Programmable low-cost DNA-based platform for viral RNA detection

Lifeng Zhou; Arun Richard Chandrasekaran; Jibin Abraham Punnoose; Gaston Bonenfant; Stephon Charles; Oksana Levchenko; Pheonah Badu; Cassandra Cavaliere; Cara T. Pager; Ken Halvorsen

Two key features of our approach are simplicity and low cost. Our DNA nanoswitches align with the goals of "frugal science" movement, where cost and accessibility to new technologies are valued alongside typical performance metrics. 59, 60 Our nanoswitches cost around 1 penny per reaction and can be stored dry at room temperature for at least one month, and can be delivered globally without transportation or biosafety concerns. The assay consists of few steps and can be performed in a matter of hours with limited laboratory needs (Fig. S17) . Our assay uses a readout by gel electrophoresis, which is relatively inexpensive and already part of the workflow in many labs, which is comparatively simpler than many nanotechnology-based assays involving multiple incubation and wash steps. Improvements to the signal readout could potentially help make this approach even more lab independent. Successful detection with a commercially available bufferless gel system (Fig. S16) takes us a step closer to enabling field deployment of our assay, and sample preparation could be aided by other frugal science approaches such as the "paperfuge" 61 and low-cost thermal cycler. 62 The programmability of our system makes it versatile for a wide variety of viruses, which can be detected with high specificity as we showed for ZIKV and DENV ( Fig. 2A-2B) and for different strains of ZIKV (Fig. 2C-2D) , even in a multiplexed fashion. Here we have focused on single-stranded RNA viruses but assays for other RNA or DNA viruses could likely be developed similarly. The fast construction and purification processes can facilitate rapid production of DNA nanoswitches to detect an emerging viral threat, potentially in as little as 1-2 days from knowledge of the target sequences, limited mostly by oligo synthesis turnaround time (Fig. S17) . Due to the low cost of the test, our assay could also be useful for monitoring viral progression over time in patients, or for testing potentially infected insects or animals. Therefore, with future optimization towards point-of-care clinical applications in resource-limiting environments, the platform we describe here has a potential to improve accuracy and ease of diagnosis in humans, non-human vectors and other animals. Ultimately this can enhance our ability to control spread of infection and more rapidly respond to emerging viral threats, working toward a reduced death toll and economic burden.