Single-Molecule High-Confidence Detection of miRNA Cancer Biomarkers

MiRNA肿瘤生物标志物的单分子高置信度检测 项目摘要/摘要 这一提议的最终目标是为高信心和健壮的单身人士开发一个技术平台 同时检测肿瘤标本中的miRNAs，in，miRNA的分子分析 不到一个小时。虽然miRNAs很短，但它们基本上调控着与人类相关的所有细胞通路 健康和疾病，包括癌症。从细胞输出后，无细胞循环的miRNAs是 被发现比其他核酸相对更稳定，使它们成为临床上高度关注的癌症 生物标志物。目前的miRNA分析方法，包括聚合酶链式反应分析，由于其固有的缺陷而面临挑战。 除了假阴性和阳性之外，日常和实验室之间的结果也不一致。我们有 最近发展出一种独特的基于荧光共振能量转移(FRET)的单分子动力学 传感器可实现对未标记DNA和miRNA靶标的高置信度和超灵敏检测 并证明了该传感平台在血清中工作，并完全区分目标和点突变 控制。利用全内反射荧光显微镜，我们证明了该传感器具有 在没有目标的情况下的静态烦躁程度。然而，在目标存在的情况下，传感器形成四个- 路连接，并因此在低和高频率状态之间进行动态切换，这是一个特征 这使得对目标的高置信度检测成为可能。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，两者都在 缓冲液和使用10%血清的添加样品。在这份提案中，我们将重点发展和 通过对非临床患者miRNAs的多路分析测试该平台的高置信度检测 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 用于检测一个样本中的DNA序列。在目标2中，我们将对检测进行表征和验证 用于同时检测针对TNBC的miRNAs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控装置对TNBC miRNAs进行研究。在目标3中，我们将确定丰富的 携带TNBC患者来源的异种移植(PDX)小鼠肿瘤组织和血清中的miRNAs MiRNA测序和应用我们的单分子多重平台检测这些TNBC miRNAs 来自PDX小鼠的血清样本。我们建议的方法提供了许多重要的创新 包括i)无差错检测miRNAs的通用平台，ii)通过单分子实现终极灵敏度 检测，三)同时检测同一样本中的多个生物标志物--允许高置信度 检测，以及iv)不需要靶标记和扩增。因此，这个多路复用的平台具有 在癌症早期诊断中成为一项变革性技术的潜力。通过提供检测 达到或超过最先进的临床分析仪器的灵敏度和信心，建议的方法 可能在临床诊断、癌症评估和个体化癌症治疗方面带来新的倡议。

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.