Ultraviolet Photodissociation and Activated Electron Photodetachment Mass Spectrometry for Top-Down Sequencing of Modified Oligoribonucleotides.

With the increased development of new RNA-based therapeutics, the need for robust analytical methods for confirming sequences and mapping modifications has accelerated. Characterizing modified ribonucleic acids using mass spectrometry is challenging because diagnostic fragmentation may be suppressed for modified nucleotides, thus hampering complete sequence coverage and the confident localization of modifications. Ultraviolet photodissociation (UVPD) has shown great potential for the characterization of nucleic acids due to extensive backbone fragmentation. Activated electron photodetachment dissociation (a-EPD) has also been used as an alternative to capitalize on the dominant charge-reduction pathway prevalent in UVPD, facilitate dissociation, and produce high abundances of fragment ions. Here, we compare higher-energy collisional activation (HCD), UVPD using 193 and 213 nm photons, and a-EPD for the top-down sequencing of modified nucleic acids, including methylated, phosphorothioate, and locked nucleic acid-modified DNA. The presence of these modifications alters the fragmentation pathways observed upon UVPD and a-EPD, and extensive backbone cleavage is observed that results in the production of fragment ions that retain the modifications and allow them to be pinpointed. LNA and 2'-O-methoxy phosphorothioate modifications caused a significant suppression of fragmentation for UVPD but not for a-EPD, whereas phosphorothioate bonds did not cause any significant suppression for either method. The incorporation of 2'-O-methyl modifications suppressed fragmentation of the antisense strand of patisiran, which resulted in some gaps in sequence coverage. However, UVPD provided the highest sequence coverage when compared to a-EPD.

Determining degradation intermediates and the pathway of 3' to 5' degradation of histone mRNA using high-throughput sequencing.

The half-life of an mRNA is an important parameter contributing to the steady-state level of the mRNA. Rapid changes in mRNA levels can result from decreasing the half-life of an mRNA. Establishing the detailed pathway of mRNA degradation for a particular class of mRNAs requires the ability to isolate mRNA degradation intermediates. High-throughput sequencing provides a method for detecting these intermediates. Here we describe a method for determining the intermediates in 3' to 5' degradation. Characterizing these intermediates requires not only determining the precise 3' end of the molecule to a single nucleotide resolution, but also the ability to detect and characterize any untemplated nucleotides present on the intermediates. We achieve this by ligating a known sequence to all the 3' termini in the cell, and then sequence the 3' termini and the ligated linker to identify any alterations to the genomic reference sequence. We have applied this method to characterize the intermediates in histone mRNA metabolism, allowing us to deduce the pathway of 3' to 5' degradation. This method can potentially be applied to any RNA, and we discuss possible strategies for extending the method to include simultaneous determination of the 3' and 5' end of the same RNA molecule.

Improved application of RNAModMapper - An RNA modification mapping software tool - For analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS) data.

Research into post-transcriptional processing and modification of RNA continues to speed forward, as their ever-emerging role in the regulation of gene expression in biological systems continues to unravel. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has proven for over two decades to be a powerful ally in the elucidation of RNA modification identity and location, but the technique has not proceeded without its own unique technical challenges. The throughput of LC-MS/MS modification mapping experiments continues to be impeded by tedious and time-consuming spectral interpretation, particularly during for the analysis of complex RNA samples. RNAModMapper was recently developed as a tool to improve the interpretation and annotation of LC-MS/MS data sets from samples containing post-transcriptionally modified RNAs. Here, we delve deeper into the methodology and practice of RNAModMapper to provide greater insight into its utility, and remaining hurdles, in current RNA modification mapping experiments.

Characterization and quantification of RNA post-transcriptional modifications using stable isotope labeling of RNA in conjunction with mass spectrometry analysis.

Mass spectrometry has emerged as an increasingly powerful tool for the identification and characterization of nucleic acids, in particular RNA post-transcriptional modifications. High mass accuracy instrumentation is often required to discriminate between compositional isomers of oligonucleotides. We have used stable isotope labeling ((15)N) of E. coli RNA in conjunction with mass spectrometry analysis of the combined heavy- and light-labeled RNA for the identification and quantification of oligoribonucleotides and post-transcriptional modifications. The number of nitrogen atoms in the oligoribonucleotide and fragment ions can readily be determined using this approach, enabling the discrimination between potential compositional isomers without the requirement of high mass accuracy mass spectrometers. In addition, the identification of specific fragment ions in both the unlabeled and labeled oligoribonucleotides can be used to gain further confidence in the assignment of RNA post-transcriptional modifications. Using this approach we have identified a range of post-transcriptional modifications of E. coli 16S rRNA. Furthermore, this method facilitates the rapid and accurate quantification of oligoribonucleotides, including cyclic phosphate intermediates and missed cleavages often generated from RNase digestions.

