Sequencing microRNAs, with single-base discrimination, using a nanopore device and an Osmium tag.

项目总结 这项工作的目标是开发一种廉价、超快速、微创的医学测试来对 对生物体液中的小RNA进行定量。短于200个核苷酸(NT)的小RNA由几个 具有保留调节功能的非编码RNA组。小RNA种类繁多，令人惊讶 血液/尿液稳定。目前的文献中充斥着确定这种小RNA是可靠的研究 大多数疾病的生物标志物。这些研究表明，一个全面的小RNA小组将反映 个人的健康/老龄化状况、对压力的反应、药物变化、发病和/或进展 疾病。目前的小RNA图谱分析方法价格昂贵，涉及广泛的基础设施，并且需要花费大量的时间- 费力且容易出错的样品制备。这些分析也会在转录后遗漏或错误识别。 已知影响RNA稳定性、特异性和功能的修饰碱基(PTM)。相反地, 基于纳米孔的技术承诺了廉价的便携式设备，包括直接RNA分析 PTM碱基的鉴定和快速的检测周转。仍然是唯一可用于商业用途的基于纳米孔的 牛津纳米孔技术公司(ONT)的设备(S)没有资格对短于200的RNA进行测序 本垒打。这种限制阻止了对生物液中发现的大多数(如果不是全部)非编码RNA进行测序，以及 限制了纳米孔技术在微创医学诊断分析中的可用性。 我们将使用商业上可用的纳米孔设备并优化测试条件，以便对 生物体液中的小RNA。Yenos Analytical LLC就是我们开发的这种努力的对象 一种专有技术，可以用笨重的Osmium标签选择性地标记RNA。笨重的标签减缓了 通过纳米孔电压驱动这些RNA替代物的移位，并产生可读的离子电流vs 时间(i-t)信号，用作特定RNA分子的指纹。由A阶段I资助的近期工作 SBIR NHGRI赠款还表明，这些标记的RNA使用 ONT设备(S)包含测序信息。为了增加慢速项目的数量，我们将 利用结合RNA的非共价探针。这种结合将减少RNA的有效负电荷。 在纳米孔进入附近的电压降的影响下，将其缓慢释放。AS 非共价探针，我们将评价阳离子多胺，如亚精胺和精胺，阳离子 多肽和RNA结合蛋白。核酸选择性标记技术与生物信息学的结合 基于纳米孔器件的检测是一种新颖的检测方法，由Yenos Analytical LLC单枪匹马完成。我们 建议优化这一技术组合，以从生物液中提取小RNA。是这样的 医学化验将直接影响医学检测、疾病的预防、诊断和治疗，成为现实 个性化医疗的承诺，降低不断膨胀的医疗保健成本，并使诊断 在基础设施最少的偏远地区进行医学测试。

PROJECT SUMMARY The goal of this work is to develop an inexpensive, ultra-fast, minimally-invasive medical test to sequence and quantitate small RNAs from biological fluids. Small RNAs, shorter than 200 nucleotides (nt) comprise several groups of non-coding RNAs with preserved regulatory functions. Small RNAs are abundant and surprisingly stable in blood/urine. Current literature is flooded with studies identifying such small RNAs as reliable biomarkers for most diseases. These studies suggest that a comprehensive small RNA panel will reflect the health/aging status of an individual, response to stress, changes in medication, onset and/or progress of disease. Current small RNA profiling assays are expensive, involve extensive infrastructure, and employ time- consuming and error-prone sample preparation. These assays also miss or misidentify post-transcriptionally modified bases (PTM) which are known to influence RNA stability, specificity, and function. On the contrary, nanopore-based technologies promise inexpensive portable devices, direct RNA profiling including identification of PTM bases, and fast assay turn-around. Still the only commercially available nanopore-based device(s) from Oxford Nanopore Technologies (ONT) are not qualified to sequence RNAs shorter than 200 bases. Such limitation prevents sequencing of most, if not all, non-coding RNAs found in biological fluids, and limits the usability of nanopore technology in minimally-invasive medical diagnostic assays. We will use commercially available nanopore devices and optimize the testing conditions in order to profile small RNAs in biological fluids. Yenos Analytical LLC is singled out for such effort, as we have developed proprietary technology to selectively tag RNAs with a bulky Osmium label. The bulky label slows down the voltage-driven translocation of these RNA surrogates via a nanopore, and yields a readable ion current vs time (i-t) signal that serves as the fingerprint of the specific RNA molecule. Recent work funded by a Phase I SBIR NHGRI grant has also shown that some of the slowest translocations of these tagged RNAs using the ONT device(s) embody sequencing information. In order to increase the number of the slow events, we will exploit non-covalent probes that bind RNA. This binding will reduce the effective negative charge of the RNA molecule, and slowly release it under the influence of the voltage drop in proximity to the nanopore’s entry. As non-covalent probes, we will evaluate cationic polyamines such as spermidine and spermine, cationic peptides, and RNA binding proteins. The combination of nucleic acid selective labeling technology with nanopore-device based detection is novel, and single-handedly carried out by Yenos Analytical LLC. We propose to optimize this combination of technologies towards profiling small RNAs from biological fluids. Such a medical assay will directly impact medical testing, prevention, diagnosis, and cure of disease, materialize the promise of personalized medicine, reduce the ballooning cost of health care, and enable diagnostic medical tests in remote locations with minimal infrastructure.