DOCSLIB.ORG

Modified Nucleosides, Nucleotides, and Nucleic

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Modified Nucleosides, Nucleotides, and Nucleic (19) TZZ ¥Z__T (11) EP 2 763 701 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 48/00 (2006.01) C07H 21/02 (2006.01) 19.12.2018 Bulletin 2018/51 C07H 21/04 (2006.01) (21) Application number: 12838676.0 (86) International application number: PCT/US2012/058519 (22) Date of filing: 03.10.2012 (87) International publication number: WO 2013/052523 (11.04.2013 Gazette 2013/15) (54) MODIFIED NUCLEOSIDES, NUCLEOTIDES, AND NUCLEIC ACIDS, AND USES THEREOF MODIFIZIERTE NUKLEOSIDE, NUKLEOTIDE UND NUKLEINSÄUREN UND VERWENDUNGEN DAVON NUCLÉOSIDES, NUCLÉOTIDES ET ACIDES NUCLÉIQUES MODIFIÉS, ET LEURS UTILISATIONS (84) Designated Contracting States: (74) Representative: Hoffmann Eitle AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Patent- und Rechtsanwälte PartmbB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Arabellastraße 30 PL PT RO RS SE SI SK SM TR 81925 München (DE) Designated Extension States: BA ME (56) References cited: WO-A2-2011/012316 US-A1- 2010 047 261 (30) Priority: 03.10.2011 US 201161542533 P US-A1- 2011 143 397 US-A1- 2012 251 618 (43) Date of publication of application: • KARIKÓ KATALIN ET AL: "Incorporation of 13.08.2014 Bulletin 2014/33 pseudouridine into mRNA yields superior nonimmunogenic vector with increased (73) Proprietor: Moderna Therapeutics, Inc. translational capacity and biological stability", Cambridge, Massachusetts 02141 (US) MOLECULAR THERAPY, NATURE PUBLISHING GROUP, GB, vol. 16, no. 11, 1 November 2008 (72) Inventors: (2008-11-01), pages 1833-1840, XP002598556, • DE FOUGEROLLES, Antonin ISSN: 1525-0024, DOI: 10.1038/MT.2008.200 Brookline, MA 02446 (US) [retrieved on 2008-09-16] •ROY,Atanu • LUIGI WARREN ET AL: "Highly efficient Stoneham, MA 02180 (US) reprogramming to pluripotency and directed • SCHRUM, Jason P. differentiation of human cells with synthetic Watertown MA 02472-2580 (US) modified mRNA", CELL STEM CELL, CELL • SIDDIQI, Suhaib PRESS, UNITED STATES, [Online] vol. 7, no. 5, 5 Burlington, MA 01803 (US) November 2010 (2010-11-05), pages 618-630, • HATALA, Paul XP002693059, ISSN: 1875-9777, DOI: Charlestown, MA 02124 (US) 10.1016/J.STEM.2010.08.012 Retrieved from the • BANCEL, Stephane Internet: Cambridge, MA 02141 (US) URL:http://www.sciencedirect.com/science/a rticle/pii/S1934590910004340> Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 763 701 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 763 701 B1 Description CROSS REFERENCE TO RELATED APPLICATIONS 5 [0001] This application claims priority to U.S. Provisional Patent Application No. 61/542,533, filed October 3, 2011, entitled Modified Nucleosides, Nucleotides, and Nucleic Acids, and Uses Thereof. TECHNICAL FIELD 10 [0002] The present disclosure provides compositions and methods using modified nucleic acids to modulate cellular function. The modified nucleic acids of the disclosure may encode peptides, polypeptides or multiple proteins. The encoded molecules may be used as therapeutics and/or diagnostics. BACKGROUND 15 [0003] Naturally occurring RNAs are synthesized from four basic ribonucleotides: ATP, CTP, UTP and GTP, but may contain post-transcriptionally modified nucleotides. Further, approximately one hundred different nucleoside modifica- tions have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). The role of nucleoside modifications on the immune-stimulatory potential 20 and on the translation efficiency of RNA, however, is unclear. [0004] There are multiple problems with prior methodologies of effecting protein expression. For example, heterologous DNA introduced into a cell can be inherited by daughter cells (whether or not the heterologous DNA has integrated into the chromosome) or by offspring. Introduced DNA can integrate into host cell genomic DNA at some frequency, resulting in alterations and/or damage to the host cell genomic DNA. In addition, multiple steps must occur before a protein is 25 made. Once inside the cell, DNA must be transported into the nucleus where it is transcribed into RNA. The RNA transcribed from DNA must then enter the cytoplasm where it is translated into protein. This need for multiple processing steps creates lag times before the generation of a protein of interest. Further, it is difficult to obtain DNA expression in cells; frequently DNA enters cells but is not expressed or not expressed at reasonable rates or concentrations. This can be a particular problem when DNA is introduced into cells such as primary cells or modified cell lines. 