Innate DNA and RNA Recognition: Methods and Protocols

199 Pages·2014·3.932 MB·English

by  Hans-Joachim Anders, Adriana Migliorini (eds.)

Methods in
Molecular Biology 1169
Hans-Joachim Anders
Adriana Migliorini Editors
Innate DNA
and RNA
Recognition
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
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Innate DNA and RNA Recognition
Methods and Protocols
Edited by
Hans-Joachim Anders
Medizinische Klinik und Poliklinik IV, Ludwig Maximilians Universität München,
München, Germany
Adriana Migliorini
Helmholtz Zentrum München German Research Center for Environmental Health,
Institute for Diabetes and Regeneration Research (IDR), Munich, Germany
Editors
Hans-Joachim Anders Adriana Migliorini
Medizinische Klinik und Poliklinik IV Helmholtz Zentrum München German
Ludwig Maximilians Universität München Research Center for Environmental Health
München, Germany Institute for Diabetes and Regeneration
Research (IDR)
Munich, Germany
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-4939-0881-3 ISBN 978-1-4939-0882-0 (eBook)
DOI 10.1007/978-1-4939-0882-0
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Prefa ce
The last decade has completely changed our understanding of pathogen recognition in
terms of how the innate immune system manages to integrate molecular patterns of com-
pletely different pathogens into rather uniform innate immune responses. This immunos-
timulatory effect of pathogen-derived nucleic acids is of great importance for host defense
as it not only initiates an immediate innate immune response but also serves as an adjuvant
for the priming of adaptive immune responses that rapidly combat the pathogen upon sub-
sequent exposures. This mechanism renders nucleic acid-based adjuvants interesting com-
pounds for vaccination strategies. Although the structures of nucleic acids are strongly
preserved from prokaryotes to mammals, the mammalian immune system has found ways
to distinguish own nucleic acids from those of bacteria and viruses. This is necessary for
immune tolerance and it is supported by compartmentalization of nucleic acids away from
nucleic acid sensors, by epigenetic DNA and RNA modifi cations, by rapid opsonization of
extracellular nuclear particles and their phagocytic clearance by macrophages. However,
unfortunate combinations of genetic variants or certain drugs can compromise these pro-
tective mechanisms. This can turn tolerogenic autoantigen presentation into aberrant acti-
vation and proliferation of auto-reactive lymphocytes, with ensuing production of
antinuclear antibodies and induction of autoimmune diseases like systemic lupus erythema-
tosus and scleroderma.
The biological signifi cance of nucleic acid immune recognition is not limited to host
defense, vaccination, and autoimmunity. The diagnostic and therapeutic use of gene target-
ing nucleic acids, e.g., siRNAs, miRNAs, or aptamers, has to consider the immunostimula-
tory potential of certain nucleic acid formats. This implies either to avoid immune
stimulation in therapeutic gene regulation with siRNA or aptamers, or to utilize immune
stimulation to overcome tumor-associated immunosuppression, e.g., by the use of bifunc-
tional siRNAs that combine a knock-down sequence with immunostimulatory structural
features in one molecule.
This edition of Methods in Molecular Biology presents validated experimental strategies
to dissect nucleic acid sensing in vitro and in vivo. It is meant as a resource for immunologists,
molecular biologists, virologists, microbiologists, and any researcher that wants to know how
the innate immune system handles nucleic acids from endogenous or foreign sources.
Several of the authors have collaborated within the Research Training Group 1202
“Oligonucleotides in cell biology and therapy” that has been funded by the Deutsche
Forschungsgemeinschaft from 2005 to 2014 at the Ludwig-Maximilians-Universität in Munich.
Munich, Germany Adriana Migliorini
Hans-Joachim Anders
v

