Toll-like receptor 8 (TLR8) is an important component of the human innate immune system that recognizes single stranded RNA (ssRNA). Recent X-ray crystal structures of TLR8 bound to ssRNA revealed a previously unrecognized binding site for a 5′-UpG-3′ dinucleotide. Here we use an atomic mutagenesis strategy coupled with a cellular TLR8 activation assay to probe the importance of specific functional groups present on the guanine base in RNA-mediated receptor agonism and antagonism. Results from RNA analogs containing 7-deazaguanosine, 2-aminopurine and inosine confirm the importance of guanine N7, O6 and N2, respectively, in TLR8 activation. Nevertheless, these RNAs each retained TLR8 antagonism activity. RNA containing 7-deaza-8-azainosine (7d8aI) was prepared from a novel phosphoramidite and found to be a weaker TLR8 activator than guanosine-containing RNA. However, 7d8aI-containing RNA also retained TLR8 antagonism activity indicating that removal of multiple TLR8 H-bonding sites on guanine is insufficient for blocking TLR8 antagonism by guanine-containing RNA. We also identified an oligoribonucleotide length dependence on both TLR8 activation and antagonism. These studies extend our understanding of the effects of nucleobase modification on immune stimulation and will inform the design of novel RNA-based therapeutics.

Toll样受体8（TLR8）是人类先天免疫系统的重要组成部分，可识别单链RNA（ssRNA）。与ssRNA结合的TLR8的最新X射线晶体结构揭示了以前无法识别的5'-UpG-3'二核苷酸结合位点。在这里，我们使用原子诱变策略与细胞TLR8激活检测相结合，以探查在RNA介导的受体激动和拮抗作用中鸟嘌呤碱基上存在的特定官能团的重要性。包含7-脱氮鸟苷，2-氨基嘌呤和肌苷的RNA类似物的结果证实了鸟嘌呤N7，O6和N2在TLR8激活中的重要性。然而，这些RNA各自保留了TLR8拮抗活性。由新型亚磷酰胺制备了含7-脱氮基8-氮杂肌苷（7d8aI）的RNA，并发现它是一种比含鸟苷的RNA弱的TLR8激活剂。但是，含7d8aI的RNA也保留了TLR8拮抗活性，这表明鸟嘌呤上多个TLR8 H结合位点的去除不足以阻断含鸟嘌呤的RNA对TLR8的拮抗作用。我们还确定了对TLR8激活和拮抗的寡核糖核苷酸长度的依赖性。这些研究扩展了我们对核碱基修饰对免疫刺激作用的理解，并将为新型基于RNA的治疗方法的设计提供参考。我们还确定了对TLR8激活和拮抗的寡核糖核苷酸长度的依赖性。这些研究扩展了我们对核碱基修饰对免疫刺激作用的理解，并将为新型基于RNA的治疗方法的设计提供参考。我们还确定了对TLR8激活和拮抗的寡核糖核苷酸长度的依赖性。这些研究扩展了我们对核碱基修饰对免疫刺激的影响的理解，并将为新型基于RNA的治疗方法的设计提供参考。