30 [0005] There is a need in the art for biological modalities to address the modulation of intracellular translation of nucleic acids. [0006] Kariko et al. (Mol Ther. 2008;16(11):1833-1840) relates to the incorporation of pseudouridine into mRNA. Reduced immunogenicity and increased translational capacity and biological stability are reported. US2011/143397A1 relates to RNA preparations comprising purified modified RNA for reprogramming cells. 35 SUMMARY [0007] The present invention is defined in the claims and provides an isolated polynucleotide encoding a polypeptide of interest, said isolated polynucleotide comprising: 40 (a) a sequence of n number of linked nucleosides or nucleotides comprising at least one modified nucleoside or nucleotide as compared to the chemical structure of an A, G, U or C nucleoside or nucleotide, (b) a 5’ UTR comprising at least one Kozak sequence, (c) a 3’ UTR, and 45 (d) at least one 5’ cap structure; wherein the at least one modified nucleoside or nucleotide comprises 5-methoxy-uracil, and is not pseudouridine or 5- methyl-cytidine, wherein the polynucleotide exhibits a reduced cellular innate immune response when introduced into a population of 50 cells, as compared to the cellular innate immune induced by a corresponding unmodified nucleic acid, wherein the polynucleotide has a Protein:Cytokine ratio of greater than 100, wherein the Protein:Cytokine ratio is the ratio of the amount of encoded polypeptide produced in a cell, tissue or organism to the amount of one or more cytokines whose expression is triggered in the cell, tissue or organism as a result of administration or contact with the polynucleotide. 55 [0008] Further aspects of the invention are also defined in the claims. The disclosure in the following description provides both information relating to subject matter covered by the definitions of the claims and additional information which is not the subject matter of the claims. In the following disclosure, where reference is made to embodiments or aspects, such embodiments or aspects are to be understood as embodiments of the disclosure in general, whereas the 2 EP 2 763 701 B1 scope of the invention is defined in the claims. The description of any subject matter not covered by the claims is provided for information only. [0009] The present disclosure provides, inter alia, modified nucleosides, modified nucleotides, and modified nucleic acids which can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and 5 ex vivo. [0010] The present disclosure provides polynucleotides which may be isolated or purified. These encode polynucle- otides encode one or more polypeptides of interest and comprise a sequence of n number of linked nucleosides or nucleotides comprising at least one modified nucleoside or nucleotide as compared to the chemical structure of an A, G, U or C nucleoside or nucleotide. The polynucleotides also comprise a 5’ UTR comprising at least one Kozak sequence, 10 a 3’ UTR, and at least one 5’ cap structure. The isolated polynucleotides may further comprise a poly-A tail and may be purified. [0011] The isolated polynucleotides of the disclosure also comprise at least one 5’ cap structure selected from the group consisting of Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2’fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. 15 [0012] Modifications of the polynucleotides of the disclosure may be on the nucleoside base and/or sugar portion of the nucleosides which comprise the polynucleotide. [0013] In some embodiments, the modification is on the nucleobase and is N1-methylpseudouridine. [0014] The modified nucleoside is not pseudouridine (ψ) or 5-methyl-cytidine (m5C). [0015] In some embodiments, multiple modifications are included in the modified nucleic acid or in one or more 20 individual nucleoside or nucleotide. For example, modifications to a nucleoside may include one or more modifications to the nucleobase and the sugar. [0016] In some embodiments are provided novel building blocks, e.g., nucleosides and nucleotides for the preparation of modified polynucleotides and their method of synthesis and manufacture. [0017] The present disclosure also provides for pharmaceutical compositions comprising the modified polynucleotides 25 described herein. These may also further include one or more pharmaceutically acceptable excipients selected from a solvent, aqueous solvent, non-aqueous solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening