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
PART I ANALYSIS OF VIRAL NUCLEIC ACID SENSING
IN-SILICO AND IN-VITRO
1 Detection of RNA Modifications by HPLC Analysis
and Competitive ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Gernot N ees, A ndreas K aufmann, and S tefan B auer
2 E nzymatic Synthesis and Purification of a Defined RIG-I Ligand. . . . . . . . . . . 1 5
Marion G oldeck, M artin S chlee, G unther Hartmann,
and Veit Hornung
3 Crystallization of Mouse RIG-I ATPase Domain: In Situ Proteolysis. . . . . . . . 2 7
Filiz C ivril and K arl-Peter H opfner
4 I solation of RIG-I-Associated RNAs from Virus-Infected Cells . . . . . . . . . . . . 3 7
Andreas Schmidt, A ndreas L inder, Nicolas L inder,
and S imon Rothenfusser
5 S tructure Modeling of Toll-Like Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 5
Jing G ong and Tiandi Wei
6 N ucleic Acid Recognition in Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5
Alexander H eiseke, Katharina E isenächer, and Anne K rug
7 Viral Nucleic Acid Recognition in Human Nonimmune Cells:
In Vitro Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Andrea Ribeiro and Markus W örnle
8 Analysis of Nucleic Acid-Induced Nonimmune Cell Death In Vitro. . . . . . . . . 7 7
Simone Romoli and A driana M igliorini
9 I n Vitro Analysis of Nucleic Acid Recognition in B Lymphocytes. . . . . . . . . . . 8 7
Saskia Ziegler and I sabelle Bekeredjian-Ding
10 M apping of Optimal CD8 T Cell Epitopes . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 7
Julia Roider, T homas V ollbrecht, and R ika Draenert
11 A Modular Approach to Suppression Assays: TLR Ligands,
Conditioned Medium, and Viral Infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Viktor H . Koelzer and David A nz
1 2 MicroRNA Methodology: Advances in miRNA Technologies . . . . . . . . . . . . . 1 21
Theresa K aeuferle, S abine B artel, S tefan Dehmel,
and Susanne K rauss-Etschmann
vii
viii Contents
PART II ANALYSIS OF NUCLEIC ACID SENSING IN-VIVO
13 Expression Profiling by Real-Time Quantitative Polymerase
Chain Reaction (RT-qPCR). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Maciej Lech and H ans-Joachim A nders
14 E valuating the Role of Nucleic Acid Antigens in Murine Models
of Systemic Lupus Erythematosus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Amanda A. Watkins, Ramon G. B . B onegio, and Ian R . Rifkin
15 Induction and Analysis of Nephrotoxic Serum Nephritis in Mice. . . . . . . . . . . 1 59
John M . Hoppe and V olker V ielhauer
16 I solation of Intratumoral Leukocytes of TLR-Stimulated
Tumor-Bearing Mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Moritz Rapp, D avid A nz, and Max S chnurr
17 B ifunctional siRNAs for Tumor Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Fanny Matheis and R obert B esch
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Contributors
HANS -JOACHIM A NDERS • Medizinische Klinik und Poliklinik IV , Ludwig Maximilians
Universität München, München, Germany
DAVID ANZ • Division of Clinical Pharmacology , D epartment of Internal Medicine,
Ludwig-Maximilian University of Munich , M unich, G ermany
SABINE BARTEL • Comprehensive Pneumology Center, Helmholtz Zentrum München ,
Ludwig-Maximilians Universität and Asklepios Clinic Gauting , M unich, G ermany
STEFAN BAUER • Institut für Immunologie, BMFZ, Philipps-Universität Marburg ,
Marburg, G ermany
ISABELLE BEKEREDJIAN-DING • Institute for Medical Microbiology, Immunology
and Parasitology (IMMIP), U niversity Hospital Bonn , Bonn, Germany
ROBERT BESCH • Department of Dermatology and Allergology , Ludwig-Maximilian
University , M unich, G ermany
RAMON G . B. BONEGIO • Renal Section, Department of Medicine, Boston University School
of Medicine , B oston , M A , U SA
FILIZ CIVRIL • Department of Biochemistry at the Gene Center, L udwig-Maximilians-
University Munich , M unich, Germany ; Graduate School for Quantivative Biosciences
Munich, Ludwig-Maximilians-University Munich , M unich, G ermany
STEFAN DEHMEL • Comprehensive Pneumology Center, Helmholtz Zentrum München ,
Ludwig-Maximilians Universität and Asklepios Clinic Gauting , M unich, G ermany
RIKA DRAENERT • Department of Infectious Diseases, Medizinische Klinik und
Poliklinik IV , K linikum der Universität München , Munich, G ermany
KATHARINA E ISENÄCHER • II. Medical Department, Klinikum rechts der Isar , T echnical
University Munich , Munich, G ermany
MARION GOLDECK • Institute for Clinical Chemistry and Clinical Pharmacology,
University Hospital, University of Bonn , B onn , G ermany
JING GONG • Cancer Research Center, School of Medicine, Shandong University , Jinan, C hina
GUNTHER HARTMANN • Institute for Clinical Chemistry and Clinical Pharmacology,
University Hospital, University of Bonn , B onn, Germany
ALEXANDER H EISEKE • II. Medical Department, Klinikum rechts der Isar, Technical University
Munich , M unich, Germany
KARL-PETER HOPFNER • Department of Biochemistry at the Gene Center ,
Ludwig-Maximilians-U niversity Munich , Munich, Germany ; G raduate School
for Quantivative Biosciences Munich, L udwig-Maximilians-University Munich ,
Munich, Germany
JOHN M . H OPPE • Nephrologisches Zentrum, Medizinische Klinik und Poliklinik IV ,
Klinikum der Universität München, Ludwig-Maximilians-University Munich ,
Munich, G ermany
VEIT HORNUNG • Institute of Molecular Medicine, U niversity Hospital, University of Bonn ,
Bonn, G ermany
THERESA K AEUFERLE • Dr. von Hauner’sche Kinderklinik, Klinikum der Universität
München , M unich, Germany
ix