Single-molecule counting of circular RNAs using phage nanoparticles as surrogates

使用噬菌体纳米颗粒作为替代物对环状 RNA 进行单分子计数

• 批准号: 10092159
• 负责人: Binrui Cao
• 金额: $ 22.79万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2022-05-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10092159
• 关键词: Affinity; Bacteria; Bacteriophage T7; Bacteriophages; Binding; Biological Assay; Biological Markers; Biomedical Engineering; Biosensing Techniques; Body Fluids; Breast Cancer Cell; Breast Cancer Patient; Buffers; Capsid; Cells; Chemicals; Clinic; Code; Color; Complementary DNA; Complex; Detection; Engineering; Eye; Fiber; Genetic; Genetic Engineering; Goals; Hour; Human; In Vitro; Letters; Magnetism; Mammary Neoplasms; Mammography; Methods; Modification; Nanotechnology; Nucleic Acids; Oligonucleotides; Peptides; Phosphate Buffer; Polymerase Chain Reaction; Proteins; RNA; Reproducibility; Resolution; Reverse Transcription; Saline; Sampling; Sensitivity and Specificity; Series; Serum; Sodium Chloride; Specificity; Structure; Tail; Techniques; Testing; Time; Tissues; Tumor Tissue; Untranslated RNA; Virus; Virus-like particle; Visual; Water; Woman; aptamer; aqueous; base; breast cancer diagnosis; cancer biomarkers; cancer diagnosis; cancer therapy; circular RNA; cost; design; detection limit; digital; early detection biomarkers; malignant breast neoplasm; millimeter; nanobiomaterial; nanomaterials; nanoparticle; novel; overexpression; particle; protein biomarkers; red fluorescent protein; single molecule

Single-molecule counting of circular RNAs using phage nanoparticles as surrogates Abstract Circular RNA (circRNA) have been considered as promising breast cancer biomarkers, but the current qRT-PCR and digital PCR techniques for quantifying them have some inherent limitations. T7 phage, a human-safe virus nanoparticle specifically infecting bacteria, can be plated to infect bacteria in a plaque-forming assay to form millimeter-scale plaques in a one to one format and at a single-particle resolution within 3 hours. Inspired from this, we propose to develop a phage plaque counting (PPC) strategy that hires bioengineered fluorescent T7 phage as a surrogate to establish a one-to-one correspondence among target circRNA, phage nanoparticles, and eye-visible phage-developed plaques, enabling us to simultaneously quantify multiple circRNA biomarkers in a single test by simply counting the corresponding plaques. Briefly, a fluorescent phage probe with an oligonucleotide (ONT-1) capable of capturing one unique segment of a target circRNA, and a magnetic microparticle (MMP) probe with an ONT-2 capable of capturing another unique segment of the same target, co- capture the target to form a sandwich complex where phages and target molecules are equimolar. Then the phages are released and plated to develop fluorescent plaques in a one to one format, thus counting the plaques at the single-particle level leads to visualized quantification of the target circRNA at a single-molecule level. A fluorescent protein on the capsid makes the corresponding plaque fluoresce a unique color and enables the simultaneous single-particle quantification of fluorescent plaques (in the same Petri dish) with each color coding one target (i.e., by displaying green and red fluorescent protein for two corresponding targets). We hypothesize that our PPC strategy with optimized conditions can simultaneously quantify a panel of two circRNAs as breast cancer biomarkers in human serum with high sensitivity, specificity, and reproducibility. Aim 1: Establish and optimize PPC strategy for quantifying single and multiple circRNA breast cancer biomarkers. We will produce and purify the target circRNAs (circ_0001785 and circ_100219) by the overexpression method and use the PPC method to quantify them with a series of dilutions in water. We will optimize the PPC and identify its detection limit. Aim 2: Validate the PPC strategy for simultaneously quantifying multiple circRNA biomarkers in breast cancer cells, tissues, and human serum. We will first use MMPs that can capture the target circRNAs to magnetically remove the pre-existing target circRNAs from the commercial human serum, which is then used to make serum samples with known concentrations of target circRNAs. Then we will employ and optimize the PPC to quantify the target circRNAs in the serum. We will also isolate total RNAs from breast cancer cells and in vitro breast tumor tissues to form aqueous RNA solutions by a commercial RNA isolation kit. We will then use the PPC to quantify the target circRNAs in the resultant RNA solutions and the commercial total RNA solutions isolated from tumor tissues of breast cancer patients. This project will lead to a new visualized single-molecule technique for quantifying circRNA biomarkers for breast cancer diagnosis.

以噬菌体纳米颗粒为替代物的环状RNA单分子计数 摘要 环状RNA（circRNA）已被认为是有前途的乳腺癌生物标志物，但目前的qRT-PCR 用于定量它们的数字PCR技术具有一些固有的局限性。T7噬菌体，一种对人类安全的病毒 专门感染细菌的纳米颗粒可以在噬菌斑形成试验中接种以感染细菌，以形成 在3小时内以一对一的格式和单粒子分辨率测量毫米级斑块。灵感来自 因此，我们建议开发一种噬菌体噬斑计数（PPC）策略， 噬菌体作为替代物，以在靶circRNA、噬菌体纳米颗粒 和肉眼可见的噬菌体形成的斑块，使我们能够同时定量多种circRNA生物标志物， 通过简单地计数相应的噬菌斑。简而言之，使用荧光噬菌体探针， 能够捕获靶circRNA的一个独特区段的寡核苷酸（ONT-1），和磁性寡核苷酸， 在一个实施方案中，本发明涉及具有ONT-2的MMP微颗粒探针，所述ONT-2能够捕获相同靶标的另一个独特区段，共 捕获靶以形成夹心复合物，其中靶分子和靶分子是等摩尔的。则 释放荧光素并铺板，以一对一的形式形成荧光噬菌斑，从而对噬菌斑计数 在单颗粒水平上的可视化导致在单分子水平上对靶circRNA的可视化定量。一 衣壳上的荧光蛋白使相应的噬菌斑发出独特的荧光颜色， 用每种颜色编码同时对荧光噬菌斑进行单颗粒定量（在同一培养皿中） 一个目标（即，通过显示两个相应靶的绿色和红色荧光蛋白）。我们假设 我们的PPC策略在优化的条件下可以同时定量一组两种circRNA作为乳腺癌的标志物， 癌症生物标志物在人血清中具有高灵敏度、特异性和可重复性。目标1：建立和 优化PPC策略，用于定量单个和多个circRNA乳腺癌生物标志物。我们将 通过过表达方法生产和纯化目标circRNA（circ_0001785和circ_100219）及其用途 PPC方法，以在水中进行一系列稀释对其进行定量。我们将优化PPC并确定其 检出限目的2：验证同时定量多种circRNA的PPC策略 乳腺癌细胞、组织和人血清中的生物标志物。我们将首先使用可以捕获 靶向circRNA以从商业人血清中磁性去除预先存在的靶circRNA， 然后将其用于制备具有已知浓度的靶circRNA的血清样品。然后我们会雇佣 并优化PPC以定量血清中的靶circRNA。我们还将从乳腺癌组织中分离总RNA， 癌细胞和体外乳腺肿瘤组织，以通过商业RNA分离试剂盒形成RNA水溶液。 然后，我们将使用PPC来定量所得RNA溶液中的靶circRNA和商业总RNA。 从乳腺癌患者的肿瘤组织分离的RNA溶液。该项目将导致一个新的可视化 用于定量circRNA生物标志物用于乳腺癌诊断的单分子技术。