Load more

Recommended publications
  • Investigation of Overhauser Effects Between Pseudouridine and Water Protons in RNA Helices
    Investigation of Overhauser effects between pseudouridine and water protons in RNA helices Meredith I. Newby* and Nancy L. Greenbaum*†‡ *Department of Chemistry and Biochemistry and †Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4390 Communicated by Michael Kasha, Florida State University, Tallahassee, FL, August 8, 2002 (received for review July 2, 2002) The inherent chemical properties of RNA molecules are expanded by posttranscriptional modification of specific nucleotides. Pseudouridine (␺), the most abundant of the modified bases, features an additional imino group, NH1, as compared with uri- dine. When ␺ forms a Watson–Crick base pair with adenine in an RNA helix, NH1 is positioned within the major groove. The pres- ence of ␺ often increases thermal stability of the helix or loop in which it is found [Hall, K. B. & McLaughlin, L. (1992) Nucleic Acids Res. 20, 1883–1889]. X-ray crystal structures of transfer RNAs [e.g., Arnez, J. & Steitz, T. (1994) Biochemistry 33, 7560–7567] have depicted water molecules bridging ␺NH1 groups and nearby phos- phate oxygen atoms, but direct evidence for this interaction in solution has not been acquired. Toward this end, we have used a rotating-frame Overhauser effect spectroscopy-type NMR pulse sequence with a CLEAN chemical-exchange spectroscopy spin-lock pulse train [Hwang, T.-L., Mori, S., Shaka, A. J. & van Zijl, P. C. M. Fig. 1. Schematic structures of U and ␺ bases. R, ribose. The ␺ base is a uracil ␺ (1997) J. Am. Chem. Soc. 119, 6203–6204] to test for NH1–water rotated about the 3–6 ring axis, so that is has a COC base–sugar linkage and cross-relaxation effects within two RNA helices: (i) a complemen- an additional protonated ring nitrogen.
    [Show full text]
  • The Human Ortholog of Archaeal Pus10 Produces Pseudouridine 54 in Select Trnas Where Its Recognition Sequence Contains a Modified Residue
    Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press The human ortholog of archaeal Pus10 produces pseudouridine 54 in select tRNAs where its recognition sequence contains a modified residue MANISHA DEOGHARIA,1 SHAONI MUKHOPADHYAY, ARCHI JOARDAR,2 and RAMESH GUPTA Department of Biochemistry and Molecular Biology, Southern Illinois University, Carbondale, Illinois 62901-4413, USA ABSTRACT The nearly conserved U54 of tRNA is mostly converted to a version of ribothymidine (T) in Bacteria and eukaryotes and to a version of pseudouridine (Ψ) in Archaea. Conserved U55 is nearly always modified to Ψ55 in all organisms. Orthologs of TrmA and TruB that produce T54 and Ψ55, respectively, in Bacteria and eukaryotes are absent in Archaea. Pus10 produces both Ψ54 and Ψ55 in Archaea. Pus10 orthologs are found in nearly all sequenced archaeal and most eukaryal genomes, but not in yeast and bacteria. This coincides with the presence of Ψ54 in most archaeal tRNAs and some animal tRNAs, but its absence from yeast and bacteria. Moreover, Ψ54 is found in several tRNAs that function as primers for retroviral DNA syn- thesis. Previously, no eukaryotic tRNA Ψ54 synthase had been identified. We show here that human Pus10 can produce Ψ54 in select tRNAs, including tRNALys3, the primer for HIV reverse transcriptase. This synthase activity of Pus10 is restrict- ed to the cytoplasm and is distinct from nuclear Pus10, which is known to be involved in apoptosis. The sequence GUUCAm1AAUC (m1A is 1-methyladenosine) at position 53–61 of tRNA along with a stable acceptor stem results in max- imum Ψ54 synthase activity.
