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Morpholino Oligomers: Methods and Protocols PDF

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Methods in Molecular Biology 1565 Hong M. Moulton Jon D. Moulton Editors Morpholino Oligomers Methods and Protocols M M B ethods in olecular iology Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Morpholino Oligomers Methods and Protocols Edited by Hong M. Moulton Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR, USA Jon D. Moulton Gene Tools, LLC, Philomath, OR, USA Editors Hong M. Moulton Jon D. Moulton Department of Biomedical Sciences Gene Tools, LLC College of Veterinary Medicine Philomath, OR, USA Oregon State University Corvallis, OR, USA ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6815-2 ISBN 978-1-4939-6817-6 (eBook) DOI 10.1007/978-1-4939-6817-6 Library of Congress Control Number: 2017937324 © Springer Science+Business Media LLC 2017 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A. Preface Morpholino phosphorodiamidate antisense oligos (Morpholinos) were originally devel- oped for therapeutic applications but have found widespread use as research reagents. They are readily available, easy-to-handle, and stable oligos that bind to complementary RNA and prevent other large molecules from accessing their bound target, like sequence-specific masking tape for RNA. Their function depends on the usual biological function of their target sequence. They have been used in animals, bacteria, protists, plants, and fungi. They don’t rely on the cells for enzymatic activity such as RNase-H or RISC. Instead the oligos simply bind to and block their complementary targets—and sometimes other partially com- plementary RNA sequences, so appropriate controls are important. Clinical trials with Morpholino oligos have had excellent safety profiles, and have led to the first approved Morpholino drug, eteplirsen (ExonDys51), for treatment of some mutations causing Duchenne muscular dystrophy. Meanwhile, their efficacy, specificity, and great versatility as antisense effector and diagnostic molecules in a broad range of biological systems bode well for their long-term place in the toolbox of molecular biology. This book begins with a historical perspective on Morpholinos by Jim Summerton, the biochemist who originally conceived of these oligos. The following three chapters present an overview of good Morpholino practices, the first publication of studies of aggregation of Morpholinos in solution, and a description of techniques for chemically modifying Morpholinos to add additional functions. Chapter 5 reviews and presents techniques for controlling Morpholino activity with light. Techniques for modulating microRNA activity in zebrafish embryos are presented in Chapter 6. Chapter 7, on probing genes during fin regeneration, presents local Morpholino delivery techniques based on injection then elec- troporation for zebrafish. Next methods for determining the structure of gene networks during development are discussed in the context of sea urchin embryos. Electroporation again appears, this time for knockdowns in chick embryos in Chapter 9. In Chapter 10 bacterial knockdowns, a potential alternative to antibiotics, are introduced using Morpholinos conjugated with cell-penetrating peptides (PPMO). Electroporation has proven effective to introduce Morpholinos into the protist Giardia lamblia, discussed in Chapter 11. A few less-commonly used RNA targets are discussed next, polyadenylation sequences in Chapter 12 and noncoding RNAs in Chapter 13. Pretargeting, an application that uses a pair of complementary Morpholinos to stick an effector (such as a radioisotope) onto a targeting moiety (such as an antibody) in vivo, is discussed in Chapter 14. Chapter 15 reviews diagnostic applications of Morpholinos and presents protocols for capacitance- based detection of nucleic acid analytes bound to complementary surface-attached Morpholinos. A PPMO is effective to inhibit a host factor that is involved in influenza infection in the lung of mice, as discussed for influenza in Chapter 16. Multiple-exon- skipping cocktails of Morpholinos have been delivered to dogs carrying a genetic model of Duchenne muscular dystrophy; protocols from these experiments are presented in Chapter 17. The retina is amenable to knockdown with Morpholinos in several organisms; protocols for knockdowns in developing mouse retina are given in Chapter 18. When Morpholinos are introduced to cerebrospinal fluid, they enter cells more effectively than from blood; v vi Preface