Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels.

Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.

Facile isolation of α-ribazole from vitamin B12 hydrolysates using boronate affinity chromatography.

Alpha-ribazole (α-R) is a unique riboside found in the nucleotide loop of coenzyme B12 (CoB12). α-R is not an intermediate of the de novo biosynthetic pathway of coenzyme B12, but some bacteria of the phylum Firmicutes have evolved a two-protein system (transporter, kinase) that scavenges α-R from the environment and converts it to the pathway intermediate α-RP. Since α-R is not commercially available, one must either synthesize α-R, or isolate it from hydrolysates of vitamin B12 (cyano-B12, CNB12), so the function of the above-mentioned proteins can be studied. Here we report a facile protocol for the isolation of α-R from CNB12 hydrolysates. CNB12 dissolved in NaOH (5 M) was heated to 85 °C for 75 min, then cooled to 4 °C for 30 min. The solution was neutralized with HCl (5 M), and the hydrolysate was diluted with an equal volume of ammonium acetate (0.3 M, pH 8.8). Alkaline phosphatase was added and the mixture was incubated at 37 °C for 16 h. After incubation, the sample was loaded onto a boronate affinity resin column, washed with ammonium sulfate (0.3 M, pH 8.8), water (to remove residual corrinoids) and finally with formic acid (0.1 M) to release (α-R). Formic acid was removed by lyophilization, and the final yield of α-R was 85% from the theoretically recoverable amount. Methods for quantifying the concentration of α-R are reported.

Immobilization of zirconium-glycerolate nanowires on magnetic nanoparticles for extraction of urinary ribonucleosides.

The authors have immobilized nanowires made from zirconium glycerolate (ZrGly) on magnetite (Fe3O4) nanoparticles by applying a solvothermal growth process using metal-glycerolate as a precursor. The structure and the dissolution-recrystallization mechanism of the resulting Fe3O4@ZrGly composite were investigated by attenuated total reflection-FTIR, energy-dispersive X-ray analysis, thermogravimetric analysis and solid-state cross polarization/magic angle spinning 13C NMR spectroscopy. The interaction between the zirconium glycerolate in Fe3O4@ZrGly and cis-diols leads to efficient adsorption of riboncleosides which then can be quantified by HPLC with UV detection. The sorbent was successfully applied to the selective enrichment of adenosine, cytidine, uridine and guanosine from spiked human urine samples. The detection limit of the method is in the range from 1.7 to 19 ng·mL-1 of nucleosides in spiked human urine, with relative standard deviations of lower than 12.4% and recoveries ranging from 90.6 to 113%. Graphical abstract Fe3O4@ZrGly with high selectivity towards ribonucleosides was designed and applied for quantitation of urinary ribonucleosides.

Polyethyleneimine-grafted boronate affinity materials for selective enrichment of cis-diol-containing compounds.

Polyethyleneimine (PEI)-grafted and 3-acrylamidophenylboronic acid (AAPBA)-functionalized SiO2 boronate affinity materials were synthesized for the selective enrichment of cis-diol-containing compounds. Characterization results of scanning electron microscopy, Fourier transform infrared spectroscopy, elemental analysis, zeta potential, and X-ray photoelectron spectroscopy indicated the successful fabrication of SiO2@PEI-AAPBA materials. Chromatographic separation of test mixtures reveals that SiO2@PEI-AAPBA has high selective enrichment ability for cis-diol-containing compounds. The binding pH between SiO2@PEI-AAPBA and catechol was found to be as low as pH 4.5, while that between SiO2@PEI-AAPBA and adenosine was only ~7.5. This difference might be attributed to the strong electrostatic repulsion between the solid phase and analytes at a low pH. Furthermore, a diphasic separation column was fabricated based on boronate affinity chromatography, C18-reversed-phase chromatography and applied in pressurized capillary electrochromatography (pCEC). Results showed that four polar nucleosides could be well captured by the boronate affinity chromatography (BAC) section and separated by reversed phase pCEC. Finally, SiO2@PEI600-AAPBA-based solid-phase extraction technology was applied to the purification of ribonucleosides in real urine samples, and results of UHPLC-MS/MS revealed that the intensities of the extracted ions (a neutral mass loss of m/z 132.04 Da) of the ribonucleosides were significantly enhanced after the enrichment.