    [Show full text]
  • Pseudouridine Synthases Modify Human Pre-Mrna Co-Transcriptionally and Affect Splicing
    bioRxiv preprint doi: https://doi.org/10.1101/2020.08.29.273565; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Pseudouridine synthases modify human pre-mRNA co-transcriptionally and affect splicing Authors: Nicole M. Martinez1, Amanda Su1, Julia K. Nussbacher2,3,4, Margaret C. Burns2,3,4, Cassandra Schaening5, Shashank Sathe2,3,4, Gene W. Yeo2,3,4* and Wendy V. Gilbert1* Authors and order TBD with final revision. Affiliations: 1Yale School of Medicine, Department of Molecular Biophysics & Biochemistry, New Haven, CT 06520, USA. 2Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92037, USA. 3Stem Cell Program, University of California, San Diego, La Jolla, CA 92037, USA. 4Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92037, USA. 5Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. *Correspondence to: [email protected], [email protected] Abstract: Eukaryotic messenger RNAs are extensively decorated with modified nucleotides and the resulting epitranscriptome plays important regulatory roles in cells 1. Pseudouridine (Ψ) is a modified nucleotide that is prevalent in human mRNAs and can be dynamically regulated 2–5. However, it is unclear when in their life cycle RNAs become pseudouridylated and what the endogenous functions of mRNA pseudouridylation are. To determine if pseudouridine is added co-transcriptionally, we conducted pseudouridine profiling 2 on chromatin-associated RNA to reveal thousands of intronic pseudouridines in nascent pre-mRNA at locations that are significantly associated with alternatively spliced exons, enriched near splice sites, and overlap hundreds of binding sites for regulatory RNA binding proteins.
    [Show full text]
  • A Dissertation Entitled Ribonucleic Acids in Disease Etiology and Drug Discovery by Immaculate Sappy Submitted to the Graduate F
    A Dissertation entitled Ribonucleic Acids in Disease Etiology and Drug Discovery by Immaculate Sappy Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Medicinal Chemistry ________________________________________ Amanda C. Bryant-Friedrich, Ph.D., Committee Chair ________________________________________ Zahoor A Shah, Ph.D., Committee Member ________________________________________ Steven M Peseckis, Ph.D., Committee Member ________________________________________ Caren Steinmiller, Ph.D., Committee Member ________________________________________ Amanda Bryant-Friedrich, PhD, Dean College of Graduate Studies The University of Toledo December 2019 Copyright 2019, Immaculate Sappy This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Ribonucleic Acids in Disease Etiology and Drug Discovery by Immaculate Sappy Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Medicinal Chemistry The University of Toledo December 2019 Pseudouridine (Ψ), the 5-ribosyl isomer of uridine (U) is the most abundant nucleic acid modification found in all domains of life and all types of RNA. Studies have shown that, urinary levels of pseudouridine are higher in Alzheimer’s Disease (AD) patients and that RNA oxidation is a major component in the pathogenesis of Alzheimer’s Disease (AD) and other neurodegenerative disorders. Therefore, there is a potential correlation between higher urinary levels of pseudouridine in AD patients and oxidative stress. Hence, subjecting pseudouridine to oxidative conditions may provide some key information about the role of this nucleoside in RNA related processes and its role in disease etiology. Besides neurodegenerative disorders, antibiotic resistance is an additional threat to human health.
    [Show full text]
  • Increased Excretion of Modified Adenine Nucleosides by Children with Adenosine Dearninase Deficiency
    Pediatr. Res. 16: 362-369 (1982) Increased Excretion of Modified Adenine Nucleosides by Children with Adenosine Dearninase Deficiency ROCHELLE HIRSCHHORN,'"~ HOWARD RATECH, ARYE RUBINSTEIN, PHOTINI PAPAGEORGIOU, HERNANT KESARWALA, ERWIN GELFAND, AND VIVIEN ROEGNER-MANISCALCO Departments of Medicine and Pathology, New York University School of Medicine, New York, New York [R.H., H.R., and V.R.-M.];Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York [A.R.]; Department of Pediatrics, Rutgers University Medical ~chool,Piscataway, New Jersey [P.P., and H.K.]; and Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada [E. G.] Summary tially in, and prevents proliferation of, irnmunocompetent cells, primarily of the T cell class (2, 5, 6, 23, 38, 49, 54). There is also We have identified seven adenine nucleosides in urines of un- in vivo and/or in vitro evidence for alternative mechanisms of treated adenosine deaminase (ADA) deficient patients, four of toxicity, which would operate via depletion of pyrimidine pools, which (adenosine, 2'-deoxyadenosine, 1-methyladenosine and N6- depletion of phosphoribosyl pyrophosphate and increases in cyclic methyladenosine) have been previously identified in urines of AMP or S-adenosyl homocysteine (16, 21, 24, 40, 46, 55). All of normals and/or ADA deficient patients. We confirm that ADA these mechanisms are dependent on accumulation of the substrates deficient patients excrete markedly increased amounts of 2'-deox- of ADA, adenosine and 2'-deoxyadenosine. yadenosine (582 k 363 versus normal of < 0.1 nmoles/mg creati- In addition to adenosine and 2'-deoxyadenosine, several other nine) and increased amounts of adenosine (29.4 & 5.7 versus modified adenine nucleosides occur naturally (17, 19) and are normal of 4.12 & 1.0 nmoles/mg creatinine).
