Single-molecule RNA sequencing for simultaneous detection of m6A and 5mC.

Epitranscriptomics is the study of RNA base modifications involving functionally relevant changes to the transcriptome. In recent years, epitranscriptomics has been an active area of research. However, a major issue has been the development of sequencing methods to map transcriptome-wide RNA base modifications. We have proposed a single-molecule quantum sequencer for mapping RNA base modifications in microRNAs (miRNAs), such as N6-methyladenosine (m6A) or 5-methylcytidine (5mC), which are related to cancer cell propagation and suppression. Here, we investigated 5mC and m6A in hsa-miR-200c-5p extracted from colorectal cancer cells and determined their methylation sites and rates; the data were comparable to those determined by mass spectrometry. Furthermore, we evaluated the methylation ratio of cytidine and adenosine at each site in the sequences and its relationship. These results suggest that the methylation ratio of cytidine and adenosine is facilitated by the presence of vicinal methylation. Our work provides a robust new tool for sequencing various types of RNA base modifications in their RNA context.

Hydrophilic organic/salt-containing aqueous two-phase solvent system for counter-current chromatography: a novel technique for separation of polar compounds.

Hydrophilic organic/salt-containing aqueous two-phase system composing of ethanol, water and ammonium sulfate for separation polar compounds was investigated on multilayer coil associated with J-type HSCCC devices. Compared to the classical polar solvent system based on 1-butanol-water or PEG1000-ammonium sulfate-water, the water content of upper phase in ethanol-ammonium sulfate-water systems was from 53.7% to 32.8% (wt%), closed to PEG1000-ammonium sulfate-water aqueous two-phase systems and higher than 1-butanol-water (22.0%, wt%). Therefore, the polarity of ethanol-ammonium sulfate-water is in the middle of 1-butanol-water and PEG-ammonium sulfate-water system, which is quite good for separating polar compounds like phenols, nucleosides and amino acids with low partition coefficient in 1-octanol-water system. The retention of stationary phase in four elution mode on type-J counter-current chromatography devices with multilayer coil column changed from 26% to 71%. Hydrodynamic trend possess both intermediate and hydrophilic solvent system property, which closely related to the composition of solvent system. The applicability of this system was demonstrated by successful separation of adenosine, uridine guanosine and cytidine.

Identification and analysis of the constituents in an aqueous extract of Tricholoma matsutake by HPLC coupled with diode array detection/electrospray ionization mass spectrometry.

The main constituents in an aqueous extract of Tricholoma matsutake (Tm) were identified by high-performance liquid chromatography coupled with diode array detection and electrospray ionization time-of-flight mass spectrometry (HPLC-DAD/TOF-MS) and ion trap mass spectrometry (HPLC-DAD/Trap-MSn). The main factors in the extraction process which affect the yields of nutrients were optimized by single-factor experiments and orthogonal experiment design. In total, 12 constituents were identified from the aqueous extract of Tm, including tyrosine, cytidine, uridine, eritadenine, phenylalanine, nicotinamide, inosine, guanosine, tryptophan, adenosine, 5'-deoxy-5'-methylthioadenosine and riboflavin. The optimized extraction conditions were: the ratio of water to sample was 10 : 1 (v/w), Tm was extracted by ultrasonic-assisted extraction for 10 min, followed by water bath heating at 60 °C for 1 h. Among these extraction factors, the heating temperature is significant based on analysis of variance (ANOVA). The yields of nutrients were affected dramatically at high temperature leading to the loss of nutrients, especially for nucleosides and some amino acids.

[Identification of 5 constituents of the aqueous extract of Isatis indigotica by HPLC-MS2].

OBJECTIVE: To identify five constituents in the aqueous extract of Isatis indigotica Fort. METHODS: After separation of the aqueous extract of Isatis indigotica Fort. by HPLC, the eluates of five peaks were collected separatively and analysed by MS2. UV spectra and MS2 were compared with those of reference standards of cytidine, uridine, guanosine xanthine and hypoxanthine. RESULTS: Each HPLC elute of the aqueous extract had same retention time, UV spectra and fragment pattern in the MS2 spectrum as the corresponding standards. CONCLUSION: Five constituents of the aqueous extract of Isatis indigotica Fort. are identified as cytidine, hypoxanthine, uridine, xanthine and guanosine.

Separation of aracytidine and cytidine by capillary electrophoretic techniques.

Aracytidine (cytarabine, 1-beta-D-arabinofuranosylcytosine) is a synthetic analog of cytidine in which ribose is substituted by arabinose; it is used as a drug for the treatment of leukemia. A fast and reliable capillary electrophoretic method for the analysis of cytarabine and cytidine is described. The procedure utilizes the interactions with sodium dodecyl sulfate (SDS) micelles and borate, present in the background electrolyte, for the mobilization and selective separation of the analytes. The detection is carried out by UV absorbance at 275 nm. The method was applied both to pharmaceutical preparations and human serum. Analysis of an untreated serum requires 15 min; the detection limit is 0.8 microgram/mL and the relative standard deviation (RSD) is 5.3%.

2'-O-methyl-5-formylcytidine (f5Cm), a new modified nucleotide at the 'wobble' of two cytoplasmic tRNAs Leu (NAA) from bovine liver.

The nucleotide analysis of a cytoplasmic tRNA(Leu) isolated from bovine liver revealed the presence of an unknown modified nucleotide N. The corresponding N nucleoside was isolated by different enzymatic and chromatographic protocols from a partially purified preparation of this tRNA(Leu). Its chemical characterization was determined from its chromatographic properties, UV-absorption spectroscopy and mass spectrometric measurements, as well as from those of the borohydride reduced N nucleoside and its etheno-trimethylsilyl derivative. The structure of N was established as 2'-O-methyl-5-formylcytidine (f5CM), and its reduced derivative as 2'-O-methyl-5-hydroxy-methylcytidine (om5Cm). By sequencing the bovine liver tRNA(Leu), the structure of the anticodon was determined as f5CmAA. In addition, the nucleotide sequence showed two primary structures differing only by the nucleotide 47c which is either uridine or adenosine. The two slightly differing bovine liver tRNAs-Leu(f5CmAA) are the only tRNAs so far sequenced which contain f5Cm. The role of such a modified cytidine at the first position of the anticodon is discussed in terms of decoding properties for the UUG and UUA leucine codons. Recently, precise evidence was obtained for the presence of f5Cm at the same position in tRNAs(Leu)(NAA) isolated from rabbit and lamb liver. Therefore, the 2'-O-methyl-5-formyl modification of cytidine at position 34 could be a general feature of cytoplasmic tRNAs(Leu)(NAA) in mammals.