n(2)-n(2)-dimethylguanosine and 1-methylguanosine

Members of the mammalian AlkB family are known to mediate nucleic acid demethylation

Topics: Adenosine; AlkB Enzymes; Cytidine; Guanosine; HEK293 Cells; HeLa Cells; Hep G2 Cells; Humans; Mitochondria; Mitochondrial Proteins; Protein Biosynthesis; RNA Processing, Post-Transcriptional; RNA, Mitochondrial; RNA, Transfer

Quantitative nontarget analysis is intended to provide a measurement of concentration of newly identified components in complex biological or environmental samples for which authentic or labeled standard do not exist. Electrospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry (ESI-FAIMS-MS) has unique advantages that allowed us to develop a novel approach for quantification of nontarget analytes. In the nontarget analysis of urinary metabolites by ESI-FAIMS-MS, we find it practical and beneficial to analyze highly diluted urine samples. We show that urine extracts can be analyzed directly at very high dilutions (up to 20,000 times) by extending MS analysis times during slow FAIMS scanning. We explore the effects of sample dilution on ionization efficiency and ionization suppression in direct electrospray of complex sample matrixes. We consistently observe two distinct regimes in ESI operation related to the limited ionization capacity of this method. In the linear dynamic concentration range below the limiting ionization capacity, the analytical sensitivity of an analyte is constant and does not depend on matrix composition and concentration. Once the capacity of ESI is exceeded, all species exhibit log-log linearity in signal response. We show how quantification can be carried out using two different approaches, one for analytes which can be detected in the linear regime and another for those only detected in the suppression regime that overcomes the effects of ionization suppression. Our new insight into ionization suppression effects in ESI is of broad interest to anyone using ESI as an ionization technique for the MS analysis of complex samples.

Topics: Guanosine; Inosine; Ions; Spectrometry, Mass, Electrospray Ionization; Urinalysis

We have systematically investigated the duplex to hairpin conversion of oligoribonucleotides under the aspect of nucleobase methylation. The first part of our study refers to the self-complementary sequence rCGCGAAUUCGCGA, which forms a stable Watson-Crick base paired duplex under various buffer conditions. It is shown that this sequence is forced to adopt a hairpin conformation if one of the central 6 nt is replaced by the corresponding methylated nucleotide, such as 1-methylguanosine N(2),N(2)-dimethylguanosine, N(6),N(6)-dimethyladenosine (m(6)(2)A) or 3-methyluridine. On the other hand, the duplex structure is retained and even stabilized by replacement of a central nucleotide with N(2)-methylguanosine (m(2)G) or N(4)-methylcytidine. A borderline case is represented by N(6)-methyladenosine (m(6)A). Although generally a duplex-preserving modification, our data indicate that m(6)A in specific strand positions and at low strand concentrations is able to effectuate duplex-hairpin conversion. Our studies also include the ssu ribosomal helix 45 sequence motif, rGACCm(2)GGm(6)(2)Am(6)(2)AGGUC. In analogy, it is demonstrated that the tandem m(6)(2)A nucleobases of this oligoribonucleotide prevent duplex formation with complementary strands. Therefore, it can be concluded that nucleobase methylations at the Watson-Crick base pairing site provide the potential not only to modulate but to substantially affect RNA structure by formation of different secondary structure motifs.

Topics: Adenosine; Base Pairing; Cytidine; Guanosine; Hydrogen Bonding; Inosine; Methylation; Nucleic Acid Conformation; Oligoribonucleotides; RNA; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thermodynamics; Uridine

Urinary excretion of modified nucleosides was measured in 15 patients with Philadelphia chromosome-positive CML to determine the correlation with activity of this disease. Resolution and quantitation of seven nucleosides were accomplished with reversed-phase HPLC. Patients in the stable phase of CML had excretion levels one to two times normal, whereas patients in the blastic phase showed elevations up to 12 times normal. The nucleosides showing the most significant differences in excretion between stable phase and blastic phase were 1-methylinosine, pseudouridine, and N2,N2-dimethylguanosine (p less than 0.01, p less than 0.001, and p less than 0.01, respectively). Nucleoside excretion was also determined in patients with bacterial pneumonia and urinary tract infection for comparison. Serial nucleoside determinations were made in two patients with CML and found to correlate closely with disease activity. The degree of elevation and the correlation with disease activity suggest the potential value of urinary nucleoside quantitation in monitoring patients with CML; in particular, nucleoside excretion may be useful in detecting early blastic transformation.

Topics: Adult; Chromatography, High Pressure Liquid; Female; Guanosine; Humans; Inosine; Leukemia, Myeloid; Longitudinal Studies; Male; Middle Aged; Nucleosides; Pneumonia; Pseudouridine; Urinary Tract Infections

The levels of urinary excretion of five modified nucleosides were quantitated by high-performance liquid chromatography for 15 normal children and 24 children with acute lymphoblastic leukemia (ALL). Excretion of each nucleoside decreased linearly with age when quantitation was based on urine creatinine content. Patients with childhood ALL at initial diagnosis or in relapse had significantly higher concentrations of 1-methylinosine, N2,N2-dimethylguanosine, 1-methylguanosine, and pseudouridine in their urine when compared to the concentrations in either patients in remission (P less than 0.001, P less than 0.001, P less than 0.01, and P less than 0.05, respectively) or normal controls (P less than 0.001, P less than 0.02, P less than 0.01, and P less than 0.001, respectively). Excretion of 2-pyridone-5-carboxamide-N'-ribofuranoside did not show significant differences. Urinary excretion of 1-methylinosine demonstrated a positive linear relationship with the percentage of blast cells in the bone marrow [correlation coefficient (r) = 0.90]; the other nucleosides had lower degrees of correlation. In comparison, the absolute blast cell count in the peripheral blood showed less correlation to the percentage of blast cells in the bone marrow (r = 0.47) than did four of the five nucleosides. The data demonstrate that excretion of modified nucleosides reflects disease activity in childhood ALL and that the urinary nucleosides could be useful clinical markers for this disease.

Topics: Adolescent; Age Factors; Bone Marrow; Child; Child, Preschool; Creatinine; Female; Guanosine; Humans; Inosine; Leukemia, Lymphoid; Male; Pseudouridine; Reference Values; Ribonucleosides

The urinary excretion of six methylated ribonucleosides was measured in a case of anorexia nervosa during the first eight weeks of therapy. Four phases can be distinguished. Highly elevated values are found in a clearly catabolic situation. Immediately after onset of therapy when catabolism is stopped but nutrition still inadequate the excretion of RNA-catabolites is lowered markedly. As soon as normocaloric nutrition is reached after two weeks, a short but clearcut peak-excretion can be observed. Finally, the excretion of methylated RNA-catabolites is stabilized on an intermediate level after three to six weeks, when nutrition is constantly normocaloric and weight continues to increase linearly.

Topics: Adenosine; Adolescent; Anorexia Nervosa; Female; Guanosine; Humans; Inosine; Nucleosides; Uridine