    [Show full text]
  • N4-Acetylcytidine and 5-Formylcytidine Are Present in Saccharomyces Cerevisiae Mrnas
    bioRxiv preprint doi: https://doi.org/10.1101/327585; this version posted May 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. N4-acetylcytidine and 5-formylcytidine are present in Saccharomyces cerevisiae mRNAs Mehmet Tardu1, Qishan Lin2, and Kristin S. Koutmou1* 1University of Michigan, Department of Chemistry, 930 N University, Ann Arbor, MI 48109, (734) 764-5650, [email protected], 2Mass Spectrometry Consortium for Epitranscriptomics, University at Albany, 1400 Washington Ave, Albany, NY 12222. Abstract Chemical modifications of RNAs have long been appreciated as key modulators of non-coding RNA structure and function. There is an emerging realization that chemical modification of protein-coding mRNAs also plays critical roles in the cell. Nonetheless, of the over 100 known RNA modifications found in biology only a handful have been identified in mRNAs. Here we use an ultra-high performance liquid chromatography- tandem mass spectrometry (UHPLC-MS/MS) method to identify and quantify modifications present in mRNAs of yeast cells. We detect the presence of four modified nucleosides in mRNAs at relatively high abundances: N7-methylguanosine, N6- methyladenosine, N4-acetylcytidine and 5-formylcytidine. Additionally, we investigate how the levels of mRNA modifications change in response to cellular stress. We find that the concentrations of nine mRNA modifications including N6-methyladenosine and N4- acetylcytidine change in response to heat stress, glucose starvation and/or oxidative stress. Our findings suggest that mRNA modification may provide a potential mechanism for cells to rapidly respond to environmental stressors.
    [Show full text]
  • Pseudouridine-Mediated Translation Control of Mrna by Methionine Aminoacyl Trna Synthetase Ofri Levi and Yoav S
    432–443 Nucleic Acids Research, 2021, Vol. 49, No. 1 Published online 10 December 2020 doi: 10.1093/nar/gkaa1178 Pseudouridine-mediated translation control of mRNA by methionine aminoacyl tRNA synthetase Ofri Levi and Yoav S. Arava * Faculty of Biology, Technion – Israel Institute of Technology, Haifa 3200003, Israel Received October 01, 2020; Revised November 06, 2020; Editorial Decision November 13, 2020; Accepted December 08, 2020 Downloaded from https://academic.oup.com/nar/article/49/1/432/6030234 by guest on 30 September 2021 ABSTRACT GRAPHICAL ABSTRACT Modification of nucleotides within an mRNA emerges as a key path for gene expression regulation. Pseu- douridine is one of the most common RNA modifica- tions; however, only a few mRNA modifiers have been identified to date, and no one mRNA pseudouridine reader is known. Here, we applied a novel genome- wide approach to identify mRNA regions that are bound by yeast methionine aminoacyl tRNAMet syn- thetase (MetRS). We found a clear enrichment to regions that were previously described to contain pseudouridine (). Follow-up in vitro and in vivo analyses on a prime target (position 1074 within YEF3 mRNA) demonstrated the importance of pseudouri- dine for MetRS binding. Furthermore, polysomal and INTRODUCTION protein analyses revealed that 1074 mediates trans- Over the past decades, >100 post-transcriptional RNA lation. Modification of this site occurs presumably by modifications have been identified in all kingdoms oflife(1). Pus6, a pseudouridine synthetase known to modify One of the most common RNA modifications in living cells MetRS cognate tRNA. Consistently, the deletion of is pseudouridine ()(2,3). The isomerization of uridine to Pus6 leads to a decrease in MetRS association with pseudouridine has a similar base pairing as uridine, yet it both tRNAMet and YEF3 mRNA.
