key: cord-0805501-ph12hixt authors: Nees, Gernot; Kaufmann, Andreas; Bauer, Stefan title: Detection of RNA Modifications by HPLC Analysis and Competitive ELISA date: 2014-03-20 journal: Innate DNA and RNA Recognition DOI: 10.1007/978-1-4939-0882-0_1 sha: 6b785877dfbda67ca020f9fa74e77ca5660a8a5e doc_id: 805501 cord_uid: ph12hixt Over 100 different RNA modifications exist that are introduced posttranscriptionally by enzymes at specific nucleotide positions. Ribosomal RNA (rRNA) and transfer RNA (tRNA) exhibit the most and diverse modifications that presumably optimize their structure and function. In contrast, oxidative damage can lead to random modifications in rRNA and messenger RNA (mRNA) that strongly impair functionality. RNA modifications have also been implicated in avoiding self-RNA recognition by the immune system or immune evasion by pathogens. Here, we describe the detection of RNA modifications by HPLC analysis and competitive ELISA. Over 100 different RNA modifi cations have been identifi ed and exist in all three kingdoms of life. A comprehensive listing of posttranscriptionally modifi ed RNA nucleosides is found in the RNA Modifi cation Database ( http://mods.rna.albany.edu/ ) [ 1 ] . Especially ribosomal and transfer RNA (rRNA and tRNA) are abundantly modifi ed. Examples are 2′-O-ribose methylation, base methylation, and the occurrence of pseudouridine. In the case of 2′-O-ribose methylation of rRNA, the methyltransferase (2′-O-MTase) fi brillarin utilizes small nucleolar RNAs (snoRNAs), so-called Box C/D snoRNAs that guide the enzyme complex to complementary regions of the rRNA for methylation [ 2 ] . Base methylation of rRNA and tRNA as well as 2′-O-ribose methylation of tRNA are carried out by position/sequence-specifi c methyltransferases independent of snoRNAs. For example, in E. coli uracil-5-methyltransferase (trmA) and guanine-7-methyltransferase (yggH/trmB) methylate specifi c bases in tRNA such as uridine 54 (m5U54) or guanosine 46 (m7G46), respectively [ 3 -5 ] . In contrast, the Gm18-2′-O-methyltransferase (spoU/trmH) methylates the 2′-O-position of a conserved guanosine at position 18 of tRNA (Gm18) [ 6 ] . Interestingly, also messenger RNA (mRNA) is internally modifi ed and carries N6-methyladenosine (m 6 A) [ 7 ] . m 6 A is present in mRNA of all higher eukaryotes tested, including mammals, plants, and insects. This modifi cation occurs on average at 1-3 residues within a defi ned sequence context (e.g., GGACU) per typical mammalian mRNA molecule [ 7 ] . Recently it has been reported that m 6 A sites are enriched near stop codons and in 3′ UTRs suggesting an important role in regulation of gene expression [ 8 , 9 ] . Random RNA modifi cations may also occur by oxidation through reactive oxygen species (ROS) which are involved in killing of bacteria and cell signaling pathways [ 10 -12 ] . Over 20 different purine and pyrimidine modifi cations formed by reactive oxygen species are known; however, 8-hydroxyguanosine (8-OHG) is the most prominent modifi cation [ 13 ] . Of note, 8-hydroxyguanosine modifi cation in mRNA leads to reduced protein levels and altered protein function due to ribosome stalling [ 14 ] . Interestingly, ageassociated oxidative damage to RNA has been demonstrated in neurons and may play a role in neurodegeneration and other diseases [ 15 ] . Bacterial and viral RNA are potent stimulators of the innate immune system leading to immune activation [ 16 ] . RNA is recognized in the endosome by Toll-like receptors (TLR). TLR3 recognizes double-stranded viral RNA and mRNA, whereas TLR7 and TLR8 sense single-stranded RNA [ 17 ] . In contrast, cytoplasmic detection of viral RNA is mediated by the RNA helicases retinoic acid inducible gene-I (RIG-I) and melanoma-differentiationassociated gene 5 (MDA5) [ 18 ] . RIG-I recognizes 5′ triphosphate RNA [ 19 , 20 ] , whereas MDA5 is activated by higher-order RNA structures generated during viral infection [ 21 ] . The effect of ribonucleoside modifi cations on immunostimulation has been investigated only recently. RNA modifi