epitranscriptome. To capture this type of information, we performed the analysis of ribonucleotide mixtures produced by hydrolysis of total RNA extracts from S. cerevisiae that was grown under hyperosmotic and heat shock conditions. Their global PTM profiles clearly indicated that the cellular responses to these types of stresses involved profound changes in the production of specific PTMs. The observed changes involved not only up-/down-regulation of typical PTMs, but also the outright induction of new ones that were absent under normal conditions, or the elimination of others that were normally present. Pointing toward the broad involvement of different classes of RNAs, many of the newly observed PTMs differed from those engaged in the known tRNA-based mechanism of translational recoding, which is induced by oxidative stress. Some of the expression effects were stress-specific, whereas others were not, thus suggesting that RNA PTMs may perform multifaceted activities in stress response, which are subjected to distinctive regulatory pathways. To explore their signaling networks, we implemented a strategy based on the systematic deletion of genes that connect established response genes with PTM biogenetic enzymes in a putative interactomic map. The results clearly identified PTMs that were under direct HOG control, a well-known protein kinase pathway involved in stress response in eukaryotes. Activation of this signaling pathway has been shown to result in the stabilization of numerous mRNAs and the induction of selected lncRNAs involved in chromatin remodeling. The fact that PTMs are capable of altering the activity of the parent RNAs suggest their possible participation in feedback mechanisms aimed at modulating the regulatory functions of such RNAs. This tantalizing hypothesis will be the object of future studies.

Quantitative analysis of intracellular nucleoside triphosphates and other polar metabolites using ion pair reversed-phase liquid chromatography coupled with tandem mass spectrometry.

Simultaneous, quantitative determination of intracellular nucleoside triphosphates and other polar metabolites using liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS) represents a bioanalytic challenge because of charged, highly hydrophilic analytes presented at a large concentration range in a complex matrix. In this study, an ion pair LC-MS/MS method using triethylamine (TEA)-hexafluoroisopropanol (HFIP) ion-pair mobile phase was optimized and validated for simultaneous and unambiguous determination of 8 nucleoside triphosphates (including ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP) in cellular samples. Compared to the the less volatile ion-pair reagent, triethylammonium acetate (100mM, pH 7.0), the combination of HFIP (100mM) and TEA (8.6mM) increased the MS signal intensity by about 50-fold, while retaining comparable chromatographic resolution. The isotope-labeled internal standard method was used for the quantitation. Lower limits of quantitation were determined at 0.5nM for CTP, UTP, dATP, dCTP, and dTTP, at 1nM for ATP, and at 5nM for GTP and dGTP. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method validation (<15%). While the present method was validated for the quantitation of intracellular nucleoside triphosphates, it had a broad application potential for quantitative profiling of nucleoside mono- and bi-phosphates as well as other polar, ionic metabolic intermediates (including carbohydrate derivatives, carboxylic acid derivatives, co-acyl A derivatives, fatty acyls, and others) in biological samples.