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肿瘤标志物的单分子高置信度检测 项目总结/摘要 本提案的最终目标是开发一个技术平台，用于高置信度和鲁棒性的单 通过同时检测miRNA，对癌症样品中的miRNA生物标志物进行分子分析， 不到一小时。尽管miRNAs很短，但它们基本上调节与人类免疫相关的所有细胞通路。 健康和疾病，包括癌症。从细胞中输出后，无细胞循环miRNA被 被发现比其他核酸相对更稳定，使得它们作为临床癌症受到高度关注。 生物标志物。目前用于miRNA分析的方法，包括PCR测定，由于固有的缺陷而面临挑战。 除了假阴性和假阳性之外，日常和实验室之间的结果不一致。我们有 最近开发了一种独特的基于荧光共振能量转移（FRET）的单分子动态 传感器，能够高置信度和超灵敏度检测未标记的DNA以及miRNA靶标 并证明该传感平台在血清中工作，并完全区分目标和点突变体 对照使用全内反射荧光显微镜，我们证明了传感器具有 在不存在靶标的情况下的静态FRET水平。然而，在目标存在的情况下，传感器形成四个- 因此，在低FRET状态和高FRET状态之间经历动态切换， 这使得能够高置信度地检测目标。我们最初使用p53 肿瘤抑制基因和后来与三阴性乳腺癌（TNBC）相关的miRNA，两者都在 缓冲液和使用10%血清的加标样品中。在本建议中，我们将重点关注发展和 通过对非临床应用中的miRNA进行多重分析，测试该平台的高置信度检测。 以及最低限度处理的癌症样本。在目标1中，我们将设计和表征传感平台 用于检测一个样本中的DNA序列。在目标2中，我们将描述和验证检测 用于同时检测特异于TNBC的miRNA的平台。我们还将建立一个快速检测 使用具有平行通道的微流体装置，在目标3中，我们将确定丰富的 携带TNBC的患者来源的异种移植物（PDX）小鼠的肿瘤组织和血清样品中的miRNA， miRNA测序并应用我们的单分子多重平台来检测TNBC中的miRNA 来自PDX小鼠的血清样品。我们提出的方法提供了一些重要的创新 包括i）用于无错误检测miRNA的通用平台，ii）通过单分子检测的最终灵敏度， 检测，iii）同时检测同一样品中的多种生物标志物-允许高置信度 检测和iv）不需要靶标记和扩增。因此，该多路复用平台具有 有可能成为癌症早期诊断的变革性技术。通过提供检测 达到或超过最先进的临床分析仪的灵敏度和置信度， 可以在临床诊断、癌症评估和个体化癌症治疗方面带来新的举措。