protocols for intracerebroventricular delivery in mice are discussed in Chapter 19. Techniques for delivering Morpholinos in utero to developing embryos have been estab- lished in sheep and are presented in Chapter 20. Pharmacokinetic, pharmacodynamic, and biodistribution studies can benefit from (1) a surface plasmon resonance-based assay that is label-free, antibody-free, and a walk-away assay for Morpholino quantification, as described in Chapter 21, and (2) in Chapter 22 an ELISA method offering rapid and inexpensive assays of Morpholino concentrations in biological extracts. Thanks to all of the authors who have contributed to this volume, creating a broad survey of the diverse applications of Morpholinos along with protocols that will assist new labs in moving the frontier. We hope that Morpholino oligos will continue to be key tools in new biological discoveries, advancing our understanding and the development of drugs and devices to better the health of humans, our associated animals, and our environment. Corvallis, OR, USA Hong M. Moulton Philomath, OR, USA Jon D. Moulton Contents Preface.......................................................... v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 Invention and Early History of Morpholinos: From Pipe Dream to Practical Products............................................ 1 James E. Summerton 2 Making a Morpholino Experiment Work: Controls, Favoring Specificity, Improving Efficacy, Storage, and Dose .............................. 17 Jon D. Moulton 3 Aggregation and Disaggregation of Morpholino Oligomers in Solution...... 31 Garrick Chow, Paul A. Morcos, and Hong M. Moulton 4 End-Modifications on Morpholino Oligos............................ 39 Yong-Fu Li 5 Inducible Inhibition of Gene Function with Photomorpholinos ........... 51 Saulius Sumanas 6 Blocking Zebrafish MicroRNAs with Morpholinos ..................... 59 Alex Sutton Flynt, Mahesh Rao, and James G. Patton 7 Using Morpholinos to Examine Gene Function During Fin Regeneration.... 79 Ryan Thummel and M. Kathryn Iovine 8 Using Morpholinos to Probe Gene Networks in Sea Urchin .............. 87 Stefan C. Materna 9 Manipulating Gene Expression in the Chick Embryo.................... 105 Octavian Voiculescu and Claudio D. Stern 10 Inhibition of Bacterial Growth by Peptide-Conjugated Morpholino Oligomers.......................................... 115 Seth M. Daly, Carolyn R. Sturge, and David E. Greenberg 11 Use of Translation Blocking Morpholinos for Gene Knockdown in Giardia lamblia ............................................. 123 Jana Krtková and Alexander R. Paredez 12 Regulation of Isoform Expression by Blocking Polyadenylation Signal Sequences with Morpholinos ................................ 141 Qiuming Gong and Zhengfeng Zhou 13 Targeting Functional Noncoding RNAs ............................. 151 Madzia P. Crossley and Torsten Krude 14 Use of Morpholino Oligomers for Pretargeting........................ 161 Guozheng Liu 15 Diagnostic Applications of Morpholinos and Label-Free Electrochemical Detection of Nucleic Acids ....................................... 181 Rastislav Levicky, Ursula Koniges, and Napoleon Tercero vii viii Contents 16 Intranasal Delivery of Peptide-Morpholinos to Knockdown Influenza Host Factors in Mice............................................ 191 Ricardo Rajsbaum 17 Systemic Delivery of Morpholinos to Skip Multiple Exons in a Dog Model of Duchenne Muscular Dystrophy.................................. 201 Rika Maruyama, Yusuke Echigoya, Oana Caluseriu, Yoshitsugu Aoki, Shin’ichi Takeda, and Toshifumi Yokota 18 In Vivo and Explant Electroporation of Morpholinos in the Developing Mouse Retina................................... 215 Devi Krishna Priya Karunakaran and Rahul Kanadia 19 Intracerebroventricular Delivery in Mice for Motor Neuron Diseases........ 229 M. Nizzardo and M. Rizzuti 20 Delivery of Morpholino Antisense Oligonucleotides to a Developing Ovine Conceptus via Luminal Injection into a Ligated Uterine Horn ............ 241 Xiaoqui Wang and Kathrin A. Dunlap 21 Surface Plasmon Resonance-Based Concentration Determination Assay: Label-Free and Antibody-Free Quantification of Morpholinos............. 251 Jordan Boutilier and Hong M. Moulton 22 Ultrasensitive Hybridization-Based ELISA Method for the Determination of Phosphorodiamidate Morpholino Oligonucleotides in Biological samples.... 