cations such as 2′-O-or base methylation and the occurrence of pseudouridine negatively modify the immunostimulatory potential of in vitro transcribed RNA with respect to TLR3, TLR7, and TLR8 [ 22 ] . Also pseudouridine-containing or 2′-O-methylated synthetic RNA loses its immunostimulatory capacity via RIG-I [ 19 ] . We and others have further demonstrated that 2′-O-methylation of synthetic RNA and tRNA not only renders TLR7 ligands nonimmunostimulatory but also converts modifi ed RNA into a TLR7 antagonist [ 23 -25 ] . Eukaryotic mRNA is not recognized by RIG-I or MDA5 due to a 5′ cap structure methylation at the N7 position of the capping guanosine residue (cap 0), and additional 2′-O-methylation(s) at the 5′-penultimate residue (cap 1) and sometimes also at adjoining residue (cap 2). Interestingly, some viruses that replicate in the cytoplasm (e.g., picornaviruses and coronaviruses) encode functions associated with the formation of a 5′ cap, which are homologous to those found in eukaryotic cells and also support immune evasion. Accordingly, 2′-O-methylation of viral mRNA cap structures by virus-encoded methyltransferases prevents recognition by MDA-5 or host restriction by interferon-induced proteins with tetratricopeptide repeats (IFIT) family members [ 26 , 27 ] . In summary, the detection and characterization of modifi ed ribonucleosides are important for understanding mechanisms of RNA-induced immune activation or immune evasion. (continued) For detection of modifi ed nucleotides in a given RNA sequence HPLC analysis of digested RNA is a suitable method with high sensitivity and specifi city. Before starting the analysis, standard mixtures of nucleosides should be analyzed with the standard protocol to yield high resolution and reproducibility ( see Note 7 ) (Fig. 1 ) . The occurrence of RNA modifi cations within a RNA sample of interest (e.g., total RNA from a eukaryotic cell line) can be judged by comparison and overlay of the individual chromatograms (Fig. 1 ). 6. Run a linear gradient from 0 to 25 % buffer B over 50 min. 7. Run 100 % buffer B for 3 column void volumes and decrease fl ow rate to 0.5 ml/min within 2 min in parallel. increase fl ow rate to 1 ml/min within 8 min. The separation of modified nucleosides can be limited when the nucleoside characteristics are very similar (e.g., 2′-Omethylguanosine and N1-methylguanosine). Therefore, the HPLC run conditions have to be optimized. By varying temperature and gradient slope it is possible to effi ciently separate 2′-Omethylguanosine and N1-methylguanosine which is not achieved by the standard protocol ( Fig. 2 ) ( see Note 8 ). 1. Set column oven to 8 °C. 2. Set fl ow rate to 0.85 ml/min. The nucleoside specifi c UV spectra are an additional characteristic that can be used to discriminate nucleosides. For example, adenosine, guanosine, and 8-hydroxyguanosine differ in the wavelength of maximum absorbance and number of peaks (Fig. 3a ) . A suitable UV detector connected to the HPLC system allows to record UV spectra and retention time simultaneously. Using the following protocol 8-hydroxyguanosine, which has two absorbance maxima at 248 and 294 nm, can be distinguished from guanosine by retention time and UV-absorbance (Fig. 3b ). 1. Set column oven to 21 °C. 2. Set fl ow rate to 1 ml/min. 9. Increase fl ow rate to 1 ml/min. Quantifi cation can be achieved using the linear standard plot method [ 28 ] . 1. Dilute 500 μM of the relevant nucleoside 1:1 for ten times. Subject these standards to HPLC analysis and plot the values of the peak areas against the concentration to create a linear standard curve. 2. The software Chromeleon 6.80 SR10 Build 2818 can determine the peak area of the relevant nucleoside in the sample of interest and calculate the absolute amount using the standard curve. For HPLC analysis the RNA of interest has to be cleaved into nucleotides by nuclease P1 and snake venom phosphodiesterase (SvP) with subsequent dephosphorylation by alkaline phosphatase (AP) to obtain nucleosides [ 29 , 30 ] . 