DOCSLIB.ORG

A Novel SV40-Based Vector Successfully Transduces and Expresses an Alpha 1-Antitrypsin Ribozyme in a Human Hepatoma-Derived Cell Line

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Novel SV40-Based Vector Successfully Transduces and Expresses an Alpha 1-Antitrypsin Ribozyme in a Human Hepatoma-Derived Cell Line Gene Therapy (1999) 6, 114–120 1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http://www.stockton-press.co.uk/gt A novel SV40-based vector successfully transduces and expresses an alpha 1-antitrypsin ribozyme in a human hepatoma-derived cell line MA Zern1,2, I Ozaki1,3, L Duan1, R Pomerantz1, S-L Liu1, and DS Strayer2 Departments of 1Medicine and 2Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, PA, USA Alpha 1-antitrypsin (␣1AT) deficiency disease is one of the ribozyme cleavage. Ribozymes were effective in inhibiting more common hereditary disorders that affects the liver ␣1AT expression in a human hepatoma cell line using a and lung. The liver disease of ␣1AT deficiency is generally newly developed simian virus (SV40) vector system. In thought to be caused by the accumulation of an abnormal addition, the hepatoma cell line was stably transduced with ␣1AT protein in hepatocytes, whereas the lung disease is a modified ␣1AT cDNA that was capable of producing wild- thought to be due to a relative lack of the normal protein type ␣1AT protein, but was not cleaved by the ribozyme in the circulation. Therefore, one possible approach to pre- that decreased endogenous ␣1AT expression. These vent and treat ␣1AT disease is to both inhibit the results suggest that ribozymes can be employed for the expression of the mutated ␣1AT gene, and to provide a specific inhibition for an abnormal ␣1AT gene product, the means of synthesizing the normal protein. To do this, we first step in designing a gene therapy for the disease. The designed specific hammerhead ribozymes that were cap- findings also suggest that the novel SV40-derived vector able of cleaving the ␣1AT mRNA at specific sites, and con- may represent a fundamental improvement in the gene structed a modified ␣1AT cDNA not susceptible to therapeutic armarmentarium. Keywords: ribozymes; SV40; gene therapy; ␣1-antitrypsin deficiency Introduction Lys substitution at amino acid 342. This mutation is thought to cause the variant protein to aggregate in the ␣ Alpha 1-antitrypsin ( 1AT) deficiency, one of the more rough endoplasmic reticulum of the liver cells.4,6 common lethal hereditary disorders in Caucasians of Eur- Although several studies have focused on the delivery opean descent, is characterized by reducing serum levels of the normal ␣1AT gene into hepatocytes or airway cells ␣ of 1AT, a 52-kDa glycoprotein that functions as an anti- to restore normal ␣1AT production and to protect lung 1 protease. The deficiency state is caused by mutations of tissue,7–9 these approaches do not affect the liver disease. ␣ 2 the 1AT gene, a pleiomorphic, 12.2-kb 7-exon gene. It would appear that the best way to treat ␣1AT ␣ ␮ 3 Normal 1AT serum levels are 20–53 m; various combi- deficiency disease is to reduce the production of the ␣ nations of at least 17 different mutations of the 1AT endogenous mutant form of ␣1AT protein and to increase ␣ Ͻ ␮ gene are associated with an 1AT level 11 m and sig- the synthesis of the normal protein. The reduction of 4 nificant risk for developing emphysema. A subset of ␣1AT expression can be achieved by several strategies, 5 mutations is associated with hepatitis and cirrhosis. such as antisense, gene-specific ribozymes, ␣1AT tran- These latter mutations all involve the production of scription factor specific-inhibitors, or intracellular abnormal proteins: they do not include null mutations. expression of antibody to the mutant form of the ␣1AT The pathogenesis of the liver disease is thought to be due protein. ␣ to the accumulation of an abnormal 1AT protein in Recently, ribozymes have been targeted to a wide var- hepatocytes, and is associated with the finding that cer- iety of substrates and tested in