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

基本信息

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
项目类别：
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

项目摘要

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置信度检测项目概要/摘要该提案的最终目标是开发一个高可信度和鲁棒性的技术平台通过同时检测 miRNA，对癌症样本中的 miRNA 生物标志物进行分子分析不到一个小时。尽管 miRNA 很短，但它们基本上调节与人类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 被发现比其他核酸相对更稳定，这使得它们作为临床癌症受到高度关注生物标志物。目前的 miRNA 分析方法，包括 PCR 检测，由于固有的原因而面临挑战。除了假阴性和假阳性之外，日常结果和实验室之间的结果也不一致。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对未标记的 DNA 以及 miRNA 目标进行高置信度和超灵敏的检测并证明该传感平台可在血清中工作并完全区分靶标和点突变体控制。使用全内反射荧光显微镜，我们证明该传感器表现出没有目标时的静态 FRET 水平。然而，在存在目标的情况下，传感器形成四路结，因此经历低 FRET 状态和高 FRET 状态之间的动态切换，这是一个特征这使得能够高置信度地检测目标。我们最初使用 p53 演示了这些功能肿瘤抑制基因和后来与三阴性乳腺癌 (TNBC) 相关的 miRNA，均在缓冲液和使用 10% 血清的掺入样品中。在本提案中，我们将重点关注发展和通过非临床中的 miRNA 多重分析对该平台进行高置信度检测测试以及经过最低限度处理的癌症样本。在目标 1 中，我们将设计并表征一个传感平台用于检测一份样本中的 DNA 序列。在目标 2 中，我们将描述并验证检测结果用于同时检测 TNBC 特异性 miRNA 的平台。我们还将建立快速检测使用具有并行通道的微流体装置来检测 TNBC miRNA。在目标 3 中，我们将确定丰富的携带 TNBC 的患者来源异种移植 (PDX) 小鼠的肿瘤组织和血清样本中的 miRNA miRNA 测序并应用我们的单分子多重平台来检测 TNBC miRNA PDX 小鼠的血清样本。我们提出的方法提供了许多重要的创新包括 i) 用于无差错检测 miRNA 的通用平台，ii) 通过单分子实现终极灵敏度检测，iii) 同时检测同一样本中的多种生物标志物 - 实现高置信度检测，以及 iv) 不需要目标标记和扩增。因此，这个多路复用平台具有有潜力成为癌症早期诊断的革命性技术。通过提供检测灵敏度和置信度达到或超过最先进的临床分析仪，建议的方法可以为临床诊断、癌症评估和个体化癌症治疗带来新的举措。