4. All buffers for RNA digestion should be sterilized by fi ltration (0.2 μm fi lter) and stored at 4 °C. Chemicals were supplied from Carl Roth, Germany. 5 . Total cellular or in vitro transcribed RNA can be used, but should be phenol-chloroform-purifi ed before digestion and HPLC analysis. RNA should be stored at −80 °C. 6. All chemicals were supplied by Carl Roth, Germany or as indicated. BSA conjugated 8-OHG was generated as described by Senapathy et al. [ 32 ] . Store conjugated protein at −20 °C. 7. HPLC equilibration, separation, and cleaning procedure: (a) Set up equilibration: Rinse the column which is stored in buffer C with at least 5 void volumes of buffer C, buffer B and buffer A to equilibrate the column. Take care to remove air bubbles thoroughly before connecting the column. (b) Separation: Set column oven temperature and run parameters according to the protocols in Subheading 3 . (c) Cleaning procedure: Wash column with least 5 void volumes of buffer B followed by the equal amount of buffer C. If the column is not used for more than 1 week, remove and seal it. 8. Change parameters of the standard protocol in the order given to optimize HPLC run such as gradient slope, fl ow rate, column temperature, eluotropic strength of elution buffer, ion strength of buffer A, pH value of buffer A, use of multidimensional buffer systems [ 33 ] . 9 . For some RNA samples digestion and dephosphorylation before 8-OHG detection may increase sensitivity. Summary: the modifi ed nucleosides of RNA Guiding ribose methylation of rRNA Transductional mapping of gene trmA responsible for the production of 5-methyluridine in transfer ribonucleic acid of Escherichia coli Transfer RNA modifi cation The yggH gene of Escherichia coli encodes a tRNA (m7G46) methyltransferase The spoU gene of Escherichia coli, the fourth gene of the spoT operon, is essential for tRNA (Gm18) 2′-O-methyltransferase activity N6-adenosine methylation in mRNA: substrate specifi city and enzyme complexity Topology of the human and mouse m 6 A RNA methylomes revealed by m 6 A-seq Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons ROS and innate immunity NLRP3 infl ammasome activation: the convergence of multiple signalling pathways on ROS production Nitric oxide and peroxynitrite in health and disease Some unusual nucleic acid bases are products of hydroxyl radical oxidation of DNA and RNA Messenger RNA oxidation is an early event preceding cell death and causes reduced protein expression Oxidative damage to RNA in aging and neurodegenerative disorders Pattern recognition receptors and infl ammation Intracellular Toll-like receptors Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses 5′-Triphosphate RNA is the ligand for RIG-I RIG-I-mediated antiviral responses to singlestranded RNA bearing 5′-phosphates Activation of MDA5 requires higher-order RNA structures generated during virus infection Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modifi cation and the evolutionary origin of RNA Alternating 2′-O-ribose methylation is a universal approach for generating nonstimulatory siRNA by acting as TLR7 antagonist The 2′-O-methylation status of a single guanosine controls transfer RNA-mediated Toll-like receptor 7 activation or inhibition 2′-O-methylmodifi ed RNAs act as TLR7 antagonists 2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5 Development of an isocratic HPLC method for catechin quantifi cation and its application to formulation studies Identity of phosphodiesterase and phosphomonoesterase activities with nuclease P1 Action of venom phosphodiesterase on deoxyribonucleic acid Urinary 8-hydroxydeoxyguanosine and its analogs as DNA marker of oxidative stress: development of an ELISA and measurement in both bladder and prostate cancers Mechanism of coupling periodate-oxidized nucleosides to proteins Changing reversed-phase high performance liquid chromatography selectivity. Which variables should be tried fi rst? These studies were funded by the German research foundation (DFG) (Grant BA 1618/5-1) and the von Behring Röntgen Stiftung (Grant 56-0034).