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1.
ACS Chem Biol ; 13(12): 3243-3250, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29313662

RESUMO

The recent discovery of reversible chemical modifications on mRNA has opened a new era of post-transcriptional gene regulation in eukaryotes. Among the 15 types of modifications identified in mRNA of eukaryotes, N7-methylguanosine (m7G) is unique owing to its presence in the 5' cap structure. It remains unknown whether m7G is also present internally in mRNA, and this is largely attributed to the lack of an appropriate analytical method to differentiate internal m7G in mRNA from that in the 5' cap. To address this analytical challenge, we developed a novel strategy of combining differential enzymatic digestion with liquid chromatography-tandem mass spectrometry analysis to quantify the levels of these two types of m7G modifications in mRNA. In particular, we found that S1 nuclease and phosphodiesterase I exhibit differential activities toward internal and 5'-terminal m7G. By using this method, we found that internal m7G was present in mRNA of cultured human cells as well as plants and rat tissue. In addition, our results showed that plants contain higher levels of internal m7G in mRNA than mammals. We also observed that exposure of rice to cadmium (Cd) stimulated marked diminution in the levels of m7G at both the 5' cap and internal positions of mRNA, which was correlated with the Cd-induced elevated expression of m7G-decapping enzymes. Taken together, we reported here a strategy to distinguish internal and 5'-terminal m7G in mRNA, and by using this method, we demonstrated the prevalence of internal m7G modification in mRNA, which we believe will stimulate future functional studies of m7G on post-transcriptional gene regulation in eukaryotes.


Assuntos
Endorribonucleases/química , Guanina/análogos & derivados , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Animais , Cádmio/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Guanina/química , Humanos , Masculino , Espectrometria de Massas/métodos , Oryza/enzimologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/síntese química , RNA Mensageiro/genética , Ratos Sprague-Dawley
2.
Talanta ; 167: 506-512, 2017 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-28340751

RESUMO

Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility. The pathogenesis of PCOS remains unclear and early diagnosis of PCOS is challenging. Follicular fluid provides a unique window in the critical processes during oocyte and follicular maturation, and the metabolic level of follicular fluid has important impact on the developmental potential of oocytes and subsequent embryos. Previous studies demonstrated some modified ribonucleosides in biological fluids were diseases related metabolites. In this respect, analysis of endogenous modified ribonucleosides in follicular fluids will facilitate the investigation of follicular development. Here, we developed a strategy for determination of ribose conjugates from follicular fluid using metal oxide-based dispersive solid-phase extraction (DSPE) coupled with liquid chromatography-multiple reaction monitoring-mass spectrometry analysis (DSPE-LC-MRM-MS/MS). Cerium dioxide (CeO2) was used to selectively recognize and capture cis-diol containing ribose conjugates from complex biological samples under basic environment. The trapped ribose conjugates were then easily released under acidic environment. The results showed that 50 potential ribose conjugates were detected in follicular fluid by the developed DSPE-LC-MRM-MS/MS method. We then further investigated the contents change of the detected ribose conjugates in follicular fluid from PCOS patients. The results indicated that the follicular fluid from healthy controls and PCOS patients can be clearly differentiated with the partial least squares-discriminate analysis (PLS-DA) based on the detected ribose conjugates. In addition, the contents of 8 ribose conjugates were significantly different between PCOS patients and healthy controls, which could potentially serve as the indicator of PCOS.


