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

The intrinsically high signal of our nanoswitches is enhanced here with a new "target multiplication" strategy where we use viral RNA fragmentation to multiply the number of targets, and thus increase the signal intensity. Using this approach, we reached near-clinical levels of detection in urine without the use of enzyme-mediated amplification strategies. This is of significance because enzymes can be key drivers of assay cost and complexity due to requirements including cold storage/transportation, special buffers and reagents, and strict operating temperatures. These factors make enzymatic assays difficult for field use or for use in low resource areas without modern lab infrastructure. Despite these challenges, most currently available techniques rely on enzyme triggered amplification. 7, 21, 57 For our assay, we demonstrated compatibility and dramatic signal improvement with an optional enzymatic preamplification step (Fig. 4C-4E) . However, we believe that further improvements should enable complete coverage of the clinical range without enzymes. A 30 mL sample of urine from a ZIKV-infected patient would contain from 10 5 to 10 9 copies of viral RNA, 58 theoretically surpassing our current detection limit. Efficient sample preparation using a viral RNA extraction kit (Fig. 4A-4B) , for example, could facilitate use with our DNA nanoswitch assay.