biological systems to ␣ tain mutations of the 1AT gene cause derangement in achieve the inhibition of cellular gene expression or viral the protein’s intracellular processing and defects in the replication.10–15 Ribozymes have an advantage in the ther- protein’s excretion, commonly associated with liver apy of chronic disease since they are not degraded when 6 injury. The molecular defect in the protease inhibitor the target RNA is cleaved, and therefore, theoretically (Pi)Z allele, the allele most commonly associated with they need not be produced at higher levels than the target liver injury, is a G to A transition resulting in a Glu to transcript. By using a hammerhead ribozyme, we should be able to target the mutant form of ␣1AT mRNA by using ␣1AT guide sequences attached to the ribozyme Correspondence: MA Zern, Jefferson Medical College, 1025 Walnut catalytic core sequence. Furthermore, if we change the Street, Room 901, Philadelphia, PA 19107, USA corresponding cloned wild-type ␣1AT cDNA GUC and 3Present address: Department of Internal Medicine, Saga Medical School, Saga, Japan guide region nucleotide sequences while maintaining the The first two authors contributed equally to this work amino acid sequence, this modified ‘wild-type’ ␣1AT Received 7 November 1997; accepted 13 August 1998 mRNA should be resistant to the ribozyme. SV40-transduced ribozymes inhibit ␣1-antitrypsin MA Zern et al 115 A major issue in designing adequate gene therapeutic approaches to diseases is the availability of an effective vector system that will both deliver the gene of interest to the target organ and allow for its adequate expression. One of our laboratories has recently engineered the simain virus (SV)40 so that it has become an effective transfer vector system.16,17 In the present study, we report the transduction of an effective ␣1AT ribozyme in this SV40 vector system to inhibit the expression of the ␣1AT gene in a human hepatoma-derived cell line. At the same time, we have constructed a modified ␣1AT cDNA that is capable of producing the normal ␣1AT protein, yet its RNA is resistant to ribozyme cleavage. Results To study the effects of ribozymes on the expression of ␣1AT in a human hepatoma-derived cell line, we trans- duced PLC/PRF/5 cells that actively produce ␣1AT with either retroviral or SV40 recombinant viruses. Both poly- Figure 1 Representative Northern blot of RNA extracted from an unselec- ted population of PLC/PRF/5 cells that were mock-infected or infected 48 h merase II and III promoters were employed to drive ␣ expression of the ribozymes. previously with an SV40-derived vector containing an 1AT ribozyme 18 driven by either the SV40 early promoter (SVP.AT589) or by a tRNA In one series of studies, retroviral vectors were used promoter [SV(⌬)AT204] or [SV(⌬).tAT589T] at an MOI of 10. The RNA to transfer ribozymes. RNA was extracted from a pooled was electrophoresed, then hybridized with ␣1AT or GAPDH probes, as population of cells that had been tranduced with the described in Materials and methods. retroviral vectors and selected with G418 for 3 weeks. Those studies indicated that whereas ribozyme expression driven by a tRNA promoter decreased ␣1AT mRNA expression by as much as 85%, no such effect on ␣1AT mRNA expression was found when the CMV pro- moter was used to drive ribozyme expression. The SV40 vector system was somewhat more effective in that ribozymes driven by a polymerase II promoter were effective when transduced with the new vector system. PLC/PRF/5 cells were transduced using three different SV40 constructs, each containing either AT204 or 589 ribozyme. One construct employed the AT204 ribozyme driven by the tRNA promoter, one used the AT589 ribozyme under the control of the SV40 early promoter, and the third used the AT589 ribozyme with the tRNA promoter. No selection was applied to these cells. Forty- eight hours following transduction, RNA was isolated from the unselected populations of cells and assayed by Northern blot hybridization analysis. ␣1AT mRNA levels were significantly decreased