Natural occurrence of 2',5'-linked heteronucleotides in marine sponges.

2',5'-oligoadenylate synthetases (OAS) as a component of mammalian interferon-induced antiviral enzymatic system catalyze the oligomerization of cellular ATP into 2',5'-linked oligoadenylates (2-5A). Though vertebrate OASs have been characterized as 2'-nucleotidyl transferases under in vitro conditions, the natural occurrence of 2',5'-oligonucleotides other than 2-5A has never been demonstrated. Here we have demonstrated that OASs from the marine sponges Thenea muricata and Chondrilla nucula are able to catalyze in vivo synthesis of 2-5A as well as the synthesis of a series 2',5'-linked heteronucleotides which accompanied high levels of 2',5'-diadenylates. In dephosphorylated perchloric acid extracts of the sponges, these heteronucleotides were identified as A2'p5'G, A2' p5'U, A2'p5'C, G2'p5'A and G2' p5'U. The natural occurrence of 2'-adenylated NAD(+) was also detected. In vitro assays demonstrated that besides ATP, GTP was a good substrate for the sponge OAS, especially for OAS from C. nucula. Pyrimidine nucleotides UTP and CTP were also used as substrates for oligomerization, giving 2',5'-linked homo-oligomers. These data refer to the substrate specificity of sponge OASs that is remarkably different from that of vertebrate OASs. Further studies of OASs from sponges may help to elucidate evolutionary and functional aspects of OASs as proteins of the nucleotidyltransferase family.

Sequencing of single and double stranded RNA oligonucleotides by acid hydrolysis and MALDI mass spectrometry.

Treatment of RNA oligonucleotides with strong acids at pH 1-2 rapidly leads to hydrolysis of the phosphodiester bonds at the 5'-position of ribose. Analysis of the resulting degradation products by MALDI coupled to an Orbitrap high resolution mass spectrometer shows almost complete mass ladders from both sides of the nucleotides without interfering fragments from base losses or internal fragments. From the mass differences between adjacent peaks of a mass ladder, the sequence can be determined. Low cleavage efficiency at the termini leads to 2mers and 3mers which can be identified by MS/MS. In this way the complete sequences of different siRNA 21mer single and double strands could be verified. This simple and fast method can be applied for controlling sequences of synthetic oligomers, as well as for de-novo sequencing. Moreover, the method is applicable for localization and identification of RNA modifications as demonstrated using the examples of an oligonucleotide with phosphorothioate backbone and of one containing 2'-methoxy-ribose modifications.

Roles for TbDSS-1 in RNA surveillance and decay of maturation by-products from the 12S rRNA locus.

The Trypanosoma brucei exoribonuclease, TbDSS-1, has been implicated in multiple aspects of mitochondrial RNA metabolism. Here, we investigate the role of TbDSS-1 in RNA processing and surveillance by analyzing 12S rRNA processing intermediates in TbDSS-1 RNAi cells. RNA fragments corresponding to leader sequence upstream of 12S rRNA accumulate upon TbDSS-1 depletion. The 5' extremity of 12S rRNA is generated by endonucleolytic cleavage, and TbDSS-1 degrades resulting upstream maturation by-products. RNAs with 5' ends at position -141 and 3' ends adjacent to the mature 5' end of 12S rRNA are common and invariably possess oligo(U) tails. 12S rRNAs with mature 3' ends and unprocessed 5' ends also accumulate in TbDSS-1 depleted cells, suggesting that these RNAs represent dead-end products normally destined for decay by TbDSS-1 in an RNA surveillance pathway. Together, these data indicate dual roles for TbDSS-1 in degradation of 12S rRNA maturation by-products and as part of a mitochondrial RNA surveillance pathway that eliminates stalled 12S processing intermediates. We further provide evidence that TbDSS-1 degrades RNAs originating upstream of the first gene on the minor strand of the mitochondrial maxicircle suggesting that TbDSS-1 also removes non-functional RNAs generated from other regions of the mitochondrial genome.

Randomized, double-blind, multicenter study of the immunogenicity and reactogenicity of 17DD and WHO 17D-213/77 yellow fever vaccines in children: implications for the Brazilian National Immunization Program.