    [Show full text]
  • Long Non-Coding RNA Epigenetics
    International Journal of Molecular Sciences Review Long Non-Coding RNA Epigenetics Marek Kazimierczyk and Jan Wrzesinski * Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Pozna´n,Poland; [email protected] * Correspondence: [email protected] Abstract: Long noncoding RNAs exceeding a length of 200 nucleotides play an important role in ensuring cell functions and proper organism development by interacting with cellular compounds such as miRNA, mRNA, DNA and proteins. However, there is an additional level of lncRNA regulation, called lncRNA epigenetics, in gene expression control. In this review, we describe the most common modified nucleosides found in lncRNA, 6-methyladenosine, 5-methylcytidine, pseudouridine and inosine. The biosynthetic pathways of these nucleosides modified by the writer, eraser and reader enzymes are important to understanding these processes. The characteristics of the individual methylases, pseudouridine synthases and adenine–inosine editing enzymes and the methods of lncRNA epigenetics for the detection of modified nucleosides, as well as the advantages and disadvantages of these methods, are discussed in detail. The final sections are devoted to the role of modifications in the most abundant lncRNAs and their functions in pathogenic processes. Keywords: lncRNA epigenetics; RNA modyfiing enzymes; detection modified nucleotides 1. General Remarks Citation: Kazimierczyk, M.; Although almost 80% of the human genome has been transcribed, only 2% of it Wrzesinski, J. Long Non-Coding RNA (mRNA) codes proteins [1,2]. All of the remaining RNAs belong to a vast group of noncod- Epigenetics. Int. J. Mol. Sci. 2021, 22, ing RNAs (ncRNAs). Based on their functions, ncRNAs can be classified as housekeeping 6166.
    [Show full text]
  • Ence of Pseudouridine (5-Ribosyluracil) and Uracil
    Journal of Clinical Investigation Vol. 41, No. 7, 1962 THE METABOLISM OF RING-LABELED OROTIC ACID IN MAN By S. M. WEISSMAN, A. Z. EISEN, H. FALLON, M. LEWIS AND M. KARON (From the Metabolism Service, Dermatology and Medicine Branches, National Cancer Institute, Bethesda, Md.) (Submitted for publication January 19, 1962; accepted March 29, 1962) Considerable knowledge concerning purine me- Total plasma and urine radioactivity was determined by tabolism has been obtained from chemical and first digesting the plasma or urine with an equal vol of isotopic studies of urinary uric acid. Correspond- 5 N KOH for 2 hours at 70° C, decolorizing with 1 to 2 drops of hydrogen peroxide, and counting 0.2 ml of this ing information in man is lacking for the closely digest in 20 ml of the toluene-methanol solution (8). In related field of pyrimidine metabolism. The avail- all cases counting efficiency was determined by the addi- ability of ring-labeled orotic acid as a specific tion of internal standards. pyrimidine precursor (1), together with the pres- Alternatively, radioactivity of urine was determined by ence of pseudouridine (5-ribosyluracil) and uracil, plating serial dilutions of each urine specimen on stainless steel cups, and extrapolating the count rates to infinite as specific end products of pyrimidine metabolism thinness. After the first 8 days, the count rate in total in man, in conveniently assayable amounts in urine was generally too low to be conveniently determined human urine (2-4), makes it possible to conduct directly. Extrapolations of total urine radioactivity were certain kinetic studies. In the present investiga- based on the assumption that total urine radioactivity had tion the metabolism of ring-labeled orotic acid and a constant ratio to urinary pseudouridine activity, since such parallelism was observed in days 3 through 8.
    [Show full text]
  • Characterization of UVA-Induced Alterations to Transfer RNA Sequences
    biomolecules Article Characterization of UVA-Induced Alterations to Transfer RNA Sequences Congliang Sun, Patrick A. Limbach and Balasubrahmanyam Addepalli * Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, USA; [email protected] (C.S.); [email protected] (P.A.L.) * Correspondence: [email protected] Received: 15 September 2020; Accepted: 5 November 2020; Published: 8 November 2020 Abstract: Ultraviolet radiation (UVR) adversely affects the integrity of DNA, RNA, and their nucleoside modifications. By employing liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based RNA modification mapping approaches, we identified the transfer RNA (tRNA) regions most vulnerable to photooxidation. Photooxidative damage to the anticodon and variable loop regions was consistently observed in both modified and unmodified sequences of tRNA upon UVA (λ 370 nm) exposure. The extent of oxidative damage measured in terms of oxidized guanosine, however, was higher in unmodified RNA compared to its modified version, suggesting an auxiliary role for nucleoside modifications. The type of oxidation product formed in the anticodon stem–loop region varied with the modification type, status, and whether the tRNA was inside or outside the cell during exposure. Oligonucleotide-based characterization of tRNA following UVA exposure also revealed the presence of novel photoproducts and stable intermediates not observed by nucleoside analysis alone. This approach provides sequence-specific information revealing potential hotspots for UVA-induced damage in tRNAs. Keywords: UVR; photooxidation; tRNA; post-transcriptional nucleoside modifications; cusativin; RNA modification mapping; RNA oxidation 1. Introduction Transfer RNAs (tRNAs) deliver amino acids to the site of ribosome-mediated protein synthesis while decoding the messenger RNA (mRNA).
