Sunday, May 11, 2025
5/11/2025
A New Class of Chemically Modified Small RNA Inhibitors against Fusobacterium nucleatum - Oral microbiome represents an exciting frontier in medicine, and early successes in the field have demonstrated the dynamic interactions among individual microbial species and highlighted the crosstalk between oral microbiota and their hosts at the mucosal interface. While the oral microbiome field has made impressive strides toward these goals, much of our knowledge is typically inferred from correlation studies between bacterial compositions and disease progression. However, there is an immediate need for targeted modulators to prove a causal relationship by selectively eliminating individual species in a multispecies community in a manner analogous to genetic tools that identify links between genotype and phenotype via targeted knockouts. The present R03 application will build on a recent fundamental study by our collaborators, which uncovered crosstalk between Fusobacterium nucleatum (Fn) and transfer RNA-derived small RNAs (tsRNAs) derived from human saliva and oral epithelial cells. Fn is a key oral commensal and opportunistic periodontal pathogen, and has garnered much attention due to its implications in periodontal diseases, preterm birth, and colon cancer. However, no tool exists that can selectively eliminate Fn to understand its role in complex diseases. In parallel, tsRNA represents a new class of small RNAs that can modulate gene expression in prokaryotes and eukaryotes, and recent work has demonstrated that host cells may employ certain tsRNAs to target Fn. Specifically, immortalized human oral epithelial cells can release tsRNA-000794 and tsRNA-020498 in response to Fn infection. Intriguingly, synthetic mimics of tsRNA-000794 and tsRNA-020498, but not scrambled RNA sequences, can kill Fn in planktonic culture, but not Porphyromonas ginigivalis, a gram-negative periodontal pathogen, or Streptococcus mitis, a health-associated gram-positive oral bacterium. However, micromolar concentrations are needed to achieve inhibition against different Fn strains, which poses a challenge for potential applications. To address this limitation, the PI has leveraged his background in RNA chemistry and delivery to chemically modify terminal nucleotides at the 5’ and 3’ ends of the two tsRNAs (MOD-tsRNAs). Impressively, this invention resulted in ~1000-fold reductions in the concentrations of MOD-tsRNA-000794 and MOD-tsRNA-020498 to achieve equivalent potency and specificity against Fn compared to natural counterparts. Motivated by the preliminary data, we will perform two independent and complementary aims towards this new class of MOD-tsRNA inhibitors. Specifically, we will demonstrate the efficacy and specificity of MOD-tsRNAs against Fn using relevant in vitro biofilm and multispecies models (Aim 1). In parallel, we will identify the targets of MOD-tsRNAs in Fn (Aim 2). While this application focuses on two specific tsRNAs and one oral microbe, the conceptual framework will pave the way for a new class of host-derived small RNA inhibitors towards genetic tool and therapeutic development, considering the already successful trajectories of other nucleic acid-based technologies.
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