Vaccines against yellow fever currently recommended by the World Health Organization contain either virus sub-strains 17D or 17DD. In adults, the 17DD vaccine demonstrated high seroconversion and similar performance to vaccines manufactured with the WHO 17D-213/77 seed-lot. In another study, 17DD vaccine showed lower seroconversion rates in children younger than 2 years. Data also suggested lower seroconversion with simultaneous application of measles vaccine. This finding in very young children is not consistent with data from studies with 17D vaccines. A multicenter, randomized, double-blind clinical trial was designed (1) to compare the immunogenicity and reactogenicity of two yellow fever vaccines: 17DD (licensed product) and 17D-213/77 (investigational product) in children aged 9-23 months; (2) to assess the effect of simultaneous administration of yellow fever and the measles-mumps-rubella vaccines; and (3) to investigate the interference of maternal antibodies in the response to yellow fever vaccination. The anticipated implications of the results are changes in vaccine sub-strains used in manufacturing YF vaccine used in several countries and changes in the yellow fever vaccination schedule recommendations in national immunization programs.

Small ncRNA transcriptome analysis from kinetoplast mitochondria of Leishmania tarentolae.

Gene expression in mitochondria of kinetoplastid protozoa requires RNA editing, a post-transcriptional process which involves insertion or deletion of uridine residues at specific sites within mitochondrial pre-mRNAs. Sequence specificity of the RNA editing process is mediated by oligo-uridylated small, non-coding RNAs, designated as guide RNAs (gRNAs). In this study, we have analyzed the small ncRNA transcriptome from kinetoplast mitochondria of Leishmania tarentolae by generating specialized cDNA libraries encoding size-selected RNA species. Through this screen, a significant number of novel oligo-uridylated RNA species, which we have termed oU-RNAs, has been identified. Most novel oU-RNAs are present as stable RNA species in mitochondria as assessed by northern blot analysis. Thereby, novel oU-RNAs show similar expression levels and sizes as previously reported for canonical gRNAs. Several oU-RNAs are transcribed from both strands of the maxicircle and minicircles components of the mitochondrial genome, from regions where up till now no transcription has been reported. Two stable oU-RNAs exhibit an anchor sequence in antisense orientation to known gRNAs and thus might regulate editing of respective pre-mRNAs. A number of oU-RNAs map in antisense orientation to non-edited protein-coding genes suggesting that they might function by a different mechanism. In addition, our screen shows that all kinetoplast-derived RNAs are prone to some degree of uridylation.

Gas-phase dissociation of oligoribonucleotides and their analogs studied by electrospray ionization tandem mass spectrometry.

Oligoribonucleotides (RNA) and modified oligonucleotides were subjected to low-energy collision-induced dissociation in a hybrid quadrupole time-of-flight mass spectrometer to investigate their fragmentation pathways. Only very restricted data are available on gas-phase dissociation of oligoribonucleotides and their analogs and the fundamental mechanistic aspects still need to be defined to develop mass spectrometry-based protocols for sequence identification. Such methods are needed, because chemically modified oligonucleotides can not be submitted to standard sequencing protocols. In contrast to the dissociation of DNA, dissociation of RNA was found to be independent of nucleobase loss and it is characterized by cleavage of the 5'-P-O bond, resulting in the formation of c- and their complementary y-type ions. To evaluate the influence of different 2'-substituents, several modified tetraribonucleotides were analyzed. Oligoribonucleotides incorporating a 2'-methoxy-ribose or a 2'-fluoro-ribose show fragmentation that does not exhibit any preferred dissociation pathway because all different types of fragment ions are generated with comparable abundance. To analyze the role of the nucleobases in the fragmentation of the phosphodiester backbone, an oligonucleotide lacking the nucleobase at one position has been studied. Experiments indicated that the dissociation mechanism of RNA is not influenced by the nucleobase, thus, supporting a mechanism where dissociation is initiated by formation of an intramolecular cyclic transition state with the 2'-hydroxyl proton bridged to the 5'-phosphate oxygen.

Analysis of biological and synthetic ribonucleic acids by liquid chromatography-mass spectrometry using monolithic capillary columns.

Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) has been evaluated as a method for the fractionation and desalting of ribonucleic acids prior to their characterization by electrospray ionization mass spectrometry. Monolithic, poly(styrene-divinylbenzene)-based capillary columns allowed the rapid and highly efficient fractionation of both synthetic and biological ribonucleic acids. The common problem of gas-phase cation adduction that is particularly prevalent in the mass spectrometric analysis of ribonucleic acids was tackled through a combination of chromatographic purification and the addition of ethylenediaminetetraacetic acid to the sample at a concentration of 25 mmol/L shortly before on-line analysis. For RNA molecules ranging in size from 10 to 120 nucleotides, the mass accuracies were typically better than 0.02%, which allowed the characterization and identification of failure sequences and byproducts with high confidence. Following injection of a 500 nL sample onto a 60 x 0.2 mm column, the limit of detection for a 120-nucleotide ribosomal RNA transcript from Escherichia coli was in the 50-80 fmol range. The method was applied to the analysis of synthetic oligoribonucleotides, transfer RNAs, and ribosomal RNA. Finally, sequence information was derived for low picomole amounts of a 32-mer RNA upon chromatographic purification and tandem mass spectrometric investigation in an ion trap mass spectrometer. Complete series of fragment ions of the c- and y-types could be assigned in the tandem mass spectrum. In conclusion, IP-RP-HPLC using monolithic capillary columns represents a very useful tool for the structural investigation and quantitative determination of RNAs of synthetic and biological origin.

Improved mass analysis of oligoribonucleotides by 13C, 15N double depletion and electrospray ionization FT-ICR mass spectrometry.

13C, 15N doubly depleted 32-ribonucleotide was synthesized enzymatically by in vitro transcription from nucleoside triphosphates isolated from E. coli grown in a minimal medium containing 12C, 14N-enriched glucose and ammonium sulfate. Following purification and desalting by reversed-phase HPLC, buffer exchange with Microcon YM-3, and ethanol precipitation, electrospray ionization Fourier transform ion cyclotron resonance mass spectra revealed greatly enhanced abundance of monoisotopic ions (by a factor of approximately 100) and a narrower isotopic distribution with higher signal-to-noise ratio. The abrupt onset and high magnitude of the monoisotopic species promise to facilitate accurate mass measurement of RNA's.

Synthesis of S6-(2,4-dinitrophenyl)-6-thioguanosine phosphoramidite and its incorporation into oligoribonucleotides.

The preparation of N(2)-phenoxylacetyl-S(6)-(2,4-dinitrophenyl)-6-thioguanosine phosphoramidite and its subsequent incorporation into oligoribonucleotides is described. The identity of the oligonucleotides was confirmed by UV spectrophotometry and nucleoside composition analysis.

Determination of platinated purines in oligoribonucleotides by limited digestion with ribonucleases T1 and U2.

Platinum complexes which are known to react preferentially with guanine (G) and adenine (A) bases of oligonucleotides can be used as tools to analyze their tertiary structures and eventually to cross-link them. However, this requires efficient methods to allow the identification and quantification of the corresponding adducts which have so far been developed only for oligodeoxyribonucleotides. Maxam-Gilbert type digestions cannot be used for RNAs and HPLC techniques would require too large amounts of expensive material for separation and further characterization. We report a method to determine platination sites on oligoribonucleotides based on the cleavage activity of ribonucleases T1 and U2. To test the method, these enzymes were first used under conditions of limited digestion on 5-mer oligoribonucleotides platinated at a single defined purine. The phosphodiester bond on the 3' side of platinated G or A appeared fully resistant to cleavage by ribonuclease T1 or U2, respectively. An inhibitory effect was also observed due to neighboring platinated purines, which decreases with their distance (-2, -1, +1, +2) from the cleavage site and with the enzyme concentration. The method allowed the identification and quantification of the platination sites of a 17-mer oligoribonucleotide, based on the analysis of the mixture of monoplatinated adducts.

Analysis of oligonucleotides and unincorporated nucleotides from in vitro transcription by capillary electrophoresis in Pluronic F127 gels.

Small functional RNAs required for structure studies are often prepared by in vitro transcription. Capillary electrophoresis in liquid crystalline gels of Pluronic F127 was used to analyze unfractionated in vitro transcription reactions and anion-exchange high-performance liquid chromatography (HPLC) fractions from transcription reactions. Guanosine monophosphate (GMP), the four nucleoside triphosphates (NTPs), abortive transcripts, and transcripts with lengths near the desired product length were simultaneously resolved and quantified in a single run. Oligonucleotides up to at least 35 nucleotides were resolved to baseline within 10 min using a moderate field (185 V/cm) and short effective capillary length (7.6 cm) for electrophoresis in 20% Pluronic F127 at pH 8.3 in Tris-borate-EDTA (TBE) buffer (30 degrees C). Nucleotide migration times were 4-5 min, in the order UTP+CTP (unresolved)

Disruption of substrate binding site in E. coli RNA polymerase by lethal alanine substitutions in carboxy terminal domain of the beta subunit.

