Amplified detection of viral RNA using catalytic DNA logic circuits

 描述(由申请人提供)：我们建议开发等温、基于分子逻辑的病毒RNA扩增检测方法，以创建一个强大、灵活的检测框架，用于临床样本中的病毒检测。该框架的优点包括能够检测多个目标并将信息组合在单个读数中以减少误报，能够将低目标浓度等温放大为可检测信号，用于现场应用的直接操作，以及能够实现快速 针对新出现的病原体菌株进行重定向。对于实用的病毒检测分析来说，这些是非常理想的特性。发展简单、恒温的病毒检测方法具有重大的医学意义，因为病毒爆发引起的疾病是全球重大的医疗负担。例如，流感病毒既是季节性疫情的罪魁祸首，也是H5N1等高致病性毒株的罪魁祸首。这些病毒株正在不断进化，因此至关重要的是，可以针对新的人类适应株快速重定向分析框架。由于气候变化，黄毒科等热带病毒的发病率增加，进一步推动了美国对实用、准确的病毒检测分析的需求。我们将开发基于逻辑的等温病毒检测方法，将我们在体外成功演示的催化分子逻辑电路与等温RNA扩增相结合。我们建议的平台将使用滚圈放大等技术来检测具有医学相关病毒滴度的多个目标，并将放大的信号馈入多输入分子逻辑电路以产生集成响应，我们将使用荧光测量来检测该响应。该平台的成功开发将在针对血清中RNA寡聚体的生物现实模型分析中得到证明。然后，这些化验将用于检测和分型临床样本中的登革热感染，并根据反应动力学得出病毒载量的估计。这些目标将需要开发具有低背景响应和高信噪比的分子逻辑器件，以抵抗生物液体中的降解，增强构建高质量分子逻辑器件的纯化方案，以及适用于临床样本的样品制备方案。如果成功，我们的检测将达到医学上相关的检测极限 而且操作简单，很容易针对新的病毒株进行重定向。除了建议的病毒检测研究的直接应用外，我们的检测框架将广泛适用于生物医学研究的其他领域。例如，通过合适的样品制备方案，我们的设备可以针对基因组DNA，从而能够检测携带志贺毒素的大肠杆菌等病原菌。此外，我们分析的分子逻辑组件将引起DNA纳米技术社区的兴趣，并在自主基因分析中应用。因此，拟议的工作将对科学和医学都具有广泛的普遍好处。

 DESCRIPTION (provided by applicant): We propose to develop isothermal, molecular logic-based assays for the amplified detection of viral RNA, to create a powerful, flexible assay framework for virus detection in clinical samples. The advantages of this framework will include the ability to sense multiple targets and combine the information in a single readout to reduce false positives, the ability to isothermally amplify low target concentrations into a detectable signal, straightforward operation for application in the field, and a simple design to enable rapid retargeting against emerging pathogen strains. These are highly desirable properties for a practical virus detection assay. The development of simple, isothermal virus detection assays has great medical significance, as the illnesses caused by viral outbreaks represent a significant global healthcare burden. For example, influenza viruses are responsible both for seasonal outbreaks and for highly pathogenic strains such as H5N1. These viral strains are continually evolving, making it essential that assay frameworks can be rapidly retargeted against new human-adapted strains. The need for practical, accurate virus detection assays in the United States is further driven by increased incidence of tropical viruses such as Flaviviridae, due to climate change. We will develop isothermal, logic-based virus detection assays by integrating catalytic molecular logic circuits, which we have successfully demonstrated in vitro, with isothermal RNA amplification. Our proposed platform will use techniques such as rolling circle amplification to sense multiple targets at medically relevant viral titers and feed the amplified signals into multi-input molecular logic circuits to produce an integrated response, which we will detect using fluorescence measurements. Successful development of this platform will be demonstrated in biologically realistic model assays against RNA oligomers in serum. The assays will then be used to detect and serotype dengue infections in the clinical samples and to derive estimates of the viral load based on the kinetics of the response. These goals will require the development of molecular logic devices with low background responses and high signal-to-noise ratios that resist degradation in biological fluids, enhanced purification protocols for the construction of high-quality molecular logic devices, and suitable sample preparation protocols for clinical samples. If successful, our assays will achieve medically relevant limits of detection and will be straightforward to perform and easy to retarget against new viral strains. Beyond the immediate applications of the proposed research for virus detection, our assay framework will be broadly applicable in other areas of biomedical research. For example, with suitable sample preparation protocols our devices could be targeted against genomic DNA, enabling the detection of pathogenic bacteria such as Shiga toxin-bearing E. coli. Furthermore, the molecular logic components of our assays will be of interest to the DNA nanotechnology community, with applications in autonomous theranostics. Thus the proposed work will have widespread general benefit both to science and to medicine.