Assuntos
Cério/química , Líquido Folicular/metabolismo , Síndrome do Ovário Policístico/metabolismo , Ribose/análise , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Estudos de Casos e Controles , Feminino , Humanos , Ribose/química , Ribose/isolamento & purificação
3.
J Chromatogr A ; 1462: 90-9, 2016 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-27495369

RESUMO

More than 140 modified ribonucleosides have been identified in RNA. Determination of endogenous modified ribonucleosides in biological fluids may serve as non-invasive disease diagnostic strategy. However, detection of the modified ribonucleosides in biological fluids is challenging, especially for the low abundant modified ribonucleosides due to the serious matrix interferences of biological fluids. Here, we developed a facile preparation strategy and successfully synthesized zirconium oxide-silica (ZrO2/SiO2) composite capillary monolithic column that exhibited excellent performance for the selective enrichment of cis-diol-containing compounds. Compared with the boronate-based affinity monolith, the ZrO2/SiO2 monolith showed ∼2 orders of magnitude higher extraction capacity and can be used under physiological pH (pH 6.5-7.5). Using the prepared ZrO2/SiO2 composite monolith as the trapping column and reversed-phase C18 column as the analytical column, we further established an online solid-phase microextraction (SPME) in combination with liquid chromatography-mass spectrometry (online SPME-LC-MS/MS) analysis for the comprehensive profiling of ribonucleosides modification in human urine. Our results showed that 68 cis-diol-containing ribosylated compounds were identified in human urine, which is, to the best of our knowledge, the highest numbers of cis-diol-containing compounds were determined in a single analysis. It is worth noting that four modified ribonucleosides were discovered in the human urine for the first time. In addition, the quantification results from the pooled urine samples showed that compared to healthy controls, the contents of sixteen ribose conjugates in the urine of gastric cancer, eleven in esophagus cancer and seven in lymphoma increased more than two folds. Among these ribose conjugates, four ribose conjugates increased more than two folds in both gastric cancer and esophagus cancer; three ribose conjugates increased more than two folds in both gastric cancer and lymphoma; one ribose conjugate increased more than two folds in both esophagus cancer and lymphoma. The developed analytical method provides a good platform to study the modified ribonucleosides in human body fluids.


Assuntos
Ribonucleosídeos/química , Ribonucleosídeos/urina , Dióxido de Silício/química , Microextração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Zircônio/química , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Humanos , Concentração de Íons de Hidrogênio , Neoplasias/urina , Ribose/química , Ribose/urina
4.
Analyst ; 140(13): 4636-41, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26030413

RESUMO

DNA methylation, catalyzed by methyltransferases, plays critical roles in various biological processes in both prokaryotes and eukaryotes. Bacterial DNA adenine methyltransferases (DAM) are associated with bacterial pathogenesis and essential for bacterial virulence and viability. Since mammals do not methylate DNA at adenine, bacterial DAM is considered to be a great candidate target for developing new therapeutics for diseases. In the current study, we developed a simple, rapid and highly sensitive fluorescence method for the detection of DAM based on exonuclease-aided signal amplification. In the proposed strategy, a liberated amplifier upon DAM methylation and Dpn I digestion of the substrate can hybridize with a reporter (FT) that contains a quencher (TAMRA) at the second base of the 3' end and a fluorophore (FAM) at the fifth base. Upon hybridization, exonuclease III degrades the reporter in the formed duplex DNA from the 3' end successively, releasing the fluorophore from the quencher and resulting in an intensive appearance of the fluorescent signal. The amplifier will hybridize with another reporter and enter a new cycle, which therefore can amplify the signal and dramatically increase the detection sensitivity even with an extremely low amount of amplifier. Using this strategy, the detection limit down to 0.0025 U mL(-1) of DAM was achieved within a short assay time of 30 min. Furthermore, the assay was applied to evaluate endogenous DAM activity in E. coli cell at different growth stages as well as the effects of inhibitors on DAM activity. Given the attractive analytical performance, the sensing strategy may find many important applications in biomedical research and clinical diagnosis.


