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

一类新型化学修饰小 RNA 抑制剂，抗具核梭杆菌

• 批准号: 10534754
• 负责人: Jiahe Li
• 金额: --
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10534754
• 关键词: 16S ribosomal RNA sequencing; Address; Affinity Chromatography; Antibiotics; Attention; Bacteria; Cells; Chemicals; Chemistry; Clustered Regularly Interspaced Short Palindromic Repeats; Colon Carcinoma; Communities; Complex; Correlation Studies; Data; Development; Disease; Disease Progression; Epithelial Cells; Eukaryota; Fluorescence; Fusobacterium nucleatum; Future; Gel; Gene Expression; Genetic; Genotype; Goals; Guide RNA; Health; Human; In Vitro; Individual; Infection; Knock-out; Knowledge; Link; Mass Spectrum Analysis; Mediating; Medicine; Microbe; Microbial Biofilms; Modeling; Modification; Molecular Target; Mucous Membrane; Mus; Nucleic Acids; Nucleotides; Oral; Pathogenesis; Pathway interactions; Periodontal Diseases; Periodontitis; Phenotype; Premature Birth; Prokaryotic Cells; Proteins; RNA; RNA Sequences; Reagent; Role; Saliva; Silver Proteins; Silver Staining; Small RNA; Sorting; Specificity; Streptococcus mitis; Streptococcus mutans; Structure-Activity Relationship; Technology; Testing; Therapeutic; Tooth Diseases; Toxic effect; Transfer RNA; Untranslated RNA; Work; antimicrobial peptide; design; fighting; frontier; improved; in vitro Model; in vivo; inhibitor; invention; microbial; oral bacteria; oral biofilm; oral cavity epithelium; oral commensal; oral infection; oral microbial community; oral microbiome; overexpression; pathogen; periodontopathogen; polymicrobial disease; public health relevance; response; success; therapeutic development; tool; tool development; transcriptome sequencing; uptake

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.

口腔微生物组代表了医学中令人兴奋的前沿，该领域的早期成功已经证明 单个微生物物种之间的动态相互作用，并强调了口腔之间的串扰 微生物群和它们的宿主在粘膜界面。虽然口腔微生物组领域已经取得了令人印象深刻的进步， 为了实现这些目标，我们的大部分知识通常是从细菌之间的相关性研究中推断出来的。 组合物和疾病进展。然而，目前迫切需要靶向调节剂来证明， 通过选择性地消除多物种群落中的个别物种的因果关系， 类似于通过靶向敲除鉴定基因型和表型之间联系的遗传工具。的 目前的R 03应用程序将建立在我们的合作者最近的基础研究上，该研究发现了串扰 具核梭杆菌（Fn）与来自人的转移RNA衍生的小RNA（tsRNA）之间的相互作用 唾液和口腔上皮细胞。Fn是一种重要的口腔黏膜和牙周条件致病菌， 由于其在牙周病、早产和结肠癌中的意义而获得了很多关注。但是，在这方面， 不存在可以选择性地消除Fn以了解其在复杂疾病中的作用的工具。与此同时，TSRNA 代表了一类新的小RNA，可以调节原核生物和真核生物中的基因表达， 最近的工作已经证明宿主细胞可以利用某些tsRNA靶向Fn。具体来说， 人口腔上皮细胞可响应于Fn感染而释放tsRNA-000794和tsRNA-020498。有趣的是， tsRNA-000794和tsRNA-020498的合成模拟物，但不是乱序RNA序列，可以杀死 牙周培养，但不是牙龈卟啉单胞菌，一种革兰氏阴性牙周病原体，或链球菌 一种与健康相关的革兰氏阳性口腔细菌。然而，需要微摩尔浓度， 实现对不同Fn菌株的抑制，这对潜在应用提出了挑战。为了解决这个 由于局限性，PI利用他在RNA化学和递送方面的背景， 在两个tsRNA的5'和3'末端的核苷酸（MOD-tsRNA）。令人印象深刻的是，这项发明导致了 MOD-tsRNA-000794和MOD-tsRNA-020498的浓度降低约1000倍，以实现 与天然对应物相比，对Fn具有等效的效力和特异性。由于初步的 数据，我们将对这类新的MOD-tsRNA抑制剂进行两个独立和互补的目标。 具体地，我们将使用相关的体外实验来证明MOD-tsRNA针对Fn的功效和特异性。 生物膜和多物种模型（目的1）。同时，我们将鉴定Fn中MOD-tsRNA的靶标（目的2）。 虽然这个应用程序的重点是两个特定的tsRNA和一个口腔微生物，概念框架将铺平 一类新的宿主衍生的小RNA抑制剂朝向遗传工具和治疗开发的方向， 考虑到其他基于核酸的技术已经成功的轨迹。