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: 10353249
• 负责人: Jiahe Li
• 金额: $ 17.38万
• 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 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10353249
• 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 - Cell Movement; Specificity; Streptococcus mitis; Streptococcus mutans; Structure-Activity Relationship; Technology; Testing; Therapeutic; Tooth Diseases; Toxic effect; Transfer RNA; Untranslated RNA; Work; antimicrobial peptide; base; 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; programs; 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.

口服微生物组代表了医学上令人兴奋的前沿，并且在田间的早期成功已经证明了 单个微生物物种之间的动态相互作用，并突出了口服之间的串扰 微生物群及其宿主处于粘膜界面。虽然口腔微生物组领域取得了令人印象深刻的进步 朝向这些目标，我们的许多知识通常是通过细菌之间的相关性研究推断出来的 组成和疾病进展。但是，有针对性调节器立即需要证明 通过以某种方式选择性地消除多种物种社区中的单个物种来有因果关系 类似于遗传工具，该工具通过靶向敲除识别基因型和表型之间的联系。这 当前的R03应用程序将基于我们的合作者最近进行的基本研究，该研究发现了Crosstalk 核杆菌（FN）和转移的RNA衍生的小RNA（TSRNA）之间 唾液和口服上皮细胞。 FN是一个关键的口服和机会性牙周病原体，具有 由于其对牙周疾病，早产和结肠癌的影响，因此引起了很多关注。然而， 没有任何工具可以选择性地消除FN以了解其在复杂疾病中的作用。并联，tsrna 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 最近的工作表明，宿主细胞可以采用某些TSRNA来靶向FN。具体而言，永生 人口腔上皮细胞可以响应FN感染而释放TSRNA-000794和TSRNA-020498。有趣的是， TSRNA-000794和TSRNA-020498的合成模仿，但不加扰的RNA序列，可以杀死FN 浮游生物培养，但不是卟啉单胞菌，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服细菌。但是，需要微摩尔浓度 实现对不同FN菌株的抑制作用，这对潜在应用构成了挑战。解决这个问题 局限性，PI利用了他的背景RNA化学和化学修饰终端的背景 两个TSRNA（MOD-TSRNA）的5'和3'端的核苷酸。令人印象深刻的是，这次事故导致 Mod-TSRNA-000794和MOD-TSRNA-020498的浓度降低了〜1000倍以实现 与天然对应物相比，针对FN的同等效力和特异性。由初步动机 数据，我们将针对这种新的Mod-TSRNA抑制剂执行两个独立和互补的目标。 具体而言，我们将使用相关的体外证明Mod-TSRNA对FN的效率和特异性 生物膜和多物种模型（AIM 1）。同时，我们将在FN中识别Mod-TSRNA的目标（AIM 2）。 虽然该应用程序侧重于两个特定的TSRNA和一个口头微生物，但概念框架将铺路 新型宿主衍生的小RNA抑制剂针对遗传工具和治疗性发育的方式， 考虑到其他基于核酸的技术已经成功的轨迹。