in cells infected with the recombinant viruses in which ribozyme expression were driven by either the SV40 early promoter or the tRNA promoter, whereas there was no difference in GAPDH expression (Figure 1). Further studies were done with ribozyme AT589 con- Figure 2 Northern blot hybridization analysis of ␣1AT ribozymes employing an SV-40 vector system. PLC/PRF/5 cells were infected in cul- structs because they generated consistently high ⌬ ⌬ ribozyme activity. Figure 2 is a series of representative ture, with SV( )CAT, SVP.AT589, or SV( ).tAT589 at MOI of approxi- mately 100, or they were mock-infected. Cells were infected and RNA Northern blots of RNA isolated from an unselected popu- extracted as in Materials and methods. SV(⌬)CAT represents a control lation of cells 48 h following transduction with a control for transduction. construct, SVCAT, or with vector constructs containing 589AT ribozymes. The blots demonstrate the effective- ness of the ribozymes and their selective and specific despite the cells having a doubling time of 3 days. This properties. suggests that the construct was integrated, although no In addition, we employed the AT589 construct in proof of integration was demonstrated. Another alterna- experiments of longer duration to demonstrate the effec- tive explanation is that the high MOI employed allows tiveness of the construct over time. The PLC/PRF/5 cells for episomal expression in daughter cells. were transduced with the SV(⌬).tAT589 construct, then To compensate for the endogenous ␣1AT expression RNA was extracted at days 2, 7 and 14 following trans- being reduced by the AT589 ribozyme, we modified the duction. Figure 3 demonstrates that the ribozyme effec- nucleotide sequences of the target site of AT589 in the tively cleaved the ␣1AT mRNA after 2 weeks of infection full-length ␣1AT cDNA, without changing amino acid SV40-transduced ribozymes inhibit ␣1-antitrypsin MA Zern et al 116 Figure 3 Northern blot hybridization analysis of ␣1AT and GAPDH mRNA in a PLC/PRF/5 cell population mock-infected or infected with an SV40-derived vector containing the AT589 ribozyme.
Load more

Recommended publications
  • How Might a Pre-Biotic Ribozyme Catalyze RNA Assembly in an RNA World?
    Science Highlight – April 2007 How Might a Pre-biotic Ribozyme Catalyze RNA Assembly in an RNA World? Which came first, nucleic acids or proteins? This question is molecular biology's version of the "chicken-or-the-egg" riddle. Genes made of nucleic acids (DNA or RNA) contain the instructions for making proteins, but enzymes made of proteins are needed to replicate genes. For those who try to understand how life origi- nated, this once seemed an intractable paradox. The discovery 25 years ago that RNA can be enzymatic permits us to speculate that pre-biotic self-replicating molecules may have been RNAs (1,2). This is known as the "RNA World" hypothesis, and with the discovery of RNA catalysis, it is now possible to imagine a prebiotic The L1 Ligase ribozyme at the moment 'RNA World' (or even one populated by early life forms) of bond creation. in which self-replicating ribozymes (RNA-based enzymes that possess the catalytic ability to copy themselves) accomplished both tasks, thus avoiding the potential “chicken-or-the-egg” conundrum (3). But there's a catch. In order to copy RNA, fragments or monomers that have 5'-triphosphates must be ligated together. This is true for modern polymerases, and is also the most likely mechanism by which a ribozyme self-replicase in an RNA World might function. Yet no one has found a modern natural ribozyme that catalyze this The RNA nucleotide triphosphate ligation reaction required reaction (pictured right). for RNA polymerization and self-replication. RNA in vitro evolution and selection has however enabled several research groups to discover RNA sequences that can in fact cata- lyze the required chemical reaction (shown above) for 5'-triphos- phate RNA fragment ligation, and one group has even produced a primitive but functional RNA-based RNA polymerase ribozyme (4).
