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

Based on our earlier observation that DNA nanoswitches can detect microRNAs (~22 nucleotides) at the sub-picomolar scale 27 without amplification, we wanted to assess the sensitivity of our approach for ZIKV RNA detection. We reacted the DNA nanoswitch mixture with different amounts of fragmented RNA in a 12-hour annealing temperature ramp from 40 °C to 25 °C. The results showed visible detection for ZIKV RNA as low as 12.5 pg (~3.5 attomole) ( Fig. 1G and S8 ) in a 10 µL reaction volume. Consistent with Fig. 1F , the approach based on using a nanoswitch mix outperformed the highest performing nanoswitch used as a single agent, which had visible detection to about 50 pg (~14 attomole) (Fig. S9) . Another key requirement for a clinical viral detection assay is specificity. Since ZIKV and Dengue virus (DENV) have overlapping geographical distributions and clinical symptoms, infection with either virus may result in clinical misdiagnosis. 34 Serological diagnostic assays are known to show antibody crossreactivity between the two viruses, and DENV has some similarity to ZIKV in its envelope protein 12 and genome sequence. 21, 31 To test the specificity of our approach, we designed a similar panel of nanoswitches to detect DENV (Fig. S10) . Using the pooled nanoswitches specific for ZIKV and DENV, we mixed each set with in vitro transcribed RNA from each virus and found perfect specificity, with each assay only detecting its correct target RNA ( Fig. 2A) . Using the programmability of the nanoswitch, we further demonstrated a multiplexed system for simultaneous detection of ZIKV and DENV. In this case we modified the DENV responsive nanoswitches to form a smaller loop size (Fig. S11) , causing two distinct detection bands to migrate to different positions in the gel. Specifically, ZIKV RNA-nanoswitch complex migrated slower/higher in the gel, while the complex of DENV RNA and the nanoswitch migrated faster/lower in the gel (Fig. 2B) . Therefore, in a single reaction our nanoswitch showed differential and specific detection of ZIKV and DENV RNA. By programming different loop sizes for different targets, this assay can be expanded for up to five viral targets. 27 The copyright holder for this preprint (which was not peer-reviewed) is the .