DREAM Sentinels: Multiplexable and programmable cell-free ADAR-mediated RNA sensing platform (cfRADAR) for quick and scalable response to emergent viral threats

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This project seeks to address a critical public health need: the rapid, precise detection of viral RNA, the genetic blueprint of many viruses, including the SARS-CoV-2 virus responsible for COVID-19. The approach is to develop a cutting-edge tool known as "cell-free reprogrammable adenosine deaminases acting on RNA" (cfRADAR). This tool functions as a highly adaptable virus detective, designed to be quickly modified to recognize new or mutating viruses. Beyond its primary function, cfRADAR is projected to have broader societal impacts. Firstly, it can significantly improve healthcare outcomes by providing real-time information on viral prevalence and spread, enabling earlier detection, prompt responses to outbreaks, and ultimately reducing the risk of transmission. This reliable, rapid diagnostic tool can enhance our capabilities to prevent, control, and treat infectious diseases. Secondly, the project will engage in outreach and educational activities, offering hands-on experiences and interactive learning for students and teachers at various levels. Therefore, cfRADAR's development stands to significantly benefit both local and global communities, serving the national interest by advancing health, prosperity, and welfare.This project's primary objective is the development of cfRADAR (cell-free reprogrammable adenosine deaminases acting on RNA), an RNA detection technology leveraging the properties of adenosine deaminases acting on RNA (ADAR). The project will tackle six main goals: 1) developing singleplex and 2) multiplex cfRADAR assays, 3) creating cfRADAR-based logic circuits for group testing, 4) formulating field deployable lyophilized cfRADAR systems, 5) designing a field deployable portable analyzer, and 6) validating cfRADAR in an infrastructure-free setting. The cfRADAR platform offers advantages such as high sensitivity, reprogrammability, modularity, and field deployability. With its single-target transcript to multiple sensor molecule editing, cfRADAR possesses high sensitivity towards low-abundance RNA targets. Its modularity and reprogrammability allow for the use of Boolean logical gates, offering the detection of multiple RNA targets simultaneously. The exploration of lyophilized cell-free reagents and portable analyzers enhances the platform's field deployment, making it an effective tool for timely response against viral threats. The project could revolutionize point-of-care diagnosis, group tests, and biosurveillance by introducing a sensitive, reprogrammable, and scalable sensing platform.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.