Single-molecule counting of cancer biomarker miRNAs in human biofluids

人体生物体液中癌症生物标志物 miRNA 的单分子计数

• 批准号: 9233284
• 负责人: NILS G WALTER
• 金额: $ 23.25万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-03 至 2020-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9233284
• 关键词: Apoptosis; Behavior; Benchmarking; Binding; Biological Assay; Biological Markers; Blood; Blood specimen; Cancer Diagnostics; Cancer Patient; Cancer Prognosis; Cells; Clinic; Clinical; Code; Complex; DNA; Detection; Development; Devices; Discrimination; Disease; Endocrine; Endopeptidase K; Equilibrium; Eukaryota; Fingerprint; Fluorescence; Fluorescence Microscopy; Foundations; Genes; Glass; Health; Human; Human Genome; Immobilization; In Situ; Individual; Kinetics; Label; Length; Malignant Neoplasms; Malignant neoplasm of prostate; Mammals; Messenger RNA; Methodology; Methods; MicroRNAs; Microfluidic Microchips; Microfluidics; Microscope; Miniaturization; Monitor; Nucleic Acids; Nucleotides; Pathway interactions; Patients; Plasma; Positioning Attribute; Preparation; Process; Proteins; Quantitative Reverse Transcriptase PCR; RNA; RNA Interference; Reader; Recovery; Regulator Genes; Regulatory Pathway; Reproducibility; Research; Sampling; Sensitivity and Specificity; Serum; Signaling Molecule; Single Nucleotide Polymorphism; Source; Specificity; Specimen; Surface; System; Technology; Time; Treatment Protocols; Untranslated RNA; Validation; Variant; Vision; Work; anticancer research; base; cancer biomarkers; cancer diagnosis; cell transformation; circulating microRNA; clinical application; clinical biomarkers; clinical development; cohort; cost; improved; innovative technologies; interest; lens; liquid biopsy; malignant breast neoplasm; microRNA biomarkers; minimally invasive; nanolitre; novel; oncology; polydimethylsiloxane; response; single molecule; tool

ABSTRACT: The ultimate vision of this proposal is to develop a technology platform for the rapid, robust single molecule analysis of microRNA (miRNA) biomarkers in cancer research that quantifies a panel of up to two hundred cancer-associated miRNAs in a patient sample in under 30 minutes. miRNAs are non-coding RNAs with pervasive gene regulatory function in higher eukaryotes. Over 1,000 miRNA genes compose ~2% of the human genome, more than all protein-coding genes combined. Although typically only 22 nucleotides (nt) in length, miRNAs regulate essentially all cellular pathways relevant to human health and disease, including cancer. Once released from cells through apoptosis or possibly as external signaling molecules, circulating, cell-free miRNAs are more stable in blood than most other nucleic acids, rendering them of high interest as clinical cancer biomarkers. The validation of blood-borne cell-free miRNA biomarkers as clinically useful has been hindered, however, by difficulties due to inherent, both pre-analytic and analytic, day-to-day and lab-to-lab variations associated with PCR assays as the state-of-the-art for miRNA biomarker detection. The resulting both false- positive and false-negative miRNA associations present a major barrier to developing miRNAs as validated clinical biomarkers. We recently invented a novel, innovative technology paradigm for the direct single-molecule identification and counting of miRNAs in crude biofluids that overcomes any need for either miRNA amplification or labeling, promising to overcome many of the current challenges. Our approach, termed Single-Molecule Recognition through Equilibrium Poisson Sampling (SiMREPS), exploits the binding of a short (9- to 10-nt), fluorescently labeled DNA reader probe to an unlabeled miRNA immobilized on a glass surface through a specific, short LNA capture probe. Using total internal reflection fluorescence (TIRF) microscopy, both specific binding to the immobilized target and non-specific surface binding are detected. However, the equilibrium binding of the reader probe to the target is distinctive in its kinetic signature, or fingerprint, a feature we have used to achieve ultrahigh-confidence discrimination against false positives. Through varying the probe length we have fine-tuned specificity, including the >500-fold discrimination between single nucleotide polymorphisms. As initial proof-of-principle, we have demonstrated the direct in situ quantification of spiked-in prostate cancer biomarker hsa-miR-141 in blood serum, after only minimal pre-treatment of a sample. We now propose to further develop SiMREPS as a platform technology, by pursuing the following two Specific Aims: (i) We will develop an optimized pre-analytic sample prep that efficiently liberates endogenous miRNAs from their serum matrix for direct SiMREPS detection, and benchmark the results against current PCR assays requiring miRNA extraction. (ii) We will develop SiMREPS toward miniaturization and multiplexing on a lens-free microscope. This project will lay the foundation for SiMREPS to have a transformative impact by breaking down the technology barriers currently limiting the successful development and validation of blood-based miRNA biomarkers for the clinic.

抽象的： 该建议的最终愿景是为快速，健壮的单分子开发技术平台 癌症研究中microRNA（miRNA）生物标志物的分析，该生物标志物量化了多达200的面板 在30分钟内，患者样品中与癌症相关的miRNA。 miRNA是非编码RNA， 较高真核生物中普遍的基因调节功能。超过1,000个miRNA基因占人类的约2％ 基因组，比所有蛋白质编码基因组合多。尽管通常只有22个核苷酸（NT），但 miRNA基本上调节与人类健康和疾病有关的所有细胞途径，包括癌症。一次 通过细胞凋亡或可能为外部信号分子释放，无细胞的miRNA循环 与大多数其他核酸相比，血液中的血液更稳定，使其引起人们的兴趣，因为临床癌症 生物标志物。作为临床上有用的无血源细胞生物标志物的验证已受到阻碍， 然而，由于固有的固定和分析，日常和实验室对象变化，由于固有的固有和分析的困难 与PCR分析作为miRNA生物标志物检测的最新方法相关。由此产生的两个错误 积极和错误的阴性miRNA关联是开发miRNA的主要障碍，如经过验证 临床生物标志物。我们最近发明了一种直接单分子的小说，创新的技术范式 在克服了对任何一种miRNA扩增的任何需求的粗体生物流体中的miRNA的识别和计数 或标签，有望克服当前许多挑战。我们的方法称为单分子 通过平衡泊松采样（SIMREPS）识别，利用短（9至10-NT）的结合 通过荧光标记DNA读取器探测到固定在玻璃表面上的未标记的miRNA 特定的短LNA捕获探针。使用总内反射荧光（TIRF）显微镜，两者均 检测到与固定靶标和非特异性表面结合的结合。但是，平衡 读取器探测器与目标的结合在其动力学签名或指纹上是独特的，这是我们具有的特征 用于实现对误报的超高信心歧视。通过改变探针长度 我们具有微调的特异性，包括单核苷酸多态性之间的> 500倍歧视。 作为初始证明，我们已经证明了尖刺的前列腺癌的原位定量 仅在最小的样品预处理后，血清中生物标志物HSA-MIR-141。我们现在建议进一步 通过追求以下两个具体目的，将SIMREP作为平台技术发展：（i）我们将开发一个 优化的预分析样品准备有效地从其血清基质中释放内源miRNA 直接SIMREP检测，并根据需要miRNA提取的当前PCR分析进行基准测试。 （ii）我们将在无镜头显微镜上开发simreps朝着微型化和多路复用。这个项目 将为Simreps打破技术障碍而产生变革性影响的基础 目前，限制了基于血液的miRNA生物标志物在诊所的成功开发和验证。