Two immunosuppressive compounds from the mushroom Rubinoboletus ballouii using human peripheral blood mononuclear cells by bioactivity-guided fractionation.

Rubinoboletus ballouii is an edible mushroom wildly grown in Yunnan province, China. Up till now, little was known about the chemical and biological properties of this mushroom. The aim of this study was to investigate the immunomodulatory effects of the ethanolic extract of Rubinoboletus ballouii and its fractions on human peripheral blood mononuclear cells (PBMCs) using bioactivity-guided fractionation. The crude extract of the fruiting bodies of RB was fractionated by high-speed counter current chromatography (HSCCC). Twelve fractions were obtained and the third fraction (Fraction C) exerted the most potent anti-inflammatory activities in mitogen-activated PBMCs. Further fractionation of fraction C led to the isolation of two single compounds which were elucidated as 1-ribofuranosyl-s-triazin-2(1H)-one and pistillarin, respectively. The results showed that both 1-ribofuranosyl-s-triazin-2(1H)-one and pistillarin exhibited significant immunosuppressive effects on phytohemagglutinin (PHA)-stimulated human PBMCs by inhibiting [methyl-(3)H]-thymidine uptake and inflammatory cytokines productions such as tumor necrosis factor (TNF)-α, interleukin (IL)-10, interferon (IFN)-γ and IL-1ß. Besides, 1-ribofuranosyl-s-triazin-2(1H)-one was firstly found in natural resources, and pistillarin was also isolated from the family Boletaceae for the first time. They exhibited great potential in developing as anti-inflammatory reagents.

Genome-wide identification of miRNA targets by PAR-CLIP.

miRNAs are short (20-23 nt) RNAs that are loaded into proteins of the Argonaute (AGO) family and guide them to partially complementary target sites on mRNAs, resulting in mRNA destabilization and/or translational repression. It is estimated that about 60% of the mammalian genes are potentially regulated by miRNAs, and therefore methods for experimental miRNA target determination have become valuable tools for the characterization of posttranscriptional gene regulation. Here we present a step-by-step protocol and guidelines for the computational analysis for the large-scale identification of miRNA target sites in cultured cells by photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP) of AGO proteins.

Chemical constituents from the seeds of Trifolium alexandrinum.

The ethanolic extract of the seeds of Trifolium alexandrinum afforded a new naturally occurring compound identified as methyl-alpha-glucose. The known compounds quercetin, kaempferol, apigenin 7-O-beta-D-glucoside and the nucleoside xanthosine were also isolated. The isolated compounds were identified by spectroscopic (UV, (1)H-, (13)C-NMR, DEPT, HMQC and HMBC) and spectrometric (ESI-MS/MS) analyses. The spectroscopic data of the isolated nucleoside were reported for the first time as a natural isolate.

Nucleoprotein-derived and unbound ribonucleosides: bioactivity and potential applications.

Naturally occurring and chemically modified ribonucleosides have interesting bioactive effects. Dietary ribonucleosides are ingested mainly as nucleoproteins and are converted in the course of intestinal digestion to monomeric compounds. Different bioactive effects of dietary ribonucleosides have been described, including the ability to enhance gut growth and maturation and to increase iron absorption. Cytochemical studies with human cells showed that several ribonucleosides can induce apoptosis in human cells, and therefore may be potentially anticancerogenic compounds. Even if suboptimal concentrations of single bioactive nucleo compounds are available from food, the total content of different bioactive ribonucleosides may reach physiologically effective concentrations in vivo where intestinal cells may represent the main target sites of a selective apoptotic activity. Modified ribonucleosides serve as valuable pathobiochemical marker molecules for cancer. Chemically modified ribonucleosides have already found interesting applications as pharmaceutically active compounds in the treatment of different illnesses including AIDS. Regarding therapeutic and pharmaceutical aspects, further studies are required to evaluate the bioactive efficacy of indigenous ribonucleosides. The findings demonstrate the great variety of potential applications of ribonucleosides, e.g. in functional foods as well as pharmaceutical preparations.