    [Show full text]
  • A Tetracycline-Dependent Ribozyme Switch Allows Conditional Induction of Gene Expression in Caenorhabditis Elegans
    ARTICLE https://doi.org/10.1038/s41467-019-08412-w OPEN A tetracycline-dependent ribozyme switch allows conditional induction of gene expression in Caenorhabditis elegans Lena A. Wurmthaler1,2, Monika Sack1,2, Karina Gense2,3, Jörg S. Hartig1,2 & Martin Gamerdinger2,3 The nematode Caenorhabditis elegans represents an important research model. Convenient methods for conditional induction of gene expression in this organism are not available. Here 1234567890():,; we describe tetracycline-dependent ribozymes as versatile RNA-based genetic switches in C. elegans. Ribozyme insertion into the 3’-UTR converts any gene of interest into a tetracycline- inducible gene allowing temporal and, by using tissue-selective promoters, spatial control of expression in all developmental stages of the worm. Using the ribozyme switches we established inducible C. elegans polyglutamine Huntington’s disease models exhibiting ligand- controlled polyQ-huntingtin expression, inclusion body formation, and toxicity. Our approach circumvents the complicated expression of regulatory proteins. Moreover, only little coding space is necessary and natural promoters can be utilized. With these advantages tetracycline-dependent ribozymes significantly expand the genetic toolbox for C. elegans. 1 Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany. 2 Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, 78457 Konstanz, Germany. 3 Department of Biology, University of Konstanz, 78457 Konstanz, Germany. Correspondence and requests for materials should be addressed to J.S.H. (email: [email protected]) or to M.G. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:491 | https://doi.org/10.1038/s41467-019-08412-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08412-w nducible regulatory systems are very powerful research tools to single A-to-G point mutation in the catalytic core of the HHR9 Iinvestigate the cellular function of individual genes.
    [Show full text]
  • Hammerhead Ribozymes Against Virus and Viroid Rnas
    Hammerhead Ribozymes Against Virus and Viroid RNAs Alberto Carbonell, Ricardo Flores, and Selma Gago Contents 1 A Historical Overview: Hammerhead Ribozymes in Their Natural Context ................................................................... 412 2 Manipulating Cis-Acting Hammerheads to Act in Trans ................................. 414 3 A Critical Issue: Colocalization of Ribozyme and Substrate . .. .. ... .. .. .. .. .. ... .. .. .. .. 416 4 An Unanticipated Participant: Interactions Between Peripheral Loops of Natural Hammerheads Greatly Increase Their Self-Cleavage Activity ........................... 417 5 A New Generation of Trans-Acting Hammerheads Operating In Vitro and In Vivo at Physiological Concentrations of Magnesium . ...... 419 6 Trans-Cleavage In Vitro of Short RNA Substrates by Discontinuous and Extended Hammerheads ........................................... 420 7 Trans-Cleavage In Vitro of a Highly Structured RNA by Discontinuous and Extended Hammerheads ........................................... 421 8 Trans-Cleavage In Vivo of a Viroid RNA by an Extended PLMVd-Derived Hammerhead ........................................... 422 9 Concluding Remarks and Outlooks ........................................................ 424 References ....................................................................................... 425 Abstract The hammerhead ribozyme, a small catalytic motif that promotes self- cleavage of the RNAs in which it is found naturally embedded, can be manipulated to recognize and cleave specifically
    [Show full text]
  • Structural Simplicity and Mechanistic Complexity in the Hammerhead Ribozyme
