Anders N.-J., Migliorini A. (eds.) Innate DNA and RNA Recognition: Methods and Protocols

Humana Press, 2014. — 360 p. — (Methods in Molecular Biology 1169).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 completely different pathogens into rather uniform innate immune responses. This immunostimulatory 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 subsequent exposures. This mechanism renders nucleic acid-based adjuvants interesting compounds 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 modifications, 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 protective mechanisms. This can turn tolerogenic autoantigen presentation into aberrant activation and proliferation of auto-reactive lymphocytes, with ensuing production of antinuclear antibodies and induction of autoimmune diseases like systemic lupus erythematosus and scleroderma.
The biological significance of nucleic acid immune recognition is not limited to host defense, vaccination, and autoimmunity. The diagnostic and therapeutic use of gene targeting nucleic acids, e.g., siRNAs, miRNAs, or aptamers, has to consider the immunostimulatory 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 bifunctional 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.Part I Analysis of Viral Nucleic Acid Sensing In-Silico and In-Vitro
Detection of RNA Modifications by HPLC Analysis and Competitive ELISA
Enzymatic Synthesis and Purification of a Defined RIG-I Ligand
Crystallization of Mouse RIG-I ATPase Domain: In Situ Proteolysis
Isolation of RIG-I-Associated RNAs from Virus-Infected Cells
Structure Modeling of Toll-Like Receptors
Nucleic Acid Recognition in Dendritic Cells
Viral Nucleic Acid Recognition in Human Nonimmune Cells: In Vitro Systems
Analysis of Nucleic Acid-Induced Nonimmune Cell Death In Vitro
In Vitro Analysis of Nucleic Acid Recognition in B Lymphocytes
Mapping of Optimal CD8 T Cell Epitopes
A Modular Approach to Suppression Assays: TLR Ligands, Conditioned Medium, and Viral Infection
MicroRNA Methodology: Advances in miRNA Technologies
Part II Analysis of Nucleic Acid Sensing In-Vivo
Expression Profiling by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
Evaluating the Role of Nucleic Acid Antigens in Murine Models of Systemic Lupus Erythematosus
Induction and Analysis of Nephrotoxic Serum Nephritis in Mice
Isolation of Intratumoral Leukocytes of TLR-Stimulated Tumor-Bearing Mice
Bifunctional siRNAs for Tumor Therapy