The 2'-O-methylation status of a single guanosine controls transfer RNA-mediated Toll-like receptor 7 activation or inhibition.

Foreign RNA serves as pathogen-associated molecular pattern (PAMP) and is a potent immune stimulator for innate immune receptors. However, the role of single bacterial RNA species in immune activation has not been characterized in detail. We analyzed the immunostimulatory potential of transfer RNA (tRNA) from different bacteria. Interestingly, bacterial tRNA induced type I interferon (IFN) and inflammatory cytokines in mouse dendritic cells (DCs) and human peripheral blood mononuclear cells (PBMCs). Cytokine production was TLR7 dependent because TLR7-deficient mouse DCs did not respond and TLR7 inhibitory oligonucleotides inhibited tRNA-mediated activation. However, not all bacterial tRNA induced IFN-α because tRNA from Escherichia coli Nissle 1917 and Thermus thermophilus were non-immunostimulatory. Of note, tRNA from an E. coli knockout strain for tRNA (Gm18)-2'-O-methyltransferase (trmH) regained immunostimulatory potential. Additionally, in vitro methylation of this immunostimulatory Gm18-negative tRNA with recombinant trmH from T. thermophilus abolished its IFN-α inducing potential. More importantly, Gm18-modified tRNA acted as TLR7 antagonist and blocked IFN-α induction of influenza A virus-infected PBMCs.

New nucleotide analogues with enhanced signal properties.

We describe synthesis and testing of a novel type of dye-modified nucleotides which we call macromolecular nucleotides (m-Nucs). Macromolecular nucleotides comprise a nucleotide moiety, a macromolecular linear linker, and a large macromolecular ligand carrying multiple fluorescent dyes. With incorporation of the nucleotide moiety into the growing nucleic acid strand during enzymatic synthesis, the macromolecular ligand together with the coupled dyes is bound to the nucleic acid. By the use of this new class of modified nucleotides, signals from multiple dye molecules can be obtained after a single enzymatic incorporation event. The modified nucleotides are considered especially useful in the fields of nanobiotechnology, where signal stability and intensity is a limiting factor.

IRF-1 gene variations influence IgE regulation and atopy.

RATIONALE: The development of atopic diseases is characterized by skewed immune responses to common allergens. Only recently, interferons have been identified to play a crucial role in these mechanisms. OBJECTIVES: Because interferon regulatory factor (IRF)-1 is critical for interferon expression, we tested the hypotheses that genetic changes in this essential transcription factor may have consequences for the development of atopy. METHODS: The IRF-1 gene locus was resequenced in 80 human chromosomes. Association and haplotype analyses were performed in a cross-sectional study population of German children from Dresden (n = 1,940), and results were replicated in a second population sample from Munich (n = 1,159), both part of the ISAAC (International Study of Asthma and Allergy in Childhood) phase II. Promoter polymorphism effects were studied using electrophoretic mobility shift assay and colorimetric binding assays. Allele-specific IRF-1 gene expression was studied in vitro using luciferase reporter assays, whereas we assessed ex vivo expression of IRF-1 by real-time polymerase chain reaction and IFN-gamma protein by Luminex technology (Bio-Rad, Hercules, CA). Statistical analyses were performed using SAS/Genetics (SAS 9.1.3; SAS Institute, Cary, NC). MEASUREMENTS AND MAIN RESULTS: By resequencing, 49 polymorphisms were identified within the IRF-1 gene. Four blocks containing 11 polymorphisms were significantly associated with atopy, total IgE levels, or specific IgE levels in both populations (P < 0.05). Two polymorphisms changed transcription factor binding of nuclear factor (NF)-kappaB and EGR1 (early growth response 1) to the IRF-1 promoter, altered gene expression in vitro (P = 0.0004), and altered IRF-1 mRNA and IFN-gamma protein expression ex vivo. CONCLUSIONS: Our results suggest that functionally relevant IRF-1 polymorphisms influence atopy risk, potentially by altering transcription factor binding, IRF-1 gene expression, and IFN-gamma regulation.