Assuntos
Ensaios Enzimáticos/métodos , Exonucleases/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Sequência de Bases , Sondas de DNA/genética , Sondas de DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Estudos de Viabilidade , Humanos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/antagonistas & inibidores , Espectrometria de Fluorescência , Fatores de Tempo
5.
Anal Chem ; 87(14): 7364-72, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26086917

RESUMO

Some modified ribonucleosides in biological fluids have been evaluated as cancer-related metabolites. Detection of endogenous modified ribonucleosides in biological fluids may serve as a noninvasive cancers diagnostic method. However, determination of modified ribonucleosides is still challenging because of their low abundance and serious matrix interferences in biological fluids. Here, we developed a novel strategy for comprehensive profiling of ribose conjugates from biological fluids using metal oxide-based dispersive solid-phase extraction (DSPE) followed with in vitro stable isotope labeling and double neutral loss scan-mass spectrometry analysis (DSPE-SIL-LC-DNLS-MS). Cerium dioxide (CeO2) was used to selectively recognize and capture ribose conjugates from complex biological samples under basic environment. The enriched ribose conjugates were subsequently labeled with a pair of isotope labeling reagents (acetone and acetone-d6). The glucosidic bond of acetone labeled ribose conjugates is readily ruptured, and the generated ribose that carries an isotope tag can be lost as a neutral fragment under collision induced dissociation (CID). Since the light (acetone) and heavy (acetone-d6) labeled compounds have the same chemical structures and can generate different neutral loss fragments (NL 172 and 178 Da), it is therefore highly convenient to profile ribose conjugates by double neutral loss scan mode in mass spectrometry analysis. In this respect, the light and heavy labeled compounds were ionized at the same condition but recorded separately on MS spectra, which can significantly improve the detection specificity and facilitate the identification of ribose conjugates. Using the developed DSPE-SIL-LC-DNLS-MS strategy, we profiled the ribose conjugates in human urine, and 49 ribose conjugates were readily identified, among which 7 ribose conjugates exhibited significant contents change between healthy controls and lymphoma patients. The DSPE-SIL-LC-DNLS-MS strategy combines the selective enrichment, stable isotope labeling, and double neutral loss scan - MS analysis, which therefore can efficiently minimize false positive results, facilitate the relative quantification, and notably increase the numbers of identified ribose conjugates in biological fluids samples. Taken together, this study established a promising strategy for the effective profiling of urinary modified ribonucleosides, and simultaneous evaluation of the contents change of multiple modified ribonucleosides should provide more accurate and conclusive results for the use of urinary modified ribonucleosides as indicators of cancers.


Assuntos
Cério/química , Marcação por Isótopo , Ribose/química , Ribose/urina , Humanos , Espectrometria de Massas , Estrutura Molecular , Ribose/metabolismo , Extração em Fase Sólida
6.
Sci Rep ; 5: 7785, 2015 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-25585609

RESUMO

RNA contains a large number of modified nucleosides. In the metabolic re-exchange of RNA, modified nucleosides cannot be recycled and are thus excreted from cells into biological fluids. Determination of endogenous modified nucleosides in biological fluids may serve as non-invasive cancers diagnostic methods. Here we prepared boronate-affinity organic-silica hybrid capillary monolithic column (BOHCMC) that exhibited excellent selectivity toward the cis-diol-containing compounds. We then used the prepared BOHCMC as the on-line solid-phase microextraction (SPME) column and developed an on-line SPME-LC-MS/MS method to comprehensively profile cis-diol-containing nucleosides and ribosylated metabolites in human urine. Forty-five cis-diol-containing nucleosides and ribosylated metabolites were successfully identified in human urine. And five ribose conjugates, for the first time, were identified existence in human urine in the current study. Furthermore, the relative quantification suggested 4 cis-diol-containing compounds (5'-deoxy-5'-methylthioadensine, N(4)-acetylcytidine, 1-ribosyl-N-propionylhistamine and N(2),N(2),7-trimethylguanosine) increased more than 1.5 folds in all the 3 types of examined cancers (lung cancer, colorectal cancer, and nasopharyngeal cancer) compared to healthy controls. The on-line SPME-LC-MS/MS method demonstrates a promising method for the comprehensive profiling of cis-diol-containing ribose conjugates in human urines, which provides an efficient strategy for the identification and discovery of biomarkers and may be used for the screening of cancers.


