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领域令人兴奋的前沿领域，该领域的早期成功已经证明 各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 粘膜界面的微生物群及其宿主，而口腔微生物组领域取得了令人瞩目的进步。 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 然而，迫切需要靶向调节剂来证明。 通过以某种方式选择性消除多物种群落中的单个物种来建立因果关系 类似于遗传工具，通过靶向敲除来识别基因型和表型之间的类似联系。 目前的 R03 应用程序将建立在我们合作者最近的一项基础研究的基础上，该研究发现了串扰 具核梭杆菌 (Fn) 与源自人类的转移 RNA 衍生小 RNA (tsRNA) 之间的关系 Fn 是一种重要的口腔共生和机会性牙周病原体。 由于其对牙周疾病、早产和结肠癌的影响而受到广泛关注。 目前还没有工具可以选择性消除 Fn 以了解其在复杂疾病中的作用。 代表一类新的小RNA，可以调节原核生物和真核生物中的基因表达，并且 最近的研究表明，宿主细胞可能会利用某些 tsRNA 来特异性地、永生化地靶向 Fn。 有趣的是，人类口腔上皮细胞可以释放 tsRNA-000794 和 tsRNA-020498 以应对 Fn 感染。 tsRNA-000794 和 tsRNA-020498 的合成模拟物（但不是乱序 RNA 序列）可以杀死 Fn 浮游培养物，但不包括牙龈卟啉单胞菌、革兰氏阴性牙周病原体或链球菌 mitis，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，这对解决这个问题的潜在应用提出了挑战。 由于局限性，PI 利用他在 RNA 化学和递送方面的背景对末端进行化学修饰 令人印象深刻的是，本发明产生了两个 tsRNA (MOD-tsRNA) 5' 和 3' 末端的核苷酸。 MOD-tsRNA-000794 和 MOD-tsRNA-020498 的浓度降低约 1000 倍，以实现 与自然信用相比，针对 Fn 的效力和特异性相同。 根据数据，我们将对这一新型 MOD-tsRNA 抑制剂执行两个独立且互补的目标。 具体来说，我们将使用相关的体外实验证明 MOD-tsRNA 针对 Fn 的功效和特异性 生物膜和多物种模型（目标 1）同时，我们将确定 Fn 中 MOD-tsRNA 的靶标（目标 2）。 虽然该应用重点关注两种特定的 tsRNA 和一种口腔微生物，但概念框架将为 新型宿主衍生小RNA抑制剂走向遗传工具和治疗开发的道路， 考虑到其他基于核酸的技术已经取得的成功轨迹。