Alanine substitution of four amino acids in two evolutionarily conserved motifs, PSRM and RFGEMIE, near the carboxy terminus of the beta subunit of E. coli RNA polymerase results in a dramatic loss of the enzyme's affinity to substrates with no apparent effect on the maximal rate of the enzymatic reaction or on binding to promoters. The magnitude and selectivity of the effect suggest that the mutations disrupt the substrate binding site of the active center.

Formation of RNA oligomers on montmorillonite: site of catalysis.

Certain montmorillonites catalyze the self condensation of the 5'-phosphorimidazolide of nucleosides in pH 8 aqueous electrolyte solutions at ambient temperatures leading to formation of RNA oligomers. In order to establish the nature of the sites on montmorillonite responsible for this catalytic activity, oligomerization reactions were run with montmorillonites which had been selectively modified (I) at the edges by (a) fluoride treatment, (b) silylation, (c) metaphosphate treatment of the anion exchange sites (II) in the interlayer by (a) saturation with quaternary alkylammonium ions of increasing size, (b) aluminum polyoxo cations. High pressure liquid chromatography, HPLC, analysis of condensation products for their chain lengths and yields indicated that modification at the edges did not affect the catalytic activity to a significant extent, while blocking the interlayer strongly inhibited product formation.

Characteristics of poly(A)-degrading factor present in the avian oocytes and early embryos.

The presence of poly(A)-degrading activity was studied in vitro in the quail and mouse oocytes and early embryos using 3H-poly(A) as a substrate. The activity was measured by adsorption of the undegraded substrate to DE-81 filter paper discs, by chromatographic separation on Sephadex G-50 column and by agarose gel electrophoresis followed by transfer onto a Zeta-probe membrane (BioRad, Richmond, CA) and autoradiography. High poly(A)-degrading activity was found in the quail previtellogenic and vitellogenic oocytes and lower activity in the early embryos from cleavage stage to gastrulation. This activity is localized predominantly in the nucleus and, to a lesser degree, in the cytoplasm and in the vitellus of vitellogenic oocytes. The length of the poly(A) degradation product was estimated to be of about (A)10. Optimum activity was at pH 8.7 and at Mn2+ concentration of 0.5 mM. This makes the deadenylating enzyme from the quail oocytes similar to endoribonuclease IV from the chick and quail oviducts (Müller [1976] Eur. J. Biochem., 70:241-248; Müller [1976], Eur. J. Biochem., 70:249-258). We suggest that the poly(A)-degrading enzyme, similar to endoribonuclease IV found in the quail oocytes, might be the "deadenylating factor" reported in Xenopus oocytes (Varnum et al. [1992] Dev. Biol., 153:283-290). Such poly(A)-degrading activity is undetectable in unfertilized mouse eggs; however, a slight, statistically insignificant tendency for poly(A) degradation was seen in two-cell embryos.

Variation in the size of nascent RNA cleavage products as a function of transcript length and elongation competence.

RNA polymerase II arrested at specific template locations can be rescued by elongation factor SII via RNA cleavage. The size of the products removed from the 3'-end of the RNA varies. The release of single nucleotides, dinucleotides, and larger oligonucleotides has been detected by different workers. Dinucleotides tend to originate from SII-independent complexes and 7-14 base products from SII-dependent complexes (Izban, M. G., and Luse, D. S. (1993) J. Biol. Chem. 268, 12874-12885). Different modes of cleavage have also been recognized for bacterial transcription complexes and are thought to represent important structural differences between functionally distinct transcription intermediates. Using an elongation complex "walking" technique, we have observed factor-independent complexes as they approach and become arrested at an arrest site. Dinucleotides or 7-9-base (large) oligonucleotides were released from SII-independent or dependent complexes, respectively. The abrupt shift between the release of dinucleotide versus larger products accompanied the change from factor-dependent to factor-independent elongation, as described by others. However, not all factor-independent complexes showed cleavage in dinucleotide intervals since oligonucleotides 2-6 bases long were also liberated from elongation-competent complexes. These were all 5'-coterminal oligonucleotides indicating that a preferred phosphodiester bond is targeted for cleavage in a series of related complexes. This is consistent with recent models postulating a large product binding site that can hold RNA chains whose size increases as a function of chain polymerization. A specific transitional complex was identified that acquired the ability to cleave in a large increment one base insertion event prior to attaining the arrested configuration.