A Novel Method for Efficiently and Robustly Retrieving Circulating miRNAs

摘要 这个直接的SBIR第二阶段项目旨在开发一种新的技术和相关的分析方法，以高效和 从血液(即血浆或血清)中提取循环中的miRNAs，这可能会改变 MiRNA检测在癌症和心血管疾病等重大疾病的诊断和监测中的应用。 我们的竞争优势在于能够更高效、更可靠地回收循环中的miRNA 目前的提取方法一直是实现miRNA临床检测的关键障碍。这 通过实现特定于序列的捕获与适当的 制作新奇的捕获珠。 循环中的miRNAs是潜在的疾病生物标志物。然而，到目前为止，还没有FDA批准的miRNA 可进行测试。一个瓶颈问题是循环miRNA提取的不可靠。现行提取方法 是基于极性分子在极性表面上的吸附，这种吸附最初是为了提取大量 DNA和RNA分子。然而，由于miRNAs非常小(~22nt)，它们与极性的相互作用 表面要弱得多，因此它们在极性表面上的吸附很容易被其他表面干扰 样品中存在的分子。由于miRNAs在极性表面的弱吸附是一个固有的问题 即使优化了所有其他条件，也不能完全解决这个问题，新的回收循环方法 显然需要基于不同机制的miRNAs。序列特定捕获(SSC)是另一个 一种提取核酸的方法，但历史上它在提取循环核酸时表现不佳 由于缺乏有效的捕获珠子，临床样本中的酸。此外，捕获的成本 目前的方法测得的珠子含量非常高，使得SSC的临床使用成本太高。 最近，我们取得了一项重大突破，开发了一种制造捕获珠子的专有方法 可以将SSC从一个概念转化为一种实用的方法来提取循环中的miRNA， 并且性价比高的方式。因此，我们使用我们的捕获珠子来开发我们自己的SSC分析，并且 进行了一项系统的研究，以检验我们的SSC检测方法在提取循环中的可行性 MiRNAs。我们的SSC分析确实被发现能高效而有力地提取循环中的miRNAs。在研究中， 我们还发现，当前试剂盒的提取效率因血浆样本的不同而变化高达60倍，进一步 确认当前的方法并不可靠。 考虑到目前方法的问题，我们SSC技术的无与伦比的特点，以及我们的成功 早期研究，我们提出这个第二阶段SBIR项目，以进一步开发和验证我们的SSC技术 从血浆/血清中提取循环miRNAs，为其商业化奠定基础。

Abstract This direct SBIR Phase II project aims to develop a novel technology and associated assays for efficiently and robustly extracting circulating miRNAs from blood (i.e. plasma or serum), which could transform the utility of miRNA testing in the diagnosis and monitoring of major diseases such as cancer and cardiovascular disease. Our competitive advantage lies in the ability to retrieve circulating miRNAs more efficiently and robustly than current extraction methods, which has been a critical barrier in implementing miRNA testing clinically. This advantage is achieved by implementing sequence specific capture in conjunction with a propriety method of making novel capture beads. Circulating miRNAs are potential disease biomarkers. However, to date, there are no FDA approved miRNA tests available. A bottleneck problem is unreliable circulating miRNA extraction. Current extraction methods are based on adsorption of polar molecules on polar surfaces, which were originally developed to extract large DNA and RNA molecules. However, because miRNAs are so small (~22nt), their interaction with polar surfaces is much weaker, therefore their adsorption on polar surfaces can be easily interrupted by other molecules present in sample. Since the weak adsorption of miRNAs on polar surfaces is an inherent problem that cannot be fully solved even if all other conditions were optimized, new methods for retrieving circulating miRNAs based on a different mechanism are clearly needed. Sequence-specific capture (SSC) is another method to extract nucleic acids, but historically it performs poorly when used to extract circulating nucleic acids from clinical samples due to the lack of effective capture beads. In addition, the cost of making capture beads by current methods is very high, making SSC too expensive for clinical use. Recently, we made a major breakthrough by developing a proprietary method of making capture beads that can transform SSC from a concept to a practical method for extracting circulating miRNAs in an efficient, robust, and cost-effective manner. Therefore, we employed our capture beads to develop our own SSC assays, and conducted a systematic study to examine the feasibility of using our SSC assay in extracting circulating miRNAs. Our SSC assay was indeed found to efficiently and robustly extract circulating miRNAs. In the study, we also found that extraction efficiency of current kits varied by as much as 60-fold by plasma sample, further confirming that current methods are not robust. Considering the problem of current methods, unparalleled features of our SSC technology, and success of our early study, we propose this Phase II SBIR project to further develop and validate our SSC technology for extracting circulating miRNAs from plasma/serum, providing a foundation for its commercialization.