    CHAPTER SEVEN Structural Simplicity and Mechanistic Complexity in the Hammerhead Ribozyme Sara M. O’Rourke, William G. Scott1 The Center for the Molecular Biology of RNA, University of California at Santa Cruz, Santa Cruz, CA, United States 1Corresponding author: e-mail address: [email protected] Contents 1. Background and Structural Overview 178 2. Fast Minimal Hammerhead Ribozymes 181 3. Acid-Base Catalysis and the Hammerhead Ribozyme 182 4. Is the Hammerhead Ligation Reaction the Reverse of the Cleavage Reaction? 191 5. Do Cooperative Interactions in the Hammerhead Ribozyme Facilitate General Base Catalysis in the Cleavage Reaction? 194 6. Summary and Concluding Remarks 195 6.1 The Structure of the Hammerhead Ribozyme May Be Much Simpler Than We Have Thought 196 6.2 The Mechanism of the Hammerhead Ribozyme May Be Much More Complicated Than We Have Thought 196 6.3 Concluding Remarks 200 References 201 Abstract Natural or full-length hammerhead ribozymes are up to 1000-fold more active than their minimal counterparts that lack a complex tertiary interaction that pre-organizes and stabilizes the ribozyme active site, positioning RNA functional groups to facilitate acid-base catalysis. The recent discovery that a single tertiary contact (an AU Hoogsteen pair) between Stems I and II confers essentially all of the enhanced activity greatly simplifies our understanding of the structural requirements for hammerhead ribozyme activity. In contrast, the simplest mechanistic interpretations are challenged with the presentation of more complex alternatives. These alternatives are elucidated and criti- cally analyzed in the context of several of the active hammerhead ribozyme structures now available. # Progress in Molecular Biology and Translational Science, Volume 159 2018 Elsevier Inc.
    [Show full text]
  • Ribozymes Targeted to the Mitochondria Using the 5S Ribosomal Rna
    RIBOZYMES TARGETED TO THE MITOCHONDRIA USING THE 5S RIBOSOMAL RNA By JENNIFER ANN BONGORNO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005 Copyright 2005 by Jennifer Bongorno To my grandmother, Hazel Traster Miller, whose interest in genealogy sparked my interest in genetics, and without whose mitochondria I would not be here ACKNOWLEDGMENTS I would like to thank all the members of the Lewin lab; especially my mentor, Al Lewin. Al was always there for me with suggestions and keeping me motivated. He and the other members of the lab were like my second family; I would not have had an enjoyable experience without them. Diana Levinson and Elizabeth Bongorno worked with me on the fourth and third mouse transfections respectively. Joe Hartwich and Al Lewin tested some of the ribozymes in vitro and cloned some of the constructs I used. James Thomas also helped with cloning and was an invaluable lab manager. Verline Justilien worked on a related project and was a productive person with whom to bounce ideas back and forth. Lourdes Andino taught me how to use the new phosphorimager for my SYBR Green-stained gels. Alan White was there through it all, like the older brother I never had. Mary Ann Checkley was with me even longer than Alan, since we both came to Florida from Ohio Wesleyan, although she did manage to graduate before me. Jia Liu and Frederic Manfredsson were there when I needed a beer.
    [Show full text]
  • One Sequence, Two Ribozymes: Could Access by Neutral Drift Every Sequence on Both Networks
    R EPORTS minus the background levels observed in the HSP in X-100 for 15 min at 4¡C with intermittent mixing, 62. M. R. Peterson, C. G. Burd, S. D. Emr, Curr. Biol. 9, 159 the control (Sar1-GDPÐcontaining) incubation that and elutes were pooled. Proteins were precipitated by (1999). prevents COPII vesicle formation. In the microsome MeOH/CH3Cl and separated by SDSÐpolyacrylamide 63. M. G. Waters, D. O. Clary, J. E. Rothman, J. Cell Biol. control, the level of p115-SNARE associations was gel electrophoresis (PAGE) followed by immunoblot- 118, 1015 (1992). less than 0.1%. ting using p115 mAb 13F12. 64. D. M. Walter, K. S. Paul, M. G. Waters, J. Biol. Chem. 46. C. M. Carr, E. Grote, M. Munson, F. M. Hughson, P. J. 51. V. Rybin et al., Nature 383, 266 (1996). 273, 29565 (1998). Novick, J. Cell Biol. 146, 333 (1999). 52. K. G. Hardwick and H. R. Pelham, J. Cell Biol. 119, 513 65. N. Hui et al., Mol. Biol. Cell 8, 1777 (1997). 47. C. Ungermann, B. J. Nichols, H. R. Pelham, W. Wick- (1992). 66. T. E. Kreis, EMBO J. 5, 931 (1986). ner, J. Cell Biol. 140, 61 (1998). 53. A. P. Newman, M. E. Groesch, S. Ferro-Novick, EMBO 67. H. Plutner, H. W. Davidson, J. Saraste, W. E. Balch, 48. E. Grote and P. J. Novick, Mol. Biol. Cell 10, 4149 J. 11, 3609 (1992). J. Cell Biol. 119, 1097 (1992). (1999). 54. A. Spang and R. Schekman, J. Cell Biol. 143, 589 (1998). 68. D. S.