N(3),5'-cycloxanthosine, the first natural occurrence of a cyclonucleoside.

An Eryus sp. of marine sponge from the Great Australian Bight has yielded the first reported natural occurrence of a cyclonucleoside, N(3),5'-cycloxanthosine. The structure of N(3),5'-cycloxanthosine was confirmed by detailed spectroscopic analysis and total synthesis.

On-line hyphenation of quantum cascade laser and capillary electrophoresis.

We report the first successful hyphenation of a Fabry Pérot quantum cascade (QC) laser to a capillary electrophoresis system. This involved use of a dedicated IR-transparent flow cell, made of CaF2, constructed by means of SU-8 based lithography and low temperature wafer bonding techniques. Adenosine, guanosine, xanthosine and adenosine-5'-monophosphate were separated in a borate-containing separation electrolyte (10 mM, pH 9.3). Functional group (carbohydrate) detection was accomplished by use of the 1080 cm(-1) emission line of the available QC-laser. The assessable optical path length could be increased, from the normally available 10-15 microm in CE-FTIR analyses, to 60 microm using this powerful mid-infrared laser and aqueous solutions.

Site-selected introduction of modified purine and pyrimidine ribonucleosides into RNA by automated phosphoramidite chemistry.

The study of modified nucleoside contributions to RNA chemistry, structure and function has been thwarted by the lack of a site-selected method of incorporation which is both versatile and adaptable to present synthetic technologies. A reproducible and versatile site-selected incorporation of nine differently modified nucleosides into hepta- and octadecamer RNAs has been achieved with automated phosphoramidite chemistry. The 5'-O-(4,4'-dimethoxytrityl-2'-O-tert-butyldimethylsilyl-ribonucleoside- 3'-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite syntheses of m5C, D, psi, riboT, s2U, mnm5U, m1G and m2A were designed for compatibility with the commercially available major and 2'OH methylated ribonucleoside phosphoramidites. The synthesis of the m5C phosphoramidite was uniquely designed, and the first syntheses and incorporation of the two modified purine ribonucleosides are reported in detail along with that of psi, s2U, and mnm5U. Cleavage of RNA product from the synthesis support column, deprotection of the RNA, its purification by HPLC and nucleoside composition analysis are described. Modified nucleoside-containing tRNA domains were synthesized and purified in mumol quantities required for biophysical, as well as biochemical, studies. The anticodon domain of yeast tRNA(Phe) was synthesized with modified nucleosides introduced at the native positions: Cm32, Gm34, m1G37 (precursor to Y), psi 39 and m5C40. The T loop and stem was synthesized with riboT54 and the D loop and stem with D16 and D17. The E coli tRNA(Glu2) anti-codon codon domain was synthesized with mnm5U at wobble position 34, but an attempt at incorporating s2U at the same position failed. The unprotected thio group was labile to the oxidation step of the cyclical process. Chemically synthesized anticodon and T domains have been used in assays of tRNA structure and function (Guenther et al (1994) Biochimie 76, 1143-1151).

Formation of ribonucleotides in DNA modified by oxidative damage in vitro and in vivo. Characterization by 32P-postlabeling.

Oxygen free radicals generated by the interaction of Fe2+ and H2O2 (Fenton reaction) are capable of reacting with DNA bases, which may induce premutagenic and precarcinogenic lesions. Products formed in DNA by such reactions have been characterized as hydroxylated derivatives of cytosine, thymine, adenine, and guanine and imidazole ring-opened derivatives of adenine and guanine. As shown here by 32P-postlabeling, incubation of DNA under Fenton reaction conditions gave rise to additional oxidation products in DNA that were characterized as putative ribonucleosides by enzymatic hydrolysis of the oxidized DNA, 32P-postlabeling, and co-chromatography in multiple systems with authentic markers. Formation of these products in DNA was enhanced by the presence of L-ascorbic acid in the reaction mixtures and their total amounts were similar to those of the major DNA oxidation product, 8-hydroxy-2'-deoxyguanosine. The ribonucleoside guanosine was also formed in kidney DNA of male rats treated with ferric nitrilotriacetate, a renal carcinogen. It is postulated that ribonucleotides alter conformation and function of DNA and thus their presence in DNA may lead to adverse health effects.