265 Umar Burki and Volker Straub Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Contributors Yoshitsugu Aoki • Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan JordAn Boutilier • Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR, USA umAr Burki • The John Walton Muscular Dystrophy Research Centre, The MRC Centre for Neuromuscular Diseases, Central Parkway, International Centre for Life, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK oAnA CAluseriu • Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Newcastle University, Edmonton, AB, Canada gArriCk Chow • Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR, USA mAdziA P. CrossleY • Department of Zoology, University of Cambridge, Cambridge, UK; Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA seth m. dAlY • Departments of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA kAthrin A. dunlAP • Department of Animal Science, Texas A&M University, College Station, TX, USA Yusuke eChigoYA • Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, AB, Canada Alex sutton FlYnt • Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, MS, USA Qiuming gong • Knight Cardiovascular Institute, Oregon Health & Science University, Portland, OR, USA dAvid e. greenBerg • Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA; Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA m. kAthrYn iovine • Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA rAhul kAnAdiA • Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, USA devi krishnA PriYA kArunAkArAn • Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, USA ursulA koniges • Department of Chemical & Biomolecular Engineering, Tandon School of Engineering, New York University, Brooklyn, NY, USA JAnA krtková • Department of Biology, University of Washington, Seattle, WA, USA; Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Prague, Czech Republic torsten krude • Department of Zoology, University of Cambridge, Cambridge, UK rAstislAv leviCkY • Department of Chemical & Biomolecular Engineering, Tandon School of Engineering, New York University, Brooklyn, NY, USA Yong-Fu li • Gene Tools, LLC, Philomath, OR, USA ix x Contributors guozheng liu • Department of Radiology, University of Massachusetts Medical School, Worcester, MA, USA rikA mAruYAmA • Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, AB, Canada steFAn C. mAternA • School of Natural Sciences, University of California Merced, CA, USA PAul A. morCos • OligoExpert, Corvallis, OR, USA Jon d. moulton • Gene Tools, LLC, Philomath, OR, USA hong m. moulton • Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR, USA m. nizzArdo • Dino Ferrari Centre, Neuroscience Section, Department of Pathophysiology and Transplantation (DEPT), University of Milan, and Neurology Unit, IRCCS Foundation Ca’ Granda, Milan, Italy AlexAnder r. PAredez • Department of Biology, University of Washington, Seattle, WA, USA JAmes g. PAtton • Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA riCArdo rAJsBAum • Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA mAhesh rAo • Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA m. rizzuti • Dino Ferrari Centre, Neuroscience Section, Department of Pathophysiology and Transplantation (DEPT), University of Milan, and Neurology Unit, IRCCS Foundation Ca’ Granda, Milan, Italy ClAudio d. stern • Department of Cell and Developmental Biology, University College London, London, UK volker strAuB • The John Walton Muscular Dystrophy Research Centre, The MRC Centre for Neuromuscular Diseases, Central Parkway, International Centre for Life, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK CArolYn r. sturge • Departments of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA sAulius sumAnAs • Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA JAmes e. summerton • Gene Tools, LLC, Philomath, OR, USA shin’iChi tAkedA • Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan nAPoleon terCero • Cytec Solvay Group, Stamford, CT, USA rYAn thummel • Department of Anatomy and Cell Biology and Opthamology, Wayne State University School of Medicine, Wayne State University, Detroit, MI, USA oCtAviAn voiCulesCu • Department of Physiology, Development and Neuroscience, Cambridge University, Cambridge, UK xiAoQui wAng • Department of Animal Science, Texas A&M University, College Station, TX, USA toshiFumi YokotA • Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, AB, Canada; The Friends of Garrett Cumming Research & Muscular Dystrophy, Canada HM Toupin Neurological Science Endowed Research Chair, Edmonton, AB, Canada zhengFeng zhou • Knight Cardiovascular Institute, Oregon Health & Science University, Portland, OR, USA

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