    [Show full text]
  • Natural Variation in the Multidrug Efflux Pump SGE1 Underlies Ionic
    | INVESTIGATION Natural Variation in the Multidrug Efflux Pump SGE1 Underlies Ionic Liquid Tolerance in Yeast Douglas A. Higgins,*,†,1 Megan K. M. Young,‡ Mary Tremaine,‡ Maria Sardi,‡,§ Jenna M. Fletcher,‡ Margaret Agnew,‡ Lisa Liu,‡ Quinn Dickinson,‡ David Peris,‡,§,2 Russell L. Wrobel,‡,§ Chris Todd Hittinger,‡,§ Audrey P. Gasch,‡,§ Steven W. Singer,*,** Blake A. Simmons,*,† Robert Landick,‡,‡‡,§§ Michael P. Thelen,*,†,3 and Trey K. Sato‡,3 *Deconstruction Division, Joint BioEnergy Institute, Emeryville, California 94608, †Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory, California 94550, ‡Department of Energy Great Lakes Bioenergy Research Center, §Laboratory of Genetics, ‡‡Department of Biochemistry, and §§Department of Bacteriology, University of Wisconsin-Madison, Wisconsin 53726, **Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, California 94720, and ††Sandia National Laboratories, Livermore, California 94550 ORCID IDs: 0000-0001-9912-8802 (D.P.); 0000-0002-5309-6718 (R.L.W.); 0000-0001-5088-7461 (C.T.H.); 0000-0002-1332-1810 (B.A.S.); 0000-0002-2479-5480 (M.P.T.); 0000-0001-6592-9337 (T.K.S.) ABSTRACT Imidazolium ionic liquids (IILs) have a range of biotechnological applications, including as pretreatment solvents that extract cellulose from plant biomass for microbial fermentation into sustainable bioenergy. However, residual levels of IILs, such as 1-ethyl-3-methylimidazolium chloride ([C2C1im]Cl), are toxic to biofuel-producing microbes, including the yeast Saccharomyces cerevisiae. S. cerevisiae strains isolated from diverse ecological niches differ in genomic sequence and in phenotypes potentially beneficial for industrial applications, including tolerance to inhibitory compounds present in hydrolyzed plant feedstocks. We evaluated .100 ge- nome-sequenced S. cerevisiae strains for tolerance to [C2C1im]Cl and identified one strain with exceptional tolerance.
