Nano-Flares for the Intracellular Detection of miRNA in Living Cells

描述（由申请人提供）：在这项小企业创新研究（SBIR）资助的赞助下，AuraSense LLC将开发一种新型纳米技术“纳米耀斑”，用于实时检测活细胞中的低丰度microRNA（miR）物种。MicroRNA是短的（长度约22个核苷酸）非蛋白质编码RNA序列，其在调节例如在发育期间或在恶性转化期间发生的多种细胞过程中起关键作用。独特的miR正在被评估为无数疾病的新生物标志物，并被探索为新的治疗靶点。没有现有的商业技术能够在活细胞中实时检测细胞内RNA靶序列，包括miR。纳米耀斑技术为检测细胞内miR靶点提供了新的机会，并将确定如何更有效地检测这些独特的RNA，提高我们对miR功能的认识，并最终靶向改善人类健康。Nano-flare技术是基于DNA寡核苷酸的独特共轭特性，其可以密集地负载在金纳米颗粒（DNA-Au NPs）的表面上。重要的是，DNA-Au纳米颗粒普遍被细胞吸收，并且可以被定制为靶向特定的细胞内RNA靶序列。当遇到靶RNA序列时，纳米耀斑打开荧光信号，该荧光信号可以使用荧光显微镜在单个细胞中检测到，或者使用荧光激活细胞分选（FACS）在数百万个细胞中进行平均。与采用聚合酶链式反应（PCR）的方法相反，使用Nano-flares技术询问的细胞是活的，因此可用于下游应用。 我们建议使用Nano-flare技术检测和定量活前列腺癌细胞中的miR和信使RNA（mRNA）序列。重要的是，我们将开发一种细胞模型，其中可以用小分子操纵特定的miR和mRNA靶标，以评估Nano-flare系统检测和定量高丰度和低丰度靶标miR相对变化的能力。使用Nano-flare系统获得的结果将直接与使用PCR技术获得的结果进行比较，PCR技术是目前检测这些目标的商业基准。项目的成功将为使用FACS和共聚焦荧光显微镜检测细胞内miR靶点提供一个强大的平台。通过使用qPCR来确认Nano-flare结果，该项目还将为活细胞内miR靶序列的实时检测设定新的分析基准，并定义单细胞和整体测量中的“低丰度”miR代表什么。最后，这项研究的完成将使这项技术更接近商业化，并增加研究人员和临床医生的潜力，最终改善患者的健康。 公共卫生相关性：尽管microRNA在人类疾病过程中的重要性已经认识到并日益增加，但目前还没有方法来实时检测活人类细胞中microRNA水平的变化。AuraSense，LLC正在开发的Nano-flare技术将提供这种能力，并且代表了一种新技术，该技术被设计用于在活细胞中显著地提供细胞内microRNA靶标的PCR样表型表征。项目的成功完成将为更广泛的研究界提供一种独特的技术工具，并最终为临床界提供改善人类健康的技术工具。

DESCRIPTION (provided by applicant): Under the auspices of this small business innovation research (SBIR) grant, AuraSense LLC will develop a novel nanotechnology, "Nano-flares", for the detection of low abundance microRNA (miR) species in living cells in real-time. MicroRNAs are short (~22 nucleotides in length) non-protein coding RNA sequences that play a key role in regulating diverse cellular processes that occur, for instance, during development or during malignant transformation. Unique miRs are being evaluated as novel biomarkers for myriad diseases, and explored as novel therapeutic targets. No existing commercial technology is capable of detecting intracellular RNA target sequences, including miRs, in real-time in living cells. Nano-flares technology provides new opportunities for the detection of intracellular miR targets and will identify how these unique RNAs can be more effectively detected, improving our knowledge of miR function, and, ultimately targeted to improve human health. Nano-flare technology is based upon the unique conjugate properties of DNA oligonucleotides which can be densely loaded on the surface of gold nanoparticles (DNA-Au NPs). Importantly, DNA-Au NPs are universally taken up by cells and can be tailored to target specific intracellular RNA target sequences. Upon encountering a target RNA sequence, Nano-flares turn on a fluorescent signal which can be detected in single cells using fluorescent microscopy, or averaged over millions of cells using fluorescence activated cell sorting (FACS). In contrast to methods which employ the polymerase chain reaction (PCR), cells interrogated using the Nano-flares technology are alive and, thus, available for downstream applications. We propose using the Nano-flare technology to detect and quantify miR and messenger RNA (mRNA) sequences in live prostate cancer cells. Importantly, we will develop a cell model where specific miR and mRNA targets can be manipulated with small molecules in order to evaluate the ability of the Nano-flare system to detect and quantify relative changes in high and low abundance target miR. Results obtained with the Nano-flare system will be directly compared to those obtained using PCR techniques, the current commercial benchmark for detection of these targets. Project success will result in a robust platform for the detection of intracellular miR targets using FACS and confocal fluorescent microscopy. By using qPCR to confirm the Nano-flare results, this project will also set new analytical benchmarks for the real-time detection of miR target sequences inside of live cells, and define what 'low abundance' miR represents in single cells and in ensemble measurements. Finally, completion of this study will bring this enabling technology closer to commercialization and increase the potential for researchers and clinicians, ultimately improving patient health. PUBLIC HEALTH RELEVANCE: Despite the realized and increasing importance of microRNAs in human disease processes, no current methodology exists to detect changes in microRNA levels in live human cells in real-time. Nano-flare technology being developed by AuraSense, LLC will provide this capability, and represents a novel technology that is designed to provide PCR-like phenotypic characterization of intracellular microRNA targets, significantly, in living cells. Successful project completion will provide a unique technological tool for the study of microRNAs to the broader research community, and, ultimately, the clinical community to improve human health.