    [Show full text]
  • Analysis of RNA Modifications by Second
    G C A T T A C G G C A T genes Review Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update Yuri Motorin 1,* and Virginie Marchand 2,* 1 Université de Lorraine, CNRS, IMoPA (UMR7365), F54000 Nancy, France 2 Université de Lorraine, CNRS, INSERM, IBSLor (UMS2008/US40), Epitranscriptomics and RNA Sequencing Core Facility, F54000 Nancy, France * Correspondence: [email protected] (Y.M.); [email protected] (V.M.); Tel.: +33-3-7274-6629 (Y.M.); +33-3-7274-6669 (V.M.) Abstract: The precise mapping and quantification of the numerous RNA modifications that are present in tRNAs, rRNAs, ncRNAs/miRNAs, and mRNAs remain a major challenge and a top priority of the epitranscriptomics field. After the keystone discoveries of massive m6A methylation in mRNAs, dozens of deep sequencing-based methods and protocols were proposed for the analysis of various RNA modifications, allowing us to considerably extend the list of detectable modified residues. Many of the currently used methods rely on the particular reverse transcription signatures left by RNA modifications in cDNA; these signatures may be naturally present or induced by an appropriate enzymatic or chemical treatment. The newest approaches also include labeling at RNA abasic sites that result from the selective removal of RNA modification or the enhanced cleavage of the RNA ribose-phosphate chain (perhaps also protection from cleavage), followed by specific adapter ligation. Classical affinity/immunoprecipitation-based protocols use either antibodies against modified RNA bases or proteins/enzymes, recognizing RNA modifications. In this survey, we review the most recent achievements in this highly dynamic field, including promising attempts to map RNA modifications by the direct single-molecule sequencing of RNA by nanopores.
    [Show full text]
  • FTO Levels Affect RNA Modification and the Transcriptome
    European Journal of Human Genetics (2013) 21, 317–323 & 2013 Macmillan Publishers Limited All rights reserved 1018-4813/13 www.nature.com/ejhg ARTICLE FTO levels affect RNA modification and the transcriptome Tea Berulava1, Matthias Ziehe2, Ludger Klein-Hitpass3, Emil Mladenov4,Ju¨rgen Thomale3, Ulrich Ru¨ther5 and Bernhard Horsthemke*,1 A block of single-nucleotide polymorphisms within intron 1 of the FTO (fat mass and obesity associated) gene is associated with variation in body weight. Previous works suggest that increased expression of FTO, which encodes a 2-oxoglutarate- dependent nucleic acid demethylase, leads to increased body weight, although the underlying mechanism has remained unclear. To elucidate the function of FTO, we examined the consequences of altered FTO levels in cultured cells and murine brain. Here we show that a knockdown of FTO in HEK293 cells affects the transcripts levels of genes involved in the response to starvation, whereas overexpression of FTO affects the transcript levels of genes related to RNA processing and metabolism. Subcellular localization of FTO further strengthens the latter notion. Using immunocytochemistry and confocal laser scanning microscopy, we detected FTO in nuclear speckles and – to a lesser and varying extent – in the nucleoplasm and nucleoli of HEK293, HeLa and MCF-7 cells. Moreover, RNA modification analyses revealed that loss of Fto affects the 3-methyluridine/ uridine and pseudouridine/uridine ratios in total brain RNA. We conclude that altered levels of FTO have multiple and diverse consequences on RNA modifications and the transcriptome. European Journal of Human Genetics (2013) 21, 317–323; doi:10.1038/ejhg.2012.168; published online 8 August 2012 Keywords: FTO; RNA modifications; nuclear speckles; transcriptome INTRODUCTION that FTO may modify rRNA.
    [Show full text]