Single-molecule counting of cancer biomarker miRNAs in human biofluids

摘要： 这项提议的最终愿景是为快速、强大的单分子开发一个技术平台 分析癌症研究中的microRNA(MiRNA)生物标记物，量化多达200人的小组 患者样本中与癌症相关的miRNAs在30分钟内完成。MiRNAs是非编码RNA，具有 高等真核生物中普遍存在的基因调控功能。超过1,000个miRNA基因占人类总数的2% 基因组，比所有蛋白质编码基因的总和还要多。尽管通常只有22个核苷酸(Nt)的长度， MiRNAs基本上调控所有与人类健康和疾病相关的细胞通路，包括癌症。一次 通过细胞凋亡或可能作为外部信号分子从细胞中释放，循环中的无细胞miRNAs 与大多数其他核酸相比，它们在血液中更稳定，因此它们作为临床癌症受到高度关注 生物标志物。血液传播的无细胞miRNA生物标记物的临床应用受到阻碍， 然而，由于固有的分析前和分析、日常和实验室之间的差异造成的困难 与聚合酶链式反应分析相关联，作为最先进的miRNA生物标记物检测。结果都是假的- 阳性和假阴性miRNA关联是开发有效miRNAs的主要障碍 临床生物标记物。我们最近发明了一种新的、创新的直接单分子技术范例 可克服任何一种miRNA扩增需要的粗生物体液中miRNA的鉴定和计数 或贴标签，承诺克服当前的许多挑战。我们的方法，称为单分子 通过平衡泊松采样识别(SiMREPS)，利用短(9-到10-nT)的结合， 通过荧光标记的DNA阅读器探针将未标记的miRNA固定在玻璃表面 特定的、短的LNA捕获探头。使用全内反射荧光(TIRF)显微镜，两者都是特定的 检测到与固定化目标的结合和非特异性表面结合。然而，均衡 阅读器探针与目标的结合在其动态签名或指纹方面是独特的，这是我们具有的特征 用于实现对假阳性的超高置信度识别。通过改变探头长度 我们具有微调的特异性，包括单核苷酸多态之间的500倍分辨能力。 作为最初的原则证明，我们已经演示了直接原位定量检测突入前列腺癌。 血清中的生物标记物hsa-miR-141，仅对样本进行最小程度的前处理。我们现在建议进一步 开发SiMREPS作为一种平台技术，通过追求以下两个具体目标：(I)我们将开发 优化的分析前样品制备，有效地将内源性miRNAs从血清基质中释放出来，用于 直接进行SiMREPS检测，并将结果与当前需要提取miRNA的聚合酶链式反应分析进行比较。 (Ii)我们将在无透镜显微镜上向微型化和多路复用方向发展SiMREPS。这个项目 将通过打破技术壁垒，为SiMREPS产生变革性影响奠定基础 目前限制了基于血液的miRNA生物标记物的成功开发和临床验证。

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.