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

Detection of RNA viruses in the clinical setting is typically performed using either immunological detection based on enzyme-linked immunosorbent assay (ELISA) to detect IgM antibodies or Nucleic acid testing (NAT) based on a reverse transcription polymerase chain reaction (RT-PCR) assay to detect viral RNA. 5-8 Diagnosing RNA viruses is made challenging by several factors including a limited time window for detection, low or varying viral load, cross-reactive IgM antibodies, and laboratory resources. The detection time windows can vary widely from as short as several days to as long as several months, 5 and molecular detection techniques are usually most reliable if performed within the first two weeks of the disease. 9, 10 Depending on the timing of testing relative to infection, even highly sensitive NAT assays may still produce false negative or positive results. 6, 11 On the other hand, results from IgM serology tests often cannot distinguish related viruses or different strains of the same virus due to cross-reactivity of IgM antibodies, thus leading to false positive results. 12, 13 These detection challenges are further exacerbated when outbreaks occur in low resource settings where infrastructure for these lab-intensive tests can be lacking, accelerating the spread of disease. 7, 14, 15 In response to some of these challenges, new techniques are being developed to detect emerging viruses. Among these are methods that adopt nanoparticles, 16, 17 graphene-based biosensors, 18, 19 and CRISPRbased methods, [20] [21] [22] to name a few. Many of these proposed strategies, although based on cutting-edge technology, require multiple reactions or signal transformation steps that may make them less suitable for a resource-limited POC setting. Here, we addressed these biosensing challenges by developing an assay that uses programmable DNA nanoswitches 23, 24 for detection of viral RNA at clinically relevant levels. We validate our strategy using ZIKV as a proof-of-concept system due to its high global health relevance and continued threat due to its re-emerging mosquito-borne nature. Although ZIKV is typically associated with mild symptoms, it has been linked to devastating birth defects associated with intrauterine infections and the appearance of Guillian-Barré syndrome in adults. 7,10 Moreover, despite significant advances in understanding the molecular biology of ZIKV, there is still a lack of antiviral drugs and vaccines, making robust detection of ZIKV vital to controlling the spread of the disease and implementing early treatments. 25 Our strategy for detecting the presence of viral RNA is based on using DNA nanoswitches that have been designed to undergo a conformational change (from linear to looped) upon binding a target viral RNA sequence. Using ZIKV as an example, the presence of the viral RNA would be indicated by shifted migration of the looped nanoswitch by gel electrophoresis (Fig. 1A) . Importantly, the system is designed to use common nucleic acid staining of the nanoswitch itself that can intercalate thousands of dye molecules to provide an inherently strong signal. Previously, we demonstrated sensitive and specific detection of DNA oligonucleotides 26 and microRNAs (~22 nucleotides long) 27 using this approach. Applied here to viral RNA detection, we solved the challenges of detecting a long viral RNA (>10,000 nucleotides), improving the signal for long RNAs with a new signal multiplication strategy, and developing wor