Assuntos
Álcoois/química , Ácidos Borônicos/química , Cromatografia de Afinidade/métodos , Cromatografia Líquida/métodos , Metaboloma , Nucleosídeos/análise , Ribose/urina , Espectrometria de Massas em Tandem/métodos , Estudos de Casos e Controles , Humanos , Dióxido de Silício/química , Microextração em Fase Sólida
7.
Anal Chem ; 86(22): 11269-74, 2014 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-25323974

RESUMO

DNA methylation plays vital roles in various biological processes in both prokaryotes and eukaryotes. In bacteria, modification of adenine at N6 can protect bacterial DNA against cleavage by restriction enzymes, and bacterial DNA adenine methyltransferases are essential for bacterial virulence and viability. DNA adenine methyltransferase (DAM) targets the sequence of 5'-GATC-3' and can convert adenine into N(6)-methyladenine (m(6)A). Because mammals do not methylate DNA at adenine, bacterial DAM represents an excellent candidate for antibiotic development. Here, we developed an exonuclease III-aided target recycling strategy to sensitively assay activity of DAM. In this method, a hairpin probe labeled with a donor fluorophore (FAM) at the 5' end and a quencher (BHQ) close to the 3' end (FQ probe) was employed as reporter. Another hairpin substrate containing sequence of GATC was used as the methylation substrate of DAM. Once the hairpin substrate was methylated by DAM, it could be recognized and cleaved by Dpn I, which allows the release of a single-stranded oligodeoxynucleotide (ssODN). The ssODN can then hybridize to the 3' protruding terminus of FQ probe, which subsequently triggers the exonuclease III-mediated target recycling reaction and therefore can significantly improve the detection sensitivity of DAM. The exonuclease-mediated target recycling strategy is extremely sensitive and as low as 0.01 U/mL DAM can be distinctly determined. Using this developed method, we evaluated DAM activity in different growth stages of E. coli cells, and we also demonstrated that the assay has the potential to screen suitable inhibitor drugs for DAM for disease(s) treatment.


Assuntos
Exonucleases/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/citologia , Escherichia coli/enzimologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/antagonistas & inibidores , Relação Estrutura-Atividade
8.
Anal Chem ; 85(12): 6129-35, 2013 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-23678980

RESUMO

5-Methylcytosine (5-mC), an important epigenetic modification involved in development, can be converted enzymatically to 5-hydroxymethylcytosine (5-hmC). 5-hmC is considered an intermediate of active DNA cytosine demethylation and makes itself serve as an epigenetic mark. 5-hmC content in most mammalian cells is low and the quantification of 5-hmC by liquid chromatography-mass spectrometry (LC-MS) frequently suffers from ion suppression by the presence of unmodified nucleosides. To circumvent this problem, we developed a method to selectively transfer a glucosyl group to the hydroxymethyl moiety of 5-hmC and form a more hydrophilic residue (ß-glucosyl-5-hydroxymethyl-2'-deoxycytidine, 5-gmdC) by using T4 ß-glucosyltransferase. The more hydrophilic 5-gmdC can be selectively enriched by using NH2-silica via hydrophilic interaction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, which eliminates the ion suppression and significantly improves the detection sensitivity and accuracy. Using this method, we successfully quantified 5-hmC content in genomic DNA of three human cell lines and seven yeast strains. To the best of our knowledge, this is the first report about the existence of 5-hmC in the model organism of yeast. In addition, the contents of 5-hmC in two yeast strains of Schizosaccharomyces pombe are even higher than those of 5-mC, indicating that 5-hmC may play important roles on the physiological functions of yeast.


Assuntos
Citosina/análogos & derivados , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas em Tandem/métodos , 5-Metilcitosina/análogos & derivados , Cromatografia Líquida/métodos , Citosina/análise , Citosina/química , Células HEK293 , Células HeLa , Humanos , Células Jurkat
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