Immune-modulating nucleotide modifications within tRNA

先天免疫系统通过模式识别受体识别保守的微生物结构，包括核酸。对外来微生物核酸的识别是基于序列和结构的差异、核苷酸修饰的存在以及各自受体的亚细胞划分。由于识别外源RNA会诱导免疫反应，因此研究和定义识别微生物核酸的结构要求是有意义的。这一结果将影响基础研究和治疗性RNA的应用。到目前为止，关于核苷酸修饰作用的实验主要是利用人工合成的寡核苷酸来完成的，而对天然RNA中序列、结构和修饰的重要性还没有深入的研究。最近的应用表明，Toll样受体7(TLR7)不仅识别单链RNA，而且还被细菌的tRNA激活。细菌和宿主tRNA的决定性区别是由于存在各种自然发生的核苷酸修饰，其中申请者发现了鸟苷18(Gm18)的抑制性2‘-O-甲基化。通过将RNA合成和分析技术与免疫生物学检测相结合，在自然tRNA背景下获得了关于抑制性修饰的新结果。新开发的技术现在将用于回答TLR7识别RNA的基本问题。它的目的是确定Gm18抑制免疫反应的分子机制。因此，将对位置效应和细胞信号转导进行分析。此外，利用LC/MS和最近开发的嵌合(修饰和未修饰)RNA的合成，将在细菌和真核细胞RNA中鉴定新的免疫调节核苷酸修饰。最后，将研究已识别的核苷酸修饰在异源/自我识别方面的功能以及细菌免疫逃避的潜在作用。实验有望深入了解RNA核苷酸修饰对免疫调节的较少研究主题。值得注意的是，这些实验将在天然RNA的背景下进行。抑制性修饰可能对先天免疫的靶向操作有用。

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.