5'-tRNAHisGUG fragment: A preferred endogenous TLR7 Ligand with reverse sequence activation insights.

Toll-like receptor 7 (TLR7), a key member of the TLR family, plays a pivotal role in innate immunity, making it an attractive therapeutic target. However, current synthetic TLR7 agonists are often associated with significant toxicity, highlighting the need for safer, naturally occurring alternatives. Our recent research identified 5'-fragments of tRNAHisGUG (5'-HisGUG) and tRNAValCAC/AAC (5'-ValCAC/AAC) as potent, naturally occurring TLR7 activators. While endogenous RNAs like 5'-HisGUG are known to activate TLR7, the molecular details of their interaction remain unclear. To address this, we performed molecular dynamics simulations and MM/GBSA binding free energy analysis to investigate how these RNA fragments engage with TLR7 in comparison to synthetic agonists. Our results revealed that 5'-HisGUG, 5'-ValCAC/AAC, and reverse sequence (5'-HisGUG-Rev) exhibit strong binding affinities, with higher energetic favorability than synthetic agonists. The free energy fluctuations suggested that endogenous RNA ligands display greater conformational variability, possibly contributing to their activation potential. Notably, 5'-HisGUG-Rev effectively activated TLR7 and enhanced cytokine mRNA expression. Comparative analysis suggests that binding affinity alone does not directly predict activation, emphasizing the importance of both strong interaction and conformational flexibility in TLR7 activation. These findings position 5'-HisGUG as a promising natural TLR7 activator with potential therapeutic applications.