    [Show full text]
  • The CPEB3 Ribozyme Modulates Hippocampal-Dependent Memory
    bioRxiv preprint doi: https://doi.org/10.1101/2021.01.23.426448; this version posted January 24, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The CPEB3 ribozyme modulates hippocampal-dependent memory Claire C. Chen1, Joseph Han2, Carlene A. Chinn2, Xiang Li2†, Mehran Nikan3, Marie Myszka4, Liqi Tong5, Timothy W. Bredy2†, Marcelo A. Wood2*, Andrej Lupták1,4,6* 1 Department of Pharmaceutical Sciences, University of California–Irvine, Irvine, California 92697, United States. 2 Department of Neurobiology and Behavior, Center for the Neurobiology of Learning and Memory, University of California–Irvine, Irvine, California 92697, United States. 3 Ionis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010, USA. 4 Department of Chemistry, University of California–Irvine, Irvine, California 92697, United States. 5 Institute for Memory Impairments and Neurological Disorders, University of California– Irvine, Irvine, California 92697, United States. 6 Department of Molecular Biology and Biochemistry, University of California–Irvine, Irvine, California 92697, United States *Correspondence to: Andrej Lupták. Department of Pharmaceutical Sciences, University of California–Irvine, Irvine, California 92697, United States. [email protected]. Marcelo A. Wood. Department of Neurobiology and Behavior, Center for the Neurobiology of Learning and Memory, University of California–Irvine, Irvine, California 92697, United States. [email protected]. † Present address: Cognitive Neuroepigenetics Laboratory, Queensland Brain Institute, The University of Queensland, Brisbane, QLD 4072, Australia. Keywords: ribozyme, splicing, self-scission, polyadenylation, local translation 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.23.426448; this version posted January 24, 2021.
    [Show full text]
  • Design and Switch of Catalytic Activity with the Dnazyme–Rnazyme Combination
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 581 (2007) 1763–1768 Design and switch of catalytic activity with the DNAzyme–RNAzyme combination Yongjie Sheng, Zhen Zeng, Wei Peng, Dazhi Jiang, Shuang Li, Yanhong Sun, Jin Zhang* Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, PR China Received 12 January 2007; revised 9 March 2007; accepted 16 March 2007 Available online 2 April 2007 Edited by Judit Ova´di backbone DNA with a regulating sequence [12]. Compared to Abstract Design and switch of catalytic activity in enzymology remains a subject of intense investigation. Here, we employ a the linear hammerhead ribozyme, the stability of the circular DNAzyme–RNAzyme combination strategy for construction of RNA–DNA enzyme was substantially enhanced. Furthering a 10–23 deoxyribozyme-hammerhead ribozyme combination that this approach, we substituted the hammerhead ribozyme with targets different sites of the b-lactamase mRNA. The 10–23 the more efficient and more stable 10–23 deoxyribozyme, while deoxyribozyme-hammerhead ribozyme combination gene was the backbone DNA was replaced by the single-stranded repli- cloned into phagemid vector pBlue-scriptIIKS (+). In vitro the cation-competent vector M13mp18 (7.25 kb), thus successfully single-strand recombinant phagemid vector containing the com- constructing a novel replicating circular deoxyribozyme, which bination sequence exhibited 10–23 deoxyribozyme activity, and displayed 10–23 deoxyribozyme activities both in vitro and in the linear transcript displayed hammerhead ribozyme activity. bacteria [13,14]. In bacteria, the 10–23 deoxyribozyme-hammerhead ribozyme Here, we report the combination of 10–23 deoxyribozyme combination inhibited the b-lactamase expression and repressed the growth of drug-resistant bacteria.
    [Show full text]
  • The RNA World” Mean to “The Origin of Life”?
    life Concept Paper What Does “the RNA World” Mean to “the Origin of Life”? Wentao Ma Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan 430072, China; [email protected] Received: 30 September 2017; Accepted: 24 November 2017; Published: 29 November 2017 Abstract: Corresponding to life’s two distinct aspects: Darwinian evolution and self-sustainment, the origin of life should also split into two issues: the origin of Darwinian evolution and the arising of self-sustainment. Because the “self-sustainment” we concern about life should be the self-sustainment of a relevant system that is “defined” by its genetic information, the self-sustainment could not have arisen before the origin of Darwinian evolution, which was just marked by the emergence of genetic information. The logic behind the idea of the RNA world is not as tenable as it has been believed. That is, genetic molecules and functional molecules, even though not being the same material, could have emerged together in the beginning and launched the evolution—provided that the genetic molecules can “simply” code the functional molecules. However, due to these or those reasons, alternative scenarios are generally much less convincing than the RNA world. In particular, when considering the accumulating experimental evidence that is supporting a de novo origin of the RNA world, it seems now quite reasonable to believe that such a world may have just stood at the very beginning of life on the Earth. Therewith, we acquire a concrete scenario for our attempts to appreciate those fundamental issues that are involved in the origin of life.
