Immune-modulating nucleotide modifications within tRNA

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The innate immune system recognizes conserved, microbial structures including nucleic acids by pattern recognition receptors. Recognition of foreign, microbial nucleic acids is based on differences in sequences and structure, presence of nucleotide modifications and subcellular compartmentalization of the respective receptors. As recognition of foreign RNA results in induction of an immune response, it is of interest to examine and define structural requirements for the recognition of microbial nucleic acids. The results will impact basic research as well as application of therapeutic RNAs. So far, experiments addressing the role of nucleotide modifications have mainly been done using synthetic oligoribonucleotides whereas the importance of sequence, structure and modifications within natural RNA has not been studied intensively.The applicants could show recently, that Toll-like receptor 7 (TLR7) not only recognizes single-stranded RNA but is also activated by bacterial tRNA. The decisive discrimination of bacterial versus host tRNA was due to the presence of various, naturally occurring nucleotide modifications, among which the applicants identified an inhibitory 2'-O-methylation at guanosine 18 (Gm18).The new results on inhibitory modifications within the natural tRNA context were obtained by combining techniques in RNA synthesis and analysis with immunobiological assays. The newly developed techniques are now to be used to answer basic questions of RNA recognition by TLR7. It is intended to identify the molecular mechanism by which Gm18 suppresses an immune response. Therefore, positional effects and cellular signal transduction will be analyzed. Furthermore, new immune-modulating nucleotide modifications will be identified within bacterial and eukaryotic RNA using LC/MS and the recently developed synthesis of chimeric (modified and unmodified) RNAs. Finally, functions of the identified nucleotide modifications with respect to foreign/self discrimination and a potential role for bacterial immune evasion will be studied.The experiments are expected to gain insight into the less well examined topic of immune-modulation by RNA nucleotide modifications. Of note, the experiments will be done within the context of natural RNA. Inhibitory modifications might be of use for the targeted manipulation of innate immunity.