Amplified detection of viral RNA using catalytic DNA logic circuits

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 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.