    [Show full text]
  • Mitochondrial Transcriptome Control and Intercompartment Cross-Talk During Plant Development
    cells Article Mitochondrial Transcriptome Control and Intercompartment Cross-Talk During Plant Development 1,2, 3, 1, 1 Adnan Khan Niazi † , Etienne Delannoy †, Rana Khalid Iqbal †, Daria Mileshina , Romain Val 1, Marta Gabryelska 4, Eliza Wyszko 4 , Ludivine Soubigou-Taconnat 3, Maciej Szymanski 5, Jan Barciszewski 4,6 , Frédérique Weber-Lotfi 1, José Manuel Gualberto 1 and André Dietrich 1,* 1 Institute of Plant Molecular Biology (IBMP), CNRS and University of Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France; [email protected] (A.K.N.); [email protected] (R.K.I.); [email protected] (D.M.); [email protected] (R.V.); lotfi[email protected] (F.W.-L.); [email protected] (J.M.G.) 2 Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad 38000, Pakistan 3 Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Evry, Université Paris-Saclay, Paris Diderot, Sorbonne Paris-Cité, 91405 Orsay, France; [email protected] (E.D.); [email protected] (L.S.-T.) 4 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Ul. Z. Noskowskiego 12/14, 61-704 Poznan, Poland; [email protected] (M.G.); [email protected] (E.W.); [email protected] (J.B.) 5 Department of Computational Biology, Institute of Molecular Biology and Biotechnology, A. Mickiewicz University Poznan, Ul. Umultowska 89, 61-614 Poznan, Poland; [email protected] 6 NanoBioMedical Centre of the Adam Mickiewicz University, Umultowska 85, 61614 Poznan, Poland * Correspondence: [email protected] These authors contributed equally to this project.
    [Show full text]
  • In Vitro Selection of Ribozyme Ligases That Use Prebiotically Plausible 2-Aminoimidazole– Activated Substrates
    In vitro selection of ribozyme ligases that use prebiotically plausible 2-aminoimidazole– activated substrates Travis Waltona,b,1, Saurja DasGuptaa,b,1, Daniel Duzdevicha,b, Seung Soo Ohc, and Jack W. Szostaka,b,2 aHoward Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114; bDepartment of Molecular Biology, Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114; and cDepartment of Materials Science and Engineering, Pohang University of Science and Technology, 37673 Pohang, Gyeongbuk, South Korea Edited by S. Altman, Yale University, New Haven, CT, and approved January 29, 2020 (received for review August 18, 2019) The hypothesized central role of RNA in the origin of life suggests monomers enhance the rate and extent of nonenzymatic that RNA propagation predated the advent of complex protein template-directed polymerization (11, 12). 2AI-activated RNA enzymes. A critical step of RNA replication is the template-directed monomers can be generated by a prebiotically relevant chemical synthesis of a complementary strand. Two experimental approaches pathway involving nucleotides, isocyanides, and aldehydes, in the have been extensively explored in the pursuit of demonstrating presence of free 2AI, and the repeated addition of isocyanide protein-free RNA synthesis: template-directed nonenzymatic RNA results in multiple cycles of monomer reactivation and sponta- polymerization using intrinsically reactive monomers and ribozyme- neous hydrolysis (13–15). These recent findings are particularly catalyzed polymerization using more stable substrates such as relevant because isocyanide is produced by a variation of the biological 5′-triphosphates. Despite significant progress in both ap- ferrocyanide chemistry that creates cyanamide, a precursor of proaches in recent years, the assembly and copying of functional RNA nucleotides and 2AI (1, 15).
    [Show full text]