Single-Molecule High-Confidence Detection of miRNA Cancer Biomarkers

miRNA 癌症生物标志物的单分子高置信度检测

• 批准号: 10612611
• 负责人: Soma Dhakal
• 金额: $ 20.48万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-13 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10612611
• 关键词: Address; Antibodies; Attention; Benchmarking; Binding; Biological; Biological Assay; Biological Markers; Buffers; Cells; Clinical; DNA; DNA Sequence; Detection; Development; Diagnosis; Diagnostic Neoplasm Staging; Diagnostic Specificity; Disease; Disease Progression; Dyes; Early Diagnosis; Enzymes; Exhibits; Face; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Goals; Health; Hour; Human; Label; Malignant Neoplasms; Measures; Methodology; Methods; MicroRNAs; Microfluidic Microchips; Monitor; Mus; Mutation; Nature; Noise; Nucleic Acids; Pathway interactions; Patient-derived xenograft models of breast cancer; Patients; Physiology; Point Mutation; Process; Prognosis; RNA Sequences; Recurrence; Reproducibility; Research; Resources; Risk; Risk Assessment; Role; Sampling; Scheme; Screening for cancer; Serum; Signal Transduction; Speed; Staging; TP53 gene; Techniques; Technology; Testing; Therapeutic; Translating; Tumor Suppressor Genes; Tumor Tissue; Uncertainty; Work; cancer biomarkers; cancer diagnosis; cancer therapy; circulating microRNA; clinical application; clinical diagnosis; clinical practice; cost; design; detection assay; detection method; detection platform; detection sensitivity; fluorophore; human disease; innovation; interest; miRNA expression profiling; microRNA biomarkers; multiplex detection; mutant; novel strategies; overexpression; patient derived xenograft model; pre-clinical; response; sensor; single cell technology; single molecule; synthetic construct; technology platform; treatment response; triple-negative invasive breast carcinoma; tumor; tumor growth; tumor xenograft

Single-Molecule High-Confidence Detection of miRNA Cancer Biomarkers PROJECT SUMMARY/ ABSTRACT The ultimate goal of this proposal is to develop a technology platform for high confidence and robust single molecule analysis of miRNA biomarkers in cancer samples through simultaneous detection of miRNAs, in under an hour. Although miRNAs are short, they regulate essentially all cellular pathways relevant to human health and disease, including cancer. After being exported from cells, the cell-free circulating miRNAs are found to be relatively more stable than other nucleic acids, making them of high interest as clinical cancer biomarkers. Current methods for miRNA analysis, including PCR assays, face challenges due to inherent inconsistencies in day-to-day and lab-to-lab results in addition to false negatives and positives. We have recently developed a unique fluorescence resonance energy transfer (FRET)-based single molecule dynamic sensor to enable high confidence and ultrasensitive detection of an unlabeled DNA as well as miRNA targets and demonstrated that the sensing platform works in serum and fully discriminates targets from point mutant controls. Using total internal reflection fluorescence microscopy, we demonstrated that the sensor exhibits a static FRET level in the absence of a target. However, in the presence of the target, the sensor forms a four- way junction and hence undergoes a dynamic switching between a low- and a high-FRET state, a feature that enables high-confidence detection of the target. We demonstrated these features initially using a p53 tumor suppressor gene and later a miRNA associated with the triple-negative breast cancer (TNBC), both in buffer and in spiked-in samples using 10% serum. In this proposal, we will focus on the development and testing of this platform for high-confidence detection through multiplexed analysis of miRNAs in non-clinical as well as minimally processed cancer samples. In Aim 1, we will design and characterize a sensing platform for the detection of DNA sequences in one sample. In Aim 2, we will characterize and validate the detection platform for simultaneous detection of miRNAs specific to TNBC. We will also establish a speedy detection of TNBC miRNAs using a microfluidic device with parallel channels. In Aim 3, we will identify the abundant miRNAs in tumor tissues and serum samples of TNBC-carrying patient-derived xenograft (PDX) mice via miRNA sequencing and apply our single-molecule multiplexed platform to detect those TNBC miRNAs in serum samples from the PDX mice. Our proposed approach offers a number of important innovations including i) a generic platform for error-free detection of miRNAs, ii) ultimate sensitivity via single-molecule detection, iii) simultaneous detection of multiple biomarkers in the same sample - allowing high-confidence detection, and iv) target labeling and amplification are not required. Therefore, this multiplexed platform has the potential to be a transformative technology in the early diagnosis of cancer. By providing detection sensitivity and confidence that meets or exceeds state-of-the-art clinical analyzers, the proposed approach could bring new initiatives in clinical diagnosis, cancer assessment, and individualized cancer treatments.

miRNA癌症生物标志物的单分子高电信检测 项目摘要/摘要 该提案的最终目标是为高度信心和强大的单身建立技术平台 通过同时检测miRNA，在癌症样品中miRNA生物标志物的分子分析，在 一个小时。尽管miRNA很短，但它们基本上调节了与人有关的所有细胞途径 健康和疾病，包括癌症。从细胞中导出后，无细胞的循环miRNA为 发现比其他核酸相对稳定，因此作为临床癌引起了人们的兴趣 生物标志物。当前用于miRNA分析的方法，包括PCR分析，由于固有而面临挑战 除了虚假负面因素和积极因素外，日常和实验室的不一致会导致。我们有 最近开发了一个独特的荧光共振能传递（FRET）的单分子动态 传感器可以使未标记的DNA以及miRNA靶标具有高置信度和超敏感性检测 并证明传感平台在血清中起作用，并完全区分了点突变体的靶标 控件。使用总内反射荧光显微镜，我们证明了传感器表现出A 在没有目标的情况下，静态货物水平。但是，在目标存在下，传感器形成四个 方式交界处，因此在低点和高率状态之间进行动态切换，一个功能 这样可以高信心检测目标。我们最初使用p53证明了这些功能 肿瘤抑制基因，后来是与三阴性乳腺癌（TNBC）相关的miRNA 缓冲液和尖刺样品中使用10％血清。在此提案中，我们将专注于发展和 通过非临床的miRNA多路复用分析来测试该平台以进行高信心检测 以及最少处理的癌症样品。在AIM 1中，我们将设计和表征一个传感平台 用于检测一个样品中的DNA序列。在AIM 2中，我们将表征和验证检测 同时检测特定于TNBC的miRNA的平台。我们还将建立一个快速检测 使用带有平行通道的微流体装置的TNBC miRNA的。在AIM 3中，我们将确定丰富的 肿瘤组织中的miRNA和由TNBC携带患者衍生异种移植（PDX）小鼠的血清样品通过 miRNA测序并应用我们的单分子多路复用平台来检测这些TNBC miRNA 来自PDX小鼠的血清样品。我们提出的方法提供了许多重要的创新 包括i）一个通用平台用于miRNA的无错误检测，ii）通过单分子的最终灵敏度 检测，iii）在同一样品中同时检测多个生物标志物 - 允许高信心 检测和iv）不需要目标标记和扩增。因此，这个多路复用平台具有 在癌症早期诊断中成为一种变革性技术的潜力。通过提供检测 符合或超过最先进的临床分析仪的敏感性和信心，该方法 可以为临床诊断，癌症评估和个性化癌症治疗带来新的举措。