Table Of ContentMethods 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:
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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
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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