Changes of nucleotide patterns in liver, muscle and blood during the growth of Ehrlich ascites cells: application of the reversed-phase and ion-pair reversed-phase high-performance liquid chromatography with radial compression column.

The pool of purine compounds was analysed in liver, skeletal muscle and blood of mice during the growth of Ehrlich ascites tumour cells. Three fast isocratic high-performance liquid chromatographic methods were used. (1) Determination of nucleotides by an isocratic ion-pair reversed-phase chromatography with a 10 mM ammonium phosphate buffer containing acetonitrile and tetrabutylammonium phosphate. (2) Separation of nucleosides and nucleobases in cell extracts by a reversed-phase system with methanol and 50 mM potassium phosphate buffer as eluent. (3) Nucleosides and nucleobases in body fluids were analysed by a reversed-phase system with 10 mM potassium phosphate containing methanol. These methods allow the rapid determination of purine compounds in small biological samples from various cell types and body fluids, with high accuracy and sensitivity. The pool of cellular nucleotides increased during the exponential phase of tumour growth. Adenosine accumulated significantly in all tissues in the stationary phase of tumour growth.

High resolution chromatography of ribonucleosides and its application to RNA analysis.

A simple and precise method was developed for the separation of nucleosides including modified nucleosides and oligonucleotides. Nineteen kinds of nucleosides were completely separated by HPLC using an ODS column (TSK-gel ODS 80TM) and aqueous mobile phases. The RNA molecule was digested by base restrictive RNase (RNase A, RNase T1) and the digests were separated chromatographically into each oligonucleotide. The nucleoside composition of an oligonucleotide was then determined by this analytical system. It is thus possible to fit the oligonucleotide in the original RNA molecule by using modified bases as markers. The reaction site of quinacrine mustard for tRNA(Phe) (from yeast) could be determined by this analytical system.

In vitro methylation of Escherichia coli 16S ribosomal RNA and 30S ribosomes.

Treatment of synthetic 30S particles lacking all of the normally methylated nucleotides with S-adenosyl-[3H]methionine and either an S100 or ribosomal high salt wash extract resulted in ribosome-dependent incorporation of [3H]methyl groups into trichloroacetic acid-insoluble material. No incorporation was observed when naturally methylated isolated 30S particles were used, showing that methylation at unnatural sites did not occur. Enzymatic hydrolysis of the labeled RNA to nucleosides followed by HPLC analysis identified the [3H]methylated residues. Activities for the formation of N6-methyladenosine, N6-dimethyladenosine, 5-methylcytidine (m5C), 3-methyluridine, and N2-methylguanosine were found. Fractionation by ammonium sulfate partially resolved the different activities. All of the fractions with m5C activity were 6-8 times more active on synthetic unmethylated 16S RNA than on synthetic 30S ribosomes, whereas the N2-methylguanosine activity preferred 30S ribosomes to 16S RNA by a factor of more than 10. The N6-methyladenosine and N6-dimethyladenosine activities were 30S ribosome-specific. The m5C activity present in the 55-85% ammonium sulfate fraction of the high salt wash yielded a maximum of 1.0 mol of m5C per mol of 16S RNA, although two m5C residues, positions 967 and 1407, are found in vivo. RNase protection by hybridization with the appropriate oligodeoxynucleotide identified the methylated residue as C-967. Methylation of m5C-967 did not require prior methylation of G-966, and methylation of A-1518 and A-1519 was not dependent on prior methylation of G-1516.