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Influenza A virus infection causes substantial morbidity and mortality in seasonal epidemic outbreaks, and more efficient treatments are urgently needed. Innate immune sensing of viral nucleic acids stimulates antiviral immunity, including cell-autonomous antiviral defense mechanisms that restrict viral replication. RNA oligonucleotide ligands that potently activate the cytoplasmic helicase retinoic-acid-inducible gene I (RIG-I) are promising candidates for the development of new antiviral therapies. Here, we demonstrate in an Mx1-expressing mouse model of influenza A virus infection that a single intravenous injection of low-dose RIG-I ligand 5′-triphosphate RNA (3pRNA) completely protected mice from a lethal challenge with influenza A virus for at least 7 days. Furthermore, systemic administration of 3pRNA rescued mice with pre-established fulminant influenza infection and prevented the fatal effects of a streptococcal superinfection. Type I interferon, but not interferon-λ, was required for the therapeutic effect. Our results suggest that the use of RIG-I activating oligonucleotide ligands has the clinical potential to confine influenza epidemics when a strain-specific vaccine is not yet available and to reduce lethality of influenza in severely infected patients.

Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3' and 5' ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1–249) and Rnl2(1–249)K227Q, for 3'-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5'-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3'-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5'-terminal 5-nt barcode extensions for a set of 20 barcoded 3' adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.

Mammalian genomic DNA complexed to the natural antimicrobial cationic peptide LL37 induces type I interferon but less than bacterial DNA or CG‐dinucleotide containing oligodeoxynucleotides. TLR9 detects DNA in endolysosomal compartments of human B cells and PDC. Recently, the concept of the CpG motif specificity of TLR9‐mediated detection, specifically of natural phosphodiester DNA, has been challenged. Unlike in human B cells, CpG specificity of natural phosphodiester DNA recognition in human PDC has not been analyzed in the literature. Here, we found that the induction of IFN‐α and TNF‐α in human PDC by phosphodiester ODNs containing one or two CG dinucleotides was reduced to a lower level when the CG dinucleotides were methylated and was abolished if the CGs were switched to GCs. Consistent with a high frequency of unmethylated CG dinucleotides, bacterial DNA induced high levels of IFN‐α in PDC; IFN‐α was reduced but not abolished upon methylation of bacterial DNA. Mammalian DNA containing low numbers of CG dinucleotides, which are frequently methylated, induced IFN‐α in PDC consistently but on a much lower level than bacterial DNA. For activation of PDC, phosphodiester ODNs and genomic DNA strictly required complexation with cationic molecules such as the keratinocyte‐derived antimicrobial peptide LL37 or a scrambled derivative. In conclusion, we demonstrate that self‐DNA complexed to cationic molecules activate PDC and thus, indeed, may function as DAMPs; nevertheless, the preference of PDC for CpG containing DNA provides the basis for the discrimination of microbial from self‐DNA even if DNA is presented in the condensed form of a complex.

The title compounds were synthesized by converting cyclic AMP with a sulfonyl chloride to the symmetrical anhydride or with diphenyl phosphorochloridate to the mixed anhydride, then aminolyzing the anhydrides without isolation. The synthesis preferentially gave Sp-amides. Characteristic differences were observed in the behavior of the unsubstituted amide and the dimethylamide under hydrolysis conditions.

Thermal stabilities of oligonucleotides containing 2′-amino-2′-deoxycytidines were determined and compared to those of the unmodified oligonucleotides. The presence of the 2′-aminonucleoside destabilised duplexes in a RNA as well as a DNA context at pH 7 as well as at pH 5. The pK, of the 2′-amino group was determined by 13C-NMR spectroscopy to be 6.2. The reactivity of an oligonucleotide containing a 2′-amino-nucleoside was exploited for the incorporation of rhodamine by its isothiocyanate derivative.