A New Class of Chemically Modified Small RNA Inhibitors against Fusobacterium nucleatum

口腔微生物组代表了医学中一个令人兴奋的前沿，该领域的早期成功已经证明 单个微生物种群之间的动态相互作用，并突出了口腔之间的串扰 位于粘膜界面的微生物区系及其宿主。虽然口腔微生物组领域取得了令人印象深刻的进展 为了实现这些目标，我们的大部分知识通常是从细菌与细菌之间的相关性研究中推断出来的。 成分和疾病进展。然而，现在迫切需要有针对性的调节剂来证明 通过以某种方式选择性地消除多物种群落中的单个物种而形成的因果关系 类似于通过有针对性的基因敲除来识别基因型和表型之间的联系的遗传工具。这个 目前的R03应用程序将建立在我们的合作者最近的一项基础研究的基础上，该研究发现了串扰 核梭杆菌(FN)与人源转移RNA小RNA(TsRNAs)之间的相互作用 唾液和口腔上皮细胞。FN是一种主要的口腔共生性和机会性牙周病原体，具有 由于其在牙周疾病、早产和结肠癌中的作用而引起了人们的广泛关注。然而， 目前还没有工具可以选择性地消除FN，以了解其在复杂疾病中的作用。同时，tsRNA 代表了一类新的小RNA，可以调节原核生物和真核生物的基因表达，以及 最近的工作表明，宿主细胞可能利用某些tsRNAs来靶向FN。具体地说，不朽的 人口腔上皮细胞在FN感染后可释放tsRNA-000794和tsRNA-020498。有趣的是， 合成的tsRNA-000794和tsRNA-020498的模拟物，而不是加扰的rna序列，可以杀死FN 浮游培养，但不包括牙龈卟啉单胞菌，一种革兰氏阴性牙周病原体，或链球菌 米蒂斯，一种与健康相关的革兰氏阳性口腔细菌。然而，微摩尔浓度是需要的 对不同的FN菌株实现抑制，这对潜在的应用提出了挑战。要解决这个问题 限制，PI利用他在RNA化学和递送方面的背景对终端进行化学修饰 两个tsRNAs的5‘和3’端的核苷酸(MOD-tsRNAs)。令人印象深刻的是，这项发明导致了 将MOD-tsRNA-000794和MOD-tsRNA-020498的浓度降低1000倍，以实现 与天然抗体相比，针对FN的效力和特异性相同。受初赛激励 数据，我们将针对这类新的MOD-tsRNA抑制剂执行两个独立和互补的目标。 具体地说，我们将在体外用相关的方法证明MOD-tsRNAs对FN的有效性和特异性 生物膜和多物种模型(目标1)。同时，我们将确定FN中MOD-tsRNAs的靶标(目标2)。 虽然这项申请的重点是两个特定的tsRNA和一种口腔微生物，但概念框架将为 一类新的宿主衍生的小RNA抑制剂朝着遗传工具和治疗开发的方向发展， 考虑到其他基于核